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1.  Does prone positioning improve oxygenation and reduce mortality in patients with acute respiratory distress syndrome? 
The emergence of computed tomography imaging more than 25 years ago led to characterization of acute respiratory distress syndrome (ARDS) as areas of relatively normal lung parenchyma juxtaposed with areas of dense consolidation and atelectasis. Given that this heterogeneity is often dorsally distributed, investigators questioned whether care for ARDS patients in the prone position would lead to improved mortality outcomes. This clinical review discusses the physiological rationale and clinical evidence supporting prone positioning in treating ARDS, in addition to its complications and contraindications.
PMCID: PMC4173887  PMID: 24927376
2.  Airway pressure and transpulmonary pressure during high-frequency oscillation for acute respiratory distress syndrome 
High-frequency oscillation is a novel form of ventilation increasingly being used to treat refractory hypoxic respiratory failure resulting from acute lung injury or acute respiratory distress syndrome. Although there is no known relationship between airway pressure and transpulmonary pressure during conventional mechanical ventilation, no study has attempted to determine transpulmonary pressure during high-frequency oscillation.
High-frequency oscillation (HFO) is used for the treatment of refractory hypoxic respiratory failure.
To demonstrate that the mean transpulmonary pressure (PL) cannot be inferred from mean airway pressure (mPaw).
In seven patients already undergoing HFO for refractory acute respiratory distress syndrome, esophageal pressure (Pes) was measured using an esophageal balloon catheter. Pleural pressure (Ppl) and PL were calculated from Pes.
In the seven patients (mean [± SD] age 59±9 years) treated with HFO at 5±1 Hz and amplitude 75±10 cmH2O, the mPaw was 27±6 cmH2O, Ppl was 9±6 cmH2O and PL was 18±11 cmH2O. Successful catheter placement and measurement of Pes occurred in 100% of subjects. There was no correlation between PL and mPaw. The majority of subjects required hemodynamic support during the use of HFO; the frequency and degree of support during the study period was no different than that before the study.
The present report is the first to describe measuring Pes and calculating Ppl during HFO for acute respiratory distress syndrome. While both current guidelines and recent trials have titrated treatment based on mPaw and oxygenation, there is wide variability in PL during HFO and PL cannot be predicted from mPaw.
PMCID: PMC4128514  PMID: 24137575
Adult; Critical care; High-frequency oscillation; Lung; Mechanical ventilation; Pneumonia
3.  Role of Cells and Mediators in Exercise-Induced Bronchoconstriction 
PMCID: PMC3763828  PMID: 23830127
Asthma; Eicosanoid; Eosinophil; Exercise-induced bronchoconstriction; Leukotriene; Mast cell; Phospholipase; Prostaglandin
4.  Regulation and Function of Epithelial Secreted Phospholipase A2 Group X in Asthma 
Rationale: Indirect airway hyperresponsiveness (AHR) is a fundamental feature of asthma that is manifest as exercise-induced bronchoconstriction (EIB). Secreted phospholipase A2 group X (sPLA2-X) plays a key role in regulating eicosanoid formation and the development of inflammation and AHR in murine models.
Objectives: We sought to examine sPLA2-X in the airway epithelium and airway wall of patients with asthma, the relationship to AHR in humans, and the regulation and function of sPLA2-X within the epithelium.
Methods: We precisely phenotyped 34 patients with asthma (19 with and 15 without EIB) and 10 normal control subjects to examine in vivo differences in epithelial gene expression, quantitative morphometry of endobronchial biopsies, and levels of secreted protein. The regulation of sPLA2-X gene (PLA2G10) expression was examined in primary airway epithelial cell cultures. The function of epithelial sPLA2-X in eicosanoid formation was examined using PLA2 inhibitors and murine tracheal epithelial cells with Pla2g10 deletion.
Measurements and Main Results: We found that sPLA2-X protein is increased in the airways of patients with asthma and that epithelial-derived sPLA2-X may be increased in association with indirect AHR. The expression of sPLA2-X increases during in vitro epithelial differentiation; is regulated by inflammatory signals including tumor necrosis factor, IL-13, and IL-17; and is both secreted from the epithelium and directly participates in the release of arachidonic acid by epithelial cells.
Conclusions: These data reveal a relationship between epithelial-derived sPLA2-X and indirect AHR in asthma and that sPLA2-X serves as an epithelial regulator of inflammatory eicosanoid formation. Therapies targeting epithelial sPLA2-X may be useful in asthma.
PMCID: PMC3735246  PMID: 23614662
airway hyperresponsiveness; asthma; eicosanoid; epithelial cell; secretory phospholipase A2
5.  Hypernatremia and intracranial pressure: more questions than answers 
Critical Care  2013;17(1):401.
The observational literature suggests that hypernatremia is associated with worse outcomes in patients with traumatic brain injury. In a previous issue of Critical Care, Wells and colleagues add to this literature by failing to show an association between hypernatremia and reduced intracranial pressure. However, we must bear in mind many limitations of observational methods before eliminating hyperosmolar therapy from our armamentarium.
PMCID: PMC4057414  PMID: 23294598
6.  Epithelial Regulation of Eicosanoid Production in Asthma 
Alterations in the airway epithelium have been associated with the development of asthma in elite athletes and in subjects that are susceptible to exercise-induced bronchoconstriction (EIB). The syndrome of EIB refers to acute airflow obstruction that is triggered by a period of physical exertion. Asthmatics who are susceptible to EIB have increased levels of cysteinyl leukotrienes (CysLTs, i.e., LTs C4, D4, and E4) in induced sputum and exhaled breath condensate, and greater shedding of epithelial cells into the airway lumen. Exercise challenge in individuals susceptible to this disorder initiates a sustained increase in CysLTs in the airways, and secreted mucin release and smooth muscle constriction, which may be mediated in part through activation of sensory nerves. We have identified a secreted phospholipase A2 (sPLA2) with increased levels in the airways of patients with EIB called sPLA2 group X (sPLA2-X). We have found that sPLA2-X is strongly expressed in the airway epithelium in asthma. Further, we discovered that transglutaminase 2 (TGM2) is expressed at increased levels in asthma and serves as a regulator of sPLA2-X. Finally, we demonstrated that sPLA2-X acts on target cells such as eosinophils to initiate cellular eicosanoid synthesis. Collectively, these studies identify a novel mechanism linking the airway epithelium to the production of inflammatory eicosanoids by leukocytes.
PMCID: PMC3627426  PMID: 23323271
7.  Hypernatremia in patients with severe traumatic brain injury: a systematic review 
Hypernatremia is common following traumatic brain injury (TBI) and occurs from a variety of mechanisms, including hyperosmotic fluids, limitation of free water, or diabetes insipidus. The purpose of this systematic review was to assess the relationship between hypernatremia and mortality in patients with TBI.
We searched the following databases up to November 2012: MEDLINE, EMBASE, and CENTRAL. Using a combination of MeSH and text terms, we developed search filters for the concepts of hypernatremia and TBI and included studies that met the following criteria: (1) compared hypernatremia to normonatremia, (2) adult patients with TBI, (3) presented adjusted outcomes for mortality or complications.
Bibliographic and conference search yielded 1,152 citations and 11 abstracts, respectively. Sixty-five articles were selected for full-text review with 5 being included in our study. All were retrospective cohort studies totaling 5,594 (range 100–4,296) patients. There was marked between-study heterogeneity. The incidence of hypernatremia ranged between 16% and 40%. Use of hyperosmolar therapy was presented in three studies (range 14-85% of patients). Hypernatremia was associated with increased mortality across all four studies that presented this outcome. Only one study considered diabetes insipidus (DI) in their analysis where hypernatremia was associated with increased mortality in patients who did not receive DDAVP.
Although hypernatremia was associated with increased mortality in the included studies, there was marked between-study heterogeneity. DI was a potential confounder in several studies. Considering these limitations, the clinical significance of hypernatremia in TBI is difficult to establish at this stage.
PMCID: PMC3826846  PMID: 24196399
Traumatic brain injury; Hypernatremia; Hyperosmolar therapy; Hypertonic saline
8.  Absence of α4 but not β2 integrins restrains development of chronic allergic asthma using mouse genetic models 
Experimental hematology  2009;37(6):715-727.e3.
Chronic asthma is characterized by ongoing recruitment of inflammatory cells and airway hyperresponsiveness leading to structural airway remodeling. Although α4β1 and β2 integrins regulate leukocyte migration in inflammatory diseases and play decisive roles in acute asthma, their role has not been explored under the chronic asthma setting. To extend our earlier studies with α4Δ/Δ and β2−/− mice, which showed that both a4 and b2 integrins have nonredundant regulatory roles in acute ovalbumin (OVA)-induced asthma, we explored to what extent these molecular pathways control development of structural airway remodeling in chronic asthma.
Materials and Methods
Control, α4Δ/Δ, and β2−/−mouse groups, sensitized by intraperitoneal OVA as allergen, received intratracheal OVA periodically over days 8 to 55 to induce a chronic asthma phenotype. Post-OVA assessment of inflammation and pulmonary function (airway hyperresponsiveness), together with airway modeling measured by goblet cell metaplasia, collagen content of lung, and transforming growth factor β1 expression in lung homogenates, were evaluated.
In contrast to control and β2−/− mice, α4Δ/Δ mice failed to develop and maintain the composite chronic asthma phenotype evaluated as mentioned and subepithelial collagen content was comparable to baseline. These data indicate that β2 integrins, although required for inflammatory migration in acute asthma, are dispensable for structural remodeling in chronic asthma.
α4 integrins appear to have a regulatory role in directing transforming growth factor β-induced collagen deposition and structural alterations in lung architecture likely through interactions of Th2 cells, eosinophils, or mast cells with endothelium, resident airway cells, and/or extracellular matrix.
PMCID: PMC3696022  PMID: 19463772
9.  An update on the role of leukotrienes in asthma 
Purpose of review
Leukotrienes (LT)s are lipid mediators involved in the pathogenesis of asthma. There is significant new information about the actions of LTs in asthma, and the evolving role of anti-LT therapies. We review recent findings on regulation of LT synthesis, biological function of LTs in disease models, and use of LT modifiers in clinical practice.
Recent findings
Our understanding of the regulation of LT synthesis at a molecular level has greatly advanced. Recent evidence indicates that genetic variation in the leukotriene synthetic pathway affects the clinical response to LT modifiers. The participation of LTB4 in the allergic sensitization process in animal models suggests a larger role for LTB4 in asthma. Preclinical and in vitro models suggest that the cysteinyl LT (CysLT)s are important in airway remodeling. LTs are key mediators of exercise-induced bronchoconstriction (EIB) with recent studies demonstrating that LT modifiers reduce the severity of EIB during short-term and long-term use.
LTs are clearly involved in airway inflammation and certain clinical features of asthma. Evolving evidence indicates that LTB4 has an important role in the development of asthma, and that CysLTs are key mediators of the airway remodeling process.
PMCID: PMC2838730  PMID: 19915456
Asthma; exercise-induced bronchoconstriction; leukotriene; pharmacogenetics; remodeling; T-cell
10.  Role of T cells in a gp91phox knockout murine model of acute allergic asthma 
Molecular regulation of inflammation, especially, the role of effector cells in NADPH oxidase-mediated redox reactions for producing O2- (superoxide anion) is a critical step. This study explores the roles of macrophages and neutrophils and their cross-talk with extra-cellular matrix components in the light of the role essayed by T cells. Materials and Methods and Treatment: To clarify the role of NADPH oxidase in the pathophysiology of T cell-initiatedmacrophage-associated allergic asthma, we induced allergen dependent inflammation in a gp91phox−/− SKO (single knockout) and a gp91phox−/− MMP-12−/− DKO (double knockout) mouse and analysed trafficking and functionality of various cell types, the T cell function and T cell-macrophage interaction being given special emphasis.
Composite asthma symptoms expressed in a more aggravated manner in both the KO (SKO and DKO) mice compared to WT indicating that some redundancy may exist in the response pathways of gp91phox and MMP-12. On the one hand, upregulation in macrophage functions such as proliferation, mixed lymphocyte reaction, and MCP-1 directed chemotaxis, may indicate that a regulatory cross-talk is switched on between T cell and macrophage and on the other, downregulation of respiratory burst response hints at a dichotomy in their signaling pathways. Increased B7.1 but reduced B7.2 and MHC class II expression on KO alveolar macrophages may suggest that a switching on-off mechanism is operative where alteration of co-stimulatory molecule expression selectively activating T cell is a critical step.
T cell mediated functions such as Th2 cytokine secretion, and T cell proliferation in response to OVA were upregulated synchronous with the overall robustness of the asthma phenotype.
As far as cell-cell interaction is concerned, the data is indicative of the existence of a plethora of networks where molecular switches may exist that selectively induce activation and deactivation of regulatory pathways that ultimately manifest in the overall response. gp91phox and MMP-12 either redundantly or synergistically but not additively, provide a regulatory checkpoint for restricting T cell cross-talk with macrophages and keep excessive tissue damage and ECM degradation during acute allergic inflammation under control.
PMCID: PMC3643823  PMID: 23390895
11.  Association of hemoglobin concentration and mortality in critically ill patients with severe traumatic brain injury 
Critical Care  2012;16(4):R128.
The critical care management of traumatic brain injury focuses on preventing secondary ischemic injury. Cerebral oxygen delivery is dependent upon the cerebral perfusion pressure and the oxygen content of blood, which is principally determined by hemoglobin. Despite its importance to the cerebral oxygen delivery, the precise hemoglobin concentration to provide adequate oxygen delivery to injured neuronal tissue in TBI patients is controversial with limited evidence to provide transfusion thresholds.
We conducted a retrospective cohort study of severe TBI patients, investigating the association between mean 7-day hemoglobin concentration and hospital mortality. Demographic, physiologic, intensive care interventions, clinical outcomes and daily hemoglobin concentrations were recorded for all patients. Patients were all cared for at a tertiary, level 1 trauma center in a mixed medical and surgical intensive unit. Patients were divided into quartiles based on their mean 7-day hemoglobin concentration: < 90 g/L, 90 - 99 g/L, 100 - 109 g/L and > 110 g/L. Multivariable log-binomial regression was used to model the association between mean daily hemoglobin concentration and hospital mortality.
Two hundred seventy-three patients with traumatic brain injury were identified and 169 were included in the analysis based on inclusion/exclusion criteria. Of these, 77% of the patients were male, with a mean age of 38 (SD 17) years and a median best GCS of 6 (IQR 5 - 7). One hundred fifteen patients (68%) received a red blood cell (RBC) transfusion. In RBCs administered in the ICU, the median pre-transfusion hemoglobin was 79 g/L (IQR 73 - 85). Thirty-seven patients (22%) died in hospital. Multivariable analysis revealed that mean 7-day hemoglobin concentration < 90 g/L was independently associated with an increased risk of hospital mortality (RR 3.1, 95% CI 1.5 - 6.3, p = 0.03). Other variables associated with increased mortality on multivariable regression were insertion of external ventricular drain, age and decreased GCS. Red blood cell transfusion was not associated with mortality following multivariable adjustment.
A mean 7-day hemoglobin concentration of < 90g/L is associated with increased hospital mortality in patients with severe traumatic brain injury.
PMCID: PMC3580711  PMID: 22817913
12.  Relationship between levels of secreted phospholipase A2 groups IIA and X in the airways and asthma severity 
Secreted phospholipase A2s (sPLA2) may be important mediators of asthma, but the specific sPLA2s involved in asthma are not known.
To evaluate sPLA2 group IIA, V, and X proteins (sPLA2-IIA, sPLA2-V and sPLA2-X) in bronchoalveolar lavage (BAL) fluid, BAL cells and airway epithelial cells of subjects with and without asthma, and examine the relationship between the levels of specific sPLA2 enzymes and airway inflammation, asthma severity, and lung function.
The expression of sPLA2-IIA, sPLA2-V and sPLA2-X in BAL cells and epithelial brushings was assessed by qPCR. The levels of these sPLA2 proteins and sPLA2 activity with and without group II and group X-specific inhibitors were measured in BAL fluid from 18 controls and 39 asthmatics.
The airway epithelium expressed sPLA2-X at higher levels than either sPLA2-IIA or sPLA2-V, whereas BAL cells expressed sPLA2-IIA and sPLA2-X at similar levels. The majority of sPLA2 activity in BAL fluid was attributed to either sPLA2-IIA or sPLA2-X. After 10-fold concentration of BAL fluid, the levels of sPLA2-X normalized to total protein were increased in asthma and were associated with lung function, the concentration of induced sputum neutrophils, and prostaglandin E2. The levels of sPLA2-IIA were elevated in asthma when normalized to total protein, but where not related to lung function, markers of airway inflammation or eicosanoid formation.
Conclusions and Clinical Relevance
These data indicate that sPLA2-IIA and sPLA2-X are the major sPLA2s in human airways, and suggest a link between the levels of sPLA2-X in the airways and several features of asthma.
PMCID: PMC3093436  PMID: 21255140
Asthma; Eicosanoid; Epithelial Cell; Leukotriene; Phospholipase
13.  Human Embryonic Stem Cells Differentiated to Lung Lineage-Specific Cells Ameliorate Pulmonary Fibrosis in a Xenograft Transplant Mouse Model 
PLoS ONE  2012;7(3):e33165.
Our aim was to differentiate human (h) embryonic stem (ES) cells into lung epithelial lineage-specific cells [i.e., alveolar epithelial type I (AEI) and type II (AEII) cells and Clara cells] as the first step in the development of cell-based strategies to repair lung injury in the bleomycin mouse model of idiopathic pulmonary fibrosis (IPF). A heterogeneous population of non-ciliated lung lineage-specific cells was derived by a novel method of embryoid body (EB) differentiation. This differentiated human cell population was used to modulate the profibrotic phenotype in transplanted animals.
Methodology and Principal Findings
Omission or inclusion of one or more components in the differentiation medium skewed differentiation of H7 hES cells into varying proportions of AEI, AEII, and Clara cells. ICG-001, a small molecule inhibitor of Wnt/β-catenin/Creb-binding protein (CBP) transcription, changed marker expression of the differentiated ES cells from an AEII-like phenotype to a predominantly AEI-like phenotype. The differentiated cells were used in xenograft transplantation studies in bleomycin-treated Rag2γC−/− mice. Human cells were detected in lungs of the transplanted groups receiving differentiated ES cells treated with or without ICG-001. The increased lung collagen content found in bleomycin-treated mice receiving saline was significantly reduced by transplantation with the lung-lineage specific epithelial cells differentiated from ES cells. A significant increase in progenitor number was observed in the airways of bleomycin-treated mice after transplantation of differentiated hES cells.
This study indicates that ES cell-based therapy may be a powerful novel approach to ameliorate lung fibrosis.
PMCID: PMC3314647  PMID: 22470441
14.  Importance of group X–secreted phospholipase A2 in allergen-induced airway inflammation and remodeling in a mouse asthma model 
Arachidonic acid metabolites, the eicosanoids, are key mediators of allergen-induced airway inflammation and remodeling in asthma. The availability of free arachidonate in cells for subsequent eicosanoid biosynthesis is controlled by phospholipase A2s (PLA2s), most notably cytosolic PLA2-α. 10 secreted PLA2s (sPLA2s) have also been identified, but their function in eicosanoid generation is poorly understood. We investigated the role of group X sPLA2 (sPLA2-X), the sPLA2 with the highest in vitro cellular phospholipolysis activity, in acute and chronic mouse asthma models in vivo. The lungs of sPLA2-X−/− mice, compared with those of sPLA2-X+/+ littermates, had significant reduction in ovalbumin-induced infiltration by CD4+ and CD8+ T cells and eosinophils, goblet cell metaplasia, smooth muscle cell layer thickening, subepithelial fibrosis, and levels of T helper type 2 cell cytokines and eicosanoids. These data direct attention to sPLA2-X as a novel therapeutic target for asthma.
PMCID: PMC2118555  PMID: 17403936
15.  Reversal of Allergen-induced Airway Remodeling by CysLT1 Receptor Blockade 
Rationale: Airway inflammation in asthma is accompanied by structural changes, including goblet cell metaplasia, smooth muscle cell layer thickening, and subepithelial fibrosis. This allergen-induced airway remodeling can be replicated in a mouse asthma model.
Objectives: The study goal was to determine whether established airway remodeling in a mouse asthma model is reversible by administration of the cysteinyl leukotriene (CysLT)1 receptor antagonist montelukast, the corticosteroid dexamethasone, or the combination montelukast + dexamethasone.
Methods: BALB/c mice, sensitized by intraperitoneal ovalbumin (OVA) as allergen, received intranasal OVA periodically Days 14–73 and montelukast or dexamethasone or placebo from Days 73–163.
Measurements and Main Results: Allergen-induced trafficking of eosinophils into the bronchoalveolar lavage fluid and lung interstitium and airway goblet cell metaplasia, smooth muscle cell layer thickening, and subepithelial fibrosis present on Day 73 persisted at Day 163, 3 mo after the last allergen challenge. Airway hyperreactivity to methacholine observed on Day 73 in OVA-treated mice was absent on Day 163. In OVA-treated mice, airway eosinophil infiltration and goblet cell metaplasia were reduced by either montelukast or dexamethasone alone. Montelukast, but not dexamethasone, reversed the established increase in airway smooth muscle mass and subepithelial collagen deposition. By immunocytochemistry, CysLT1 receptor expression was significantly increased in airway smooth muscle cells in allergen-treated mice compared with saline-treated controls and was reduced by montelukast, but not dexamethasone, administration.
Conclusions: These data indicate that established airway smooth muscle cell layer thickening and subepithelial fibrosis, key allergen-induced airway structural changes not modulated by corticosteroids, are reversible by CysLT1 receptor blockade therapy.
PMCID: PMC2662952  PMID: 16387808
eosinophils; fibrosis; mucus; smooth muscle
16.  Defining the molecular role of gp91phox in the immune manifestation of acute allergic asthma using a preclinical murine model 
The phenomena manifested during inflammation require interplay between circulating effector cells, local resident cells, soluble mediators and genetic host factors to establish, develop and maintain itself. Of the molecues involed in the initiation and perpetuation of acute allergic inflammation in asthma, the involvement of effector cells in redox reactions for producing O2- (superoxide anion) through the mediation of NADPH oxidase is a critical step. Prior data suggest that reactive oxygen species (ROS) produced by NADPH oxidase homologues in non-phagocytic cells play an important role in the regulation of signal transduction, while macrophages use a membrane-associated NADPH oxidase to generate an array of oxidizing intermediates which inactivate MMPs on or near them.
Materials and Methods and Treatment
To clarify the role of gp91phox subunit of NADPH oxidase in the development and progression of an acute allergic asthma phenotype, we induced allergen dependent inflammation in a gp91phox-/- single knockout and a gp91phox-/-MMP-12-/- double knockout mouse models.
In the knockout mice, both inflammation and airway hyperreactivity were more extensive than in wildtype mice post-OVA. Although OVA-specific IgE in plasma were comparable in wildtype and knockout mice, enhanced inflammatory cell recruitment from circulation and cytokine release in lung and BALf, accompanied by higher airway resistance as well as Penh in response to methacholine, indicate a regulatory role for NADPH oxidase in development of allergic asthma. While T cell mediated functions like Th2 cytokine secretion, and proliferation to OVA were upregulated synchronous with the overall robustness of the asthma phenotype, macrophage upregulation in functions such as proliferation, and mixed lymphocyte reaction indicate a regulatory role for gp91phox and an overall non-involvement or synergistic involvement of MMP12 in the response pathway (comparing data from gp91phox-/- and gp91phox-/-MMP-12-/- mice).
PMCID: PMC3266200  PMID: 22216879
17.  Clinical review: Guyton - the role of mean circulatory filling pressure and right atrial pressure in controlling cardiac output 
Critical Care  2010;14(6):243.
Arthur Guyton's concepts of the determinative role of right heart filling in cardiac output continue to be controversial. This paper reviews his seminal experiments in detail and clarifies the often confusing concepts underpinning his model. One primary criticism of Guyton's model is that the parameters describing venous return had not been measured in a functioning cardiovascular system in humans. Thus, concerns have been expressed in regard to the ability of Guyton's simplistic model, with few parameters, to model the complex human circulation. Further concerns have been raised in regard to the artificial experimental preparations that Guyton used. Recently reported measurements in humans support Guyton's theoretical and animal work.
PMCID: PMC3220048  PMID: 21144008
18.  Transglutaminase 2, a Novel Regulator of Eicosanoid Production in Asthma Revealed by Genome-Wide Expression Profiling of Distinct Asthma Phenotypes 
PLoS ONE  2010;5(1):e8583.
A frequent manifestation of asthma, exercise-induced bronchoconstriction (EIB), occurs in 30–50% of asthmatics and is characterized by increased release of inflammatory eicosanoids. The objective of this study was to identify genes differentially expressed in EIB and to understand the function of these genes in the biology of asthma.
Methodology/Principal Findings
Genome-wide expression profiling of airway leukocytes and epithelial cells obtained by induced sputum was conducted in two groups of subjects with asthma with and without EIB (n = 7 per group), at baseline and following exercise challenge. Based on the results of the gene expression study, additional comparisons were made with a normal control group (n = 10). Localization studies were conducted on epithelial brushings and biopsies from an additional group of asthmatics with EIB (n = 3). Genes related to epithelial repair and mast cell infiltration including β-tryptase and carboxypeptidase A3 were upregulated by exercise challenge in the asthma group with EIB. A gene novel to asthma pathogenesis, transglutaminase 2 (TGM2), was the most differentially expressed at baseline between the groups. In vivo studies confirmed the increased expression of TGM2 in airway cells and airway lining fluid, and demonstrate that TGM2 is avidly expressed in the asthmatic airway epithelium. In vitro studies using recombinant human enzymes reveal that TGM2 augments the enzymatic activity of secreted phospholipase A2 (PLA2) group X (sPLA2-X), an enzyme recently implicated in asthma pathogenesis.
This study found that TGM2, a mediator that is novel to asthma pathogenesis, is overexpressed in asthmatic airways and functions to increase sPLA2-X enzymatic activity. Since PLA2 serves as the first rate-limiting step leading to eicosanoid formation, these results suggest that TGM2 may be a key initiator of the airway inflammatory cascade in asthma.
PMCID: PMC2797392  PMID: 20052409
19.  Intensive insulin therapy and mortality among critically ill patients: a meta-analysis including NICE-SUGAR study data 
Hyperglycemia is associated with increased mortality in critically ill patients. Randomized trials of intensive insulin therapy have reported inconsistent effects on mortality and increased rates of severe hypoglycemia. We conducted a meta-analysis to update the totality of evidence regarding the influence of intensive insulin therapy compared with conventional insulin therapy on mortality and severe hypoglycemia in the intensive care unit (ICU).
We conducted searches of electronic databases, abstracts from scientific conferences and bibliographies of relevant articles. We included published randomized controlled trials conducted in the ICU that directly compared intensive insulin therapy with conventional glucose management and that documented mortality. We included in our meta-analysis the data from the recent NICE-SUGAR (Normoglycemia in Intensive Care Evaluation — Survival Using Glucose Algorithm Regulation) study.
We included 26 trials involving a total of 13 567 patients in our meta-analysis. Among the 26 trials that reported mortality, the pooled relative risk (RR) of death with intensive insulin therapy compared with conventional therapy was 0.93 (95% confidence interval [CI] 0.83–1.04). Among the 14 trials that reported hypoglycemia, the pooled RR with intensive insulin therapy was 6.0 (95% CI 4.5–8.0). The ICU setting was a contributing factor, with patients in surgical ICUs appearing to benefit from intensive insulin therapy (RR 0.63, 95% CI 0.44–0.91); patients in the other ICU settings did not (medical ICU: RR 1.0, 95% CI 0.78–1.28; mixed ICU: RR 0.99, 95% CI 0.86–1.12). The different targets of intensive insulin therapy (glucose level ≤ 6.1 mmol/L v. ≤ 8.3 mmol/L) did not influence either mortality or risk of hypoglycemia.
Intensive insulin therapy significantly increased the risk of hypoglycemia and conferred no overall mortality benefit among critically ill patients. However, this therapy may be beneficial to patients admitted to a surgical ICU.
PMCID: PMC2665940  PMID: 19318387
20.  Activating transcription factor 3 is a negative regulator of allergic pulmonary inflammation 
The Journal of Experimental Medicine  2008;205(10):2349-2357.
We recently demonstrated the pivotal role of the transcription factor (TF) activating TF 3 (ATF3) in dampening inflammation. We demonstrate that ATF3 also ameliorates allergen-induced airway inflammation and hyperresponsiveness in a mouse model of human asthma. ATF3 expression was increased in the lungs of mice challenged with ovalbumin allergen, and this was associated with its recruitment to the promoters of genes encoding Th2-associated cytokines. ATF3-deficient mice developed significantly increased airway hyperresponsiveness, pulmonary eosinophilia, and enhanced chemokine and Th2 cytokine responses in lung tissue and in lung-derived CD4+ lymphocytes. Although several TFs have been associated with enhanced inflammatory responses in the lung, ATF3 attenuates the inflammatory responses associated with allergic airway disease.
PMCID: PMC2556774  PMID: 18794337
21.  Secreted Phospholipase A2 Group X Overexpression in Asthma and Bronchial Hyperresponsiveness 
Rationale: Secreted phospholipase A2 enzymes (sPLA2s) play key regulatory roles in the biosynthesis of eicosanoids, such as the cysteinyl leukotrienes, but the role of these enzymes in the pathogenesis of asthma is not known.
Objectives: To establish if sPLA2s are overexpressed in the airways of patients with asthma, and to determine if these enzymes may play a role in the generation of eicosanoids in exercise-induced bronchoconstriction.
Methods: Induced sputum samples were obtained from subjects with asthma with exercise-induced bronchoconstriction and nonasthmatic control subjects at baseline, and on a separate day 30 minutes after exercise challenge. The expression of the PLA2s in induced sputum cells and supernatant was determined by quantitative polymerase chain reaction, immunocytochemistry, and Western blot.
Measurements and Main Results: The sPLA2s expressed at the highest levels in airway cells of subjects with asthma were groups X and XIIA. Group X sPLA2 (sPLA2-X) was differentially overexpressed in asthma and localized to airway epithelial cells and bronchial macrophages. The gene expression, immunostaining in airway epithelial cells and bronchial macrophages, and the level of the extracellular sPLA2-X protein in the airways increased in response to exercise challenge in the asthma group, whereas the levels were lower and unchanged after challenge in nonasthmatic control subjects.
Conclusions: Increased expression of sPLA2-X may play a key role in the dysregulated eicosanoid synthesis in asthma.
PMCID: PMC2176098  PMID: 17901411
asthma; eicosanoid; epithelial cell; leukotriene; macrophage
22.  Role of MUC5AC in the pathogenesis of exercise-induced bronchoconstriction 
The pathogenesis of exercise-induced bronchoconstriction (EIB) involves the release of mediators from several airway cells in response to exercise challenge, but the mechanism leading to airflow obstruction during EIB is incompletely understood.
To evaluate the role of secreted mucin in the pathogenesis of EIB.
Induced sputum was collected at baseline and 30 minutes after exercise challenge in patients with asthma with EIB. The expression of gel-forming mucins and epidermal growth factor receptor ligands were assessed by quantitative polymerase chain reaction. Secreted mucin 5AC (MUC5AC), the eicosanoids cysteinyl leukotrienes (cysLTs) and 15S-hydroxyeicosatetraenoic acid (15S-HETE), and tachykinins neurokinin A (NKA) and substance P (SP) were measured in induced sputum supernatant.
Among the gel-forming mucins, MUC5AC was expressed at the highest level. The gene expression of MUC5AC increased after exercise challenge compared with baseline and was associated with EIB severity by regression analysis. The relative levels of MUC5AC in induced sputum increased from a geometric mean of 9.5 at baseline to 18.4 postexercise challenge. Associations between the levels of MUC5AC and cysLTs and between the levels of cysLTs and NKA postexercise challenge were identified by regression analysis.
These data indicate that (1) the predominant gel-forming mucin expressed in induced sputum of patients with asthma with EIB is MUC5AC; (2) an increase in MUC5AC gene expression and release of MUC5AC protein occurs after exercise challenge; and (3) MUC5AC release may occur through the cysLT-associated activation of sensory airway nerves.
PMCID: PMC2049009  PMID: 17321575
Asthma; exercise-induced bronchoconstriction; epithelial cell; goblet cell; mucin
23.  Inflammatory Basis of Exercise-induced Bronchoconstriction 
Exercise-induced bronchoconstriction (EIB) is a highly prevalent condition with unclear pathogenesis. Two competing theories of the pathogenesis of EIB differ regarding the inflammatory basis of this condition.
Our goals were to establish whether epithelial cell and mast cell activation with release of inflammatory mediators occurs during EIB and how histamine and cysteinyl leukotriene antagonists alter the airway events occurring during EIB.
Induced sputum was used to measure mast cell mediators and eicosanoids at baseline and 30 minutes after exercise challenge in 25 individuals with asthma with EIB. In a randomized, double-blind crossover study, the cysteinyl leukotriene antagonist montelukast and antihistamine loratadine or two matched placebos were administered for two doses before exercise challenge.
Main Results
The percentage of columnar epithelial cells in induced sputum at baseline was associated with the severity of EIB. After exercise challenge, histamine, tryptase, and cysteinyl leukotrienes significantly increased and prostaglandin E2 and thromboxane B2 significantly decreased in the airways, and there was an increase in columnar epithelial cells in the airways. The concentration of columnar epithelial cells was associated with the levels of histamine and cysteinyl leukotrienes in the airways. Treatment with montelukast and loratadine inhibited the release of cysteinyl leukotrienes and histamine into the airways, but did not inhibit the release of columnar epithelial cells into the airways.
These data indicate that epithelial cells, mast cell mediators, and eicosanoids are released into the airways during EIB, supporting an inflammatory basis for EIB.
PMCID: PMC2041799  PMID: 15947280
asthma; eicosanoid; epithelial cell; exercise-induced bronchoconstriction; mast cell
24.  Airway immunopathology of asthma with exercise-induced bronchoconstriction 
Exercise-induced bronchoconstriction (EIB) is a common cause of symptoms in a subgroup of asthmatic subjects. The pathobiology that makes this group of asthmatic subjects susceptible to bronchoconstriction after a brief period of exercise remains poorly understood.
We sought to determine whether there are differences in lower airway inflammation and production of cytokines and eicosanoids between asthmatic subjects with and without EIB.
Two distinct groups of asthmatic subjects based on a priori definitions were identified, one with moderate-to-severe EIB and the other without significant bronchoconstriction after exercise challenge. Both groups met the definition of asthma on the basis of bronchodilator response, bronchial hyperresponsiveness, or both. A comparative immunopathology study was conducted by using induced sputum to identify differences in lower airway inflammation and production of cytokines and eicosanoids.
The groups had similar baseline lung function and bronchodilator response and did not have any asthma exacerbations within the prior year. The concentration of columnar epithelial cells was markedly higher in the group with EIB (1.4 × 105 vs 2.9 × 104 cells/mL, P = .01). The concentration of eosinophils was higher in the group with EIB (3.6 × 104 vs 4.9 × 103 cells/mL P = .04). Cysteinyl leukotrienes (CysLTs; 727.7 vs 151.9 pg/mL, P = .01) and the ratio of CysLTs to prostaglandin E2 (1.85 vs 1.04, P = .002) in the airways were higher in the group with EIB.
Injury to the airway epithelium, overexpression of CysLTs, relative underproduction of prostaglandin E2, and greater airway eosinophilia are distinctive immunopathologic features of asthma with EIB. (J Allergy Clin Immunol 2005;116:586-93.)
PMCID: PMC2040056  PMID: 16159628
Asthma; exercise-induced bronchoconstriction; epithelial cell; leukotriene; prostaglandin; eosinophil; mast cell
25.  Inflammatory Basis of Exercise-induced Bronchoconstriction 
Rationale: Exercise-induced bronchoconstriction (EIB) is a highly prevalent condition with unclear pathogenesis. Two competing theories of the pathogenesis of EIB differ regarding the inflammatory basis of this condition. Objectives: Our goals were to establish whether epithelial cell and mast cell activation with release of inflammatory mediators occurs during EIB and how histamine and cysteinyl leukotriene antagonists alter the airway events occurring during EIB. Methods: Induced sputum was used to measure mast cell mediators and eicosanoids at baseline and 30 minutes after exercise challenge in 25 individuals with asthma with EIB. In a randomized, double-blind crossover study, the cysteinyl leukotriene antagonist montelukast and antihistamine loratadine or two matched placebos were administered for two doses before exercise challenge. Main Results: The percentage of columnar epithelial cells in induced sputum at baseline was associated with the severity of EIB. After exercise challenge, histamine, tryptase, and cysteinyl leukotrienes significantly increased and prostaglandin E2 and thromboxane B2 significantly decreased in the airways, and there was an increase in columnar epithelial cells in the airways. The concentration of columnar epithelial cells was associated with the levels of histamine and cysteinyl leukotrienes in the airways. Treatment with montelukast and loratadine inhibited the release of cysteinyl leukotrienes and histamine into the airways, but did not inhibit the release of columnar epithelial cells into the airways. Conclusions: These data indicate that epithelial cells, mast cell mediators, and eicosanoids are released into the airways during EIB, supporting an inflammatory basis for EIB.
PMCID: PMC2041799  PMID: 15947280
asthma; eicosanoid; epithelial cell; exercise-induced bronchoconstriction; mast cell

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