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1.  Survey of Innate Immune Responses to Burkholderia pseudomallei in Human Blood Identifies a Central Role for Lipopolysaccharide 
PLoS ONE  2013;8(11):e81617.
B. pseudomallei is a gram-negative bacterium that causes the tropical infection melioidosis. In northeast Thailand, mortality from melioidosis approaches 40%. As exemplified by the lipopolysaccharide-Toll-like receptor 4 interaction, innate immune responses to invading bacteria are precipitated by activation of host pathogen recognition receptors by pathogen associated molecular patterns. Human melioidosis is characterized by up-regulation of pathogen recognition receptors and pro-inflammatory cytokine release. In contrast to many gram-negative pathogens, however, the lipopolysaccharide of B. pseudomallei is considered only weakly inflammatory. We conducted a study in 300 healthy Thai subjects to investigate the ex vivo human blood response to various bacterial pathogen associated molecular patterns, including lipopolysaccharide from several bacteria, and to two heat-killed B. pseudomallei isolates. We measured cytokine levels after stimulation of fresh whole blood with a panel of stimuli. We found that age, sex, and white blood cell count modulate the innate immune response to B. pseudomallei. We further observed that, in comparison to other stimuli, the innate immune response to B. pseudomallei is most highly correlated with the response to lipopolysaccharide. The magnitude of cytokine responses induced by B. pseudomallei lipopolysaccharide was significantly greater than those induced by lipopolysaccharide from Escherichia coli and comparable to many responses induced by lipopolysaccharide from Salmonella minnesota despite lower amounts of lipid A in the B. pseudomallei lipopolysaccharide preparation. In human monocytes stimulated with B. pseudomallei, addition of polymyxin B or a TLR4/MD-2 neutralizing antibody inhibited the majority of TNF-α production. Challenging existing views, our data indicate that the innate immune response to B. pseudomallei in human blood is largely driven by lipopolysaccharide, and that the response to B. pseudomallei lipopolysaccharide in blood is greater than the response to other lipopolysaccharide expressing isolates. Our findings suggest that B. pseudomallei lipopolysaccharide may play a central role in stimulating the host response in melioidosis.
doi:10.1371/journal.pone.0081617
PMCID: PMC3841221  PMID: 24303060
2.  On-time clinical phenotype prediction based on narrative reports 
In this paper we describe a natural language processing system which is able to predict whether or not a patient exhibits a specific phenotype using the information extracted from the narrative reports associated with the patient. Furthermore, the phenotypic annotations from our report dataset were performed at the report level which allows us to perform the prediction of the clinical phenotype at any point in time during the patient hospitalization period. Our experiments indicate that an important factor in achieving better results for this problem is to determine how much information to extract from the patient reports in the time interval between the patient admission time and the current prediction time.
PMCID: PMC3900127  PMID: 24551325
3.  Pneumonia identification using statistical feature selection 
Objective
This paper describes a natural language processing system for the task of pneumonia identification. Based on the information extracted from the narrative reports associated with a patient, the task is to identify whether or not the patient is positive for pneumonia.
Design
A binary classifier was employed to identify pneumonia from a dataset of multiple types of clinical notes created for 426 patients during their stay in the intensive care unit. For this purpose, three types of features were considered: (1) word n-grams, (2) Unified Medical Language System (UMLS) concepts, and (3) assertion values associated with pneumonia expressions. System performance was greatly increased by a feature selection approach which uses statistical significance testing to rank features based on their association with the two categories of pneumonia identification.
Results
Besides testing our system on the entire cohort of 426 patients (unrestricted dataset), we also used a smaller subset of 236 patients (restricted dataset). The performance of the system was compared with the results of a baseline previously proposed for these two datasets. The best results achieved by the system (85.71 and 81.67 F1-measure) are significantly better than the baseline results (50.70 and 49.10 F1-measure) on the restricted and unrestricted datasets, respectively.
Conclusion
Using a statistical feature selection approach that allows the feature extractor to consider only the most informative features from the feature space significantly improves the performance over a baseline that uses all the features from the same feature space. Extracting the assertion value for pneumonia expressions further improves the system performance.
doi:10.1136/amiajnl-2011-000752
PMCID: PMC3422830  PMID: 22539080
4.  Variation in the TLR10/TLR1/TLR6 Locus is the Major Genetic Determinant of Inter-Individual Difference in TLR1/2-Mediated Responses 
Genes and immunity  2012;14(1):52-57.
Toll-like receptor (TLR)-mediated innate immune responses are important in early host defense. Using a candidate gene approach, we previously identified genetic variation within TLR1 that is associated with hyper-responsiveness to a TLR1/2 agonist in vitro and with death and organ dysfunction in patients with sepsis. Here we report a genome-wide association study designed to identify genetic loci controlling whole blood cytokine responses to the TLR1/2 lipopeptide agonist, Pam3CSK4 ex vivo. We identified a very strong association (p<1×10−27) between genetic variation within the TLR10/1/6 locus on chromosome 4, and Pam3CSK4-induced cytokine responses. This was the predominant association explaining over 35% of the population variance for this phenotype. Notably, strong associations were observed within TLR10 suggesting genetic variation in TLR10 may influence bacterial lipoprotein-induced responses. These findings establish the TLR10/1/6 locus as the dominant common genetic factor controlling inter-individual variability in Pam3CSK4-induced whole blood responses in the healthy population.
doi:10.1038/gene.2012.53
PMCID: PMC3554851  PMID: 23151486
TLR; polymorphism; genomics; innate immunity
5.  Automated Tools for Phenotype Extraction from Medical Records  
Clinical research studying critical illness phenotypes relies on the identification of clinical syndromes defined by consensus definitions. Historically, identifying phenotypes has required manual chart review, a time and resource intensive process. The overall research goal of C ritical I llness PH enotype E xt R action (deCIPHER) project is to develop automated approaches based on natural language processing and machine learning that accurately identify phenotypes from EMR. We chose pneumonia as our first critical illness phenotype and conducted preliminary experiments to explore the problem space. In this abstract, we outline the tools we built for processing clinical records, present our preliminary findings for pneumonia extraction, and describe future steps.
PMCID: PMC3845784  PMID: 24303281
6.  Impaired TLR5 Functionality Is Associated with Survival in Melioidosis 
Melioidosis is infection caused by the flagellated saprophyte Burkholderia pseudomallei. TLR5 is a pathogen recognition receptor activated by bacterial flagellin. We studied a genetic variant that encodes a defective TLR5 protein, TLR51174C>T, to elucidate the role of TLR5 in melioidosis. We measured NF-κB activation induced by B. pseudomallei in human embryonic kidney–293 cells transfected with TLR5 and found that B. pseudomallei induced TLR51174C- but not TLR51174T-dependent activation of NF-κB. We tested the association of TLR51174C>T with outcome in 600 Thai subjects with melioidosis. In a dominant model, TLR51174C>T was associated with protection against in-hospital death (adjusted odds ratio: 0.20; 95% confidence interval: 0.08–0.50; p = 0.001) and organ failure (adjusted odds ratio: 0.37; 95% confidence interval: 0.19–0.71; p = 0.003). We analyzed blood cytokine production induced by flagellin or heat-killed B. pseudomallei by TLR51174C>T genotype in healthy subjects. Flagellin induced lower monocyte-normalized levels of IL-6, IL-8, TNF-α, IL-10, MCP-1, IL-1ra, G-CSF, and IL-1β in carriers of TLR51174T compared with carriers of TLR51174C. B. pseudomallei induced lower monocyte-normalized levels of IL-10 in carriers of TLR51174T. We conclude that the hypofunctional genetic variant TLR51174C>T is associated with reduced organ failure and improved survival in melioidosis. This conclusion suggests a deleterious immunoregulatory effect of TLR5 that may be mediated by IL-10 and identifies this receptor as a potential therapeutic target in melioidosis.
doi:10.4049/jimmunol.1202974
PMCID: PMC3607401  PMID: 23447684
7.  Assessing Pneumonia Identification from Time-Ordered Narrative Reports 
AMIA Annual Symposium Proceedings  2012;2012:1119-1128.
In this paper, we present a natural language processing system that can be used in hospital surveillance applications with the purpose of identifying patients with pneumonia. For this purpose, we built a sequence of supervised classifiers, where the dataset corresponding to each classifier consists of a restricted set of time-ordered narrative reports. In this way the pneumonia surveillance application will be able to invoke the most suitable classifier for each patient based on the period of time that has elapsed since the patient was admitted into the hospital. Our system achieves significantly better results when compared with a baseline previously proposed for pneumonia identification.
PMCID: PMC3540463  PMID: 23304388
8.  A Nonsynonymous Polymorphism of IRAK4 Associated with Increased Prevalence of Gram-Positive Infection and Decreased Response to Toll-Like Receptor Ligands 
Journal of Innate Immunity  2011;3(5):447-458.
Mutations in IRAK4 have been associated with recurrent Gram-positive infections in children. Given the central role of IRAK4 in innate immunity signaling, we hypothesized that common genetic variants of IRAK4 may be associated with prevalence of Gram-positive infection in critically ill adults. Haplotype clade tag single nucleotide polymorphisms (SNPs) of the IRAK4 gene were selected and genotyped in a cohort of 1,029 critically ill patients with systemic inflammatory response syndrome (SIRS). We found that a haplotype clade tagged by the A allele of the htSNP G29429A (Ala428Thr) was associated with increased relative risk of Gram-positive infection at admission to ICU (RR = 1.2, p < 0.05). Furthermore, the 29429A allele was associated with decreased lymphoblastoid cell response to CpG (as measured by IL-6 production) (raw values ± 95% CI 40.3 ± 32.3 vs. 85.8 ± 29.4 pg/ml; log-transformed values ± 95% CI 1.13 ± 0.37 vs. 1.55 ± 0.18, p < 0.04). We also found that IRAK4-deficient fibroblasts transfected with an IRAK4 expression plasmid containing the 29429A allele produced less IL-6 in response to lipopolysaccharide (p = 0.07). Our data suggest that the IRAK4 haplotype clade marked by 29429A (428Thr) alters susceptibility to Gram-positive bacteria, by decreasing cellular response to TLR ligands.
Copyright © 2011 S. Karger AG, Basel
doi:10.1159/000323880
PMCID: PMC3186712  PMID: 21576904
Bacterial infections; Protein kinase; Inflammation
9.  Microarray-based Analysis of Ventilator-induced Lung Injury 
Acute respiratory distress syndrome (ARDS) and acute lung injury (ALI) are a frequent cause of intensive care unit admission, affecting over 200,000 patients in the United States each year. Mechanical ventilation is a life-saving intervention in the setting of ARDS and ALI, but clinical trials have demonstrated that mechanical ventilation with excessive tidal volumes plays a role in promoting and perpetuating lung injury and leads to excess mortality. This process has been labeled ventilator-induced lung injury (VILI), but the molecular mechanisms driving this process and its interactions with predisposing risk factors such as sepsis and chemical injury remain incompletely understood. Genome-wide measurements of gene expression using microarray technology represent a powerful tool to examine the pathophysiology of VILI. Several recent studies have used this approach to study VILI in isolation and associated with endotoxin instillation or saline lavage. These studies and others examining gene expression profiles in epithelial cells subjected to cyclic stretch have provided novel insights on the molecular mechanisms underlying VILI. This review will summarize these findings and discuss implications for future studies.
doi:10.1513/pats.200608-149JG
PMCID: PMC2647617  PMID: 17202295
microarray analysis; lung injury; mechanical ventilation; gene expression; genomics
10.  Genetic Variation in the FAS Gene and Associations with Acute Lung Injury 
Rationale: Fas (CD95) modulates apoptosis and inflammation and is believed to play an important role in lung injury.
Objectives: To determine if common genetic variation in FAS is associated with acute lung injury (ALI) susceptibility, risk of death, and FAS gene expression.
Methods: We genotyped 14 single nucleotide polymorphisms (tagSNPS) in FAS in samples from healthy white volunteers (control subjects, n = 294) and patients with ALI (cases, n = 324) from the ARDSnet Fluid and Catheter Treatment Trial (FACTT). FAS genotypes associated with ALI in the discovery study were confirmed in a nested case-control validation study of critically ill patients at risk for ALI (n = 657). We also tested for associations between selected tagSNPS and FAS mRNA levels in whole blood from healthy control subjects exposed to media alone or LPS ex vivo.
Measurements and Main Results: We identified associations between four tagSNPs in FAS (FAS−11341A>T [rs17447140], FAS9325G>A [rs2147420], FAS21541C>T [rs2234978], and FAS24484A>T [rs1051070]) and ALI case status. Haplotype-based analyses suggested that three of the tagSNPs (FAS9325G>A, FAS21541C>T, and FAS24484A>T) function as a unit. The association with this haplotype and ALI was validated in a nested case-control study of at-risk subjects (P = 0.05). This haplotype was also associated with increased FAS mRNA levels in response to LPS stimulation. There was no association between FAS polymorphisms and risk of death among ALI cases.
Conclusions: Common genetic variants in FAS are associated with ALI susceptibility. This is the first genetic evidence supporting a role for FAS in ALI.
doi:10.1164/rccm.201003-0351OC
PMCID: PMC3056231  PMID: 20813889
adult respiratory distress syndrome; FAS receptor; genetic predisposition; apoptosis
11.  Genome Wide Association Identifies PPFIA1 as a Candidate Gene for Acute Lung Injury Risk Following Major Trauma 
PLoS ONE  2012;7(1):e28268.
Acute Lung Injury (ALI) is a syndrome with high associated mortality characterized by severe hypoxemia and pulmonary infiltrates in patients with critical illness. We conducted the first investigation to use the genome wide association (GWA) approach to identify putative risk variants for ALI. Genome wide genotyping was performed using the Illumina Human Quad 610 BeadChip. We performed a two-stage GWA study followed by a third stage of functional characterization. In the discovery phase (Phase 1), we compared 600 European American trauma-associated ALI cases with 2266 European American population-based controls. We carried forward the top 1% of single nucleotide polymorphisms (SNPs) at p<0.01 to a replication phase (Phase 2) comprised of a nested case-control design sample of 212 trauma-associated ALI cases and 283 at-risk trauma non-ALI controls from ongoing cohort studies. SNPs that replicated at the 0.05 level in Phase 2 were subject to functional validation (Phase 3) using expression quantitative trait loci (eQTL) analyses in stimulated B-lymphoblastoid cell lines (B-LCL) in family trios. 159 SNPs from the discovery phase replicated in Phase 2, including loci with prior evidence for a role in ALI pathogenesis. Functional evaluation of these replicated SNPs revealed rs471931 on 11q13.3 to exert a cis-regulatory effect on mRNA expression in the PPFIA1 gene (p = 0.0021). PPFIA1 encodes liprin alpha, a protein involved in cell adhesion, integrin expression, and cell-matrix interactions. This study supports the feasibility of future multi-center GWA investigations of ALI risk, and identifies PPFIA1 as a potential functional candidate ALI risk gene for future research.
doi:10.1371/journal.pone.0028268
PMCID: PMC3266233  PMID: 22295056
12.  Identification and Characterization of a Loss-of-Function Human MPYS Variant 
Genes and immunity  2011;12(4):263-269.
MPYS, also known as STING and MITA, is an IFNβ stimulator essential for host defense against RNA, DNA viruses and intracellular bacteria. MPYS also facilitates the adjuvant activity of DNA vaccines. Here we report identification of a distinct human MPYS haplotype that contains three non-synonymous SNPs, R71H-G230A-R293Q (thus named the HAQ haplotype). We estimate, in two cohorts (1074 individuals), that ~3% of Americans are homozygous for this HAQ haplotype. HAQ MPYS exhibits a >90% loss in the ability to stimulate IFNβ production. Furthermore, fibroblasts and macrophage cells expressing HAQ are defective in Listeria monocytogenes infection-induced IFNβ production. Lastly, we find that the loss of IFNβ activity is due primarily to the R71H and R293Q SNPs in HAQ. We hypothesize that individuals carrying HAQ may exhibit heightened susceptibility to viral infection and respond poorly to DNA vaccines.
doi:10.1038/gene.2010.75
PMCID: PMC3107388  PMID: 21248775
SNP; IFNβ; anti-viral signaling; MPYS/STING/MITA
13.  Automated Classification of Radiology Reports for Acute Lung Injury: Comparison of Keyword and Machine Learning Based Natural Language Processing Approaches 
This paper compares the performance of keyword and machine learning-based chest x-ray report classification for Acute Lung Injury (ALI). ALI mortality is approximately 30 percent. High mortality is, in part, a consequence of delayed manual chest x-ray classification. An automated system could reduce the time to recognize ALI and lead to reductions in mortality. For our study, 96 and 857 chest x-ray reports in two corpora were labeled by domain experts for ALI. We developed a keyword and a Maximum Entropy-based classification system. Word unigram and character n-grams provided the features for the machine learning system. The Maximum Entropy algorithm with character 6-gram achieved the highest performance (Recall=0.91, Precision=0.90 and F-measure=0.91) on the 857-report corpus. This study has shown that for the classification of ALI chest x-ray reports, the machine learning approach is superior to the keyword based system and achieves comparable results to highest performing physician annotators.
doi:10.1109/BIBMW.2009.5332081
PMCID: PMC2998031  PMID: 21152268
14.  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.
Background
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.
Conclusions/Significance
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.
doi:10.1371/journal.pone.0008583
PMCID: PMC2797392  PMID: 20052409
15.  Toll-like Receptor 1 Polymorphisms Affect Innate Immune Responses and Outcomes in Sepsis 
Rationale: Polymorphisms affecting Toll-like receptor (TLR)–mediated responses could predispose to excessive inflammation during an infection and contribute to an increased risk for poor outcomes in patients with sepsis.
Objectives: To identify hypermorphic polymorphisms causing elevated TLR-mediated innate immune cytokine and chemokine responses and to test whether these polymorphisms are associated with increased susceptibility to death, organ dysfunction, and infections in patients with sepsis.
Methods: We screened single-nucleotide polymorphisms (SNPs) in 43 TLR-related genes to identify variants affecting TLR-mediated inflammatory responses in blood from healthy volunteers ex vivo. The SNP associated most strongly with hypermorphic responses was tested for associations with death, organ dysfunction, and type of infection in two studies: a nested case–control study in a cohort of intensive care unit patients with sepsis, and a case–control study using patients with sepsis, patients with sepsis-related acute lung injury, and healthy control subjects.
Measurements and Main Results: The SNP demonstrating the most hypermorphic effect was the G allele of TLR1−7202A/G (rs5743551), which associated with elevated TLR1-mediated cytokine production (P < 2 × 10−20). TLR1−7202G marked a coding SNP that causes higher TLR1-induced NF-κB activation and higher cell surface TLR1 expression. In the cohort of patients with sepsis TLR1−7202G predicted worse organ dysfunction and death (odds ratio, 1.82; 95% confidence interval, 1.07–3.09). In the case-control study TLR1−7202G was associated with sepsis-related acute lung injury (odds ratio, 3.40; 95% confidence interval, 1.59–7.27). TLR1−7202G also associated with a higher prevalence of gram-positive cultures in both clinical studies.
Conclusions: Hypermorphic genetic variation in TLR1 is associated with increased susceptibility to organ dysfunction, death, and gram-positive infection in sepsis.
doi:10.1164/rccm.200803-462OC
PMCID: PMC2556453  PMID: 18635889
innate immunity; genetic variation; genetic predisposition
16.  Proteomic and Computational Analysis of Bronchoalveolar Proteins during the Course of the Acute Respiratory Distress Syndrome 
Rationale: Acute lung injury causes complex changes in protein expression in the lungs. Whereas most prior studies focused on single proteins, newer methods allowing the simultaneous study of many proteins could lead to a better understanding of pathogenesis and new targets for treatment.
Objectives: The purpose of this study was to examine the changes in protein expression in the bronchoalveolar lavage fluid (BALF) of patients during the course of the acute respiratory distress syndrome (ARDS).
Methods: Using two-dimensional difference gel electrophoresis (DIGE), the expression of proteins in the BALF from patients on Days 1 (n = 7), 3 (n = 8), and 7 (n = 5) of ARDS were compared with findings in normal volunteers (n = 9). The patterns of protein expression were analyzed using principal component analysis (PCA). Biological processes that were enriched in the BALF proteins of patients with ARDS were identified using Gene Ontology (GO) analysis. Protein networks that model the protein interactions in the BALF were generated using Ingenuity Pathway Analysis.
Measurements and Main Results: An average of 991 protein spots were detected using DIGE. Of these, 80 protein spots, representing 37 unique proteins in all of the fluids, were identified using mass spectrometry. PCA confirmed important differences between the proteins in the ARDS and normal samples. GO analysis showed that these differences are due to the enrichment of proteins involved in inflammation, infection, and injury. The protein network analysis showed that the protein interactions in ARDS are complex and redundant, and revealed unexpected central components in the protein networks.
Conclusions: Proteomics and protein network analysis reveals the complex nature of lung protein interactions in ARDS. The results provide new insights about protein networks in injured lungs, and identify novel mediators that are likely to be involved in the pathogenesis and progression of acute lung injury.
doi:10.1164/rccm.200712-1895OC
PMCID: PMC2556452  PMID: 18658106
acute respiratory distress syndrome; acute lung injury; proteomic analysis; bronchoalveolar lavage; 2D gel electrophoresis
17.  Polymorphisms of the IL1-receptor antagonist gene (IL1RN) are associated with multiple markers of systemic inflammation 
Background
Circulating levels of acute phase reactant proteins such as plasma C-reactive protein (CRP) are likely influenced by multiple genes regulating the innate immune response.
Methods
We screened a set of 16 inflammation-related genes for association with CRP in a large, population-based study of healthy young adults (n=1,627). Results were validated in two independent studies (n=1,208 and n=4,310), including a pooled analysis of all 3 studies.
Results
In the pooled analysis, the minor allele of IL1RN 1018 (rs4251961) within the gene encoding interleukin-1 receptor antagonist (IL-1RA) was significantly associated with higher mean plasma log(CRP) level (p < 1 × 10−4). The same IL1RN 1018 allele was associated with higher mean plasma log(IL-6) levels (p=0.004). In the pooled analysis, the minor allele of IL1RN 13888 (rs2232354) was associated with higher fibrinogen, (p = 0.001). The IL1RN 1018 and 13888 variant alleles tag a clade of IL1RN haplotypes linked to allele 1 of a 86 bp VNTR polymorphism. We confirmed that the IL1RN 1018 variant (rs4251961) was associated with decreased cellular IL-1RA production ex vivo.
Conclusions
Common functional polymorphisms of the IL1RN gene are associated with several markers of systemic inflammation.
doi:10.1161/ATVBAHA.108.167437
PMCID: PMC2748384  PMID: 18451331
IL-1receptor antagonist; C-reactive protein; inflammation; fibrinogen
18.  The Duffy Antigen Modifies Systemic and Local Tissue Chemokine Responses following Lipopolysaccharide Stimulation1 
The Duffy blood group Ag (dfy) binds selective CXC and CC chemokines at high affinity and is expressed on erythrocytes and endothelial cells. However, it does not transmit a signal via G proteins, as occurs with other seven-transmembrane receptors. We hypothesized that dfy functions as a chemokine reservoir and regulates inflammation by altering soluble chemokine concentrations in the blood and tissue compartments. We determined whether Duffy Ag “loss-of-function” phenotypes (human and murine) are associated with alterations in plasma chemokine concentrations during the innate inflammatory response to LPS. Plasma CXCL8 and CCL2 concentrations from humans homozygous for the GATA-1 box polymorphism, a dfy polymorphism that abrogates erythrocyte chemokine binding, were higher than in heterozygotes following LPS stimulation of their whole blood in vitro. Similarly, dfy−/− mice showed higher plasma MIP-2 concentrations than dfy+/+ mice following LPS stimulation of whole blood in vitro. We then determined the relative contributions of erythrocyte and endothelial Duffy Ag in modifying chemokine concentrations and neutrophil recruitment in the lungs following intratracheal LPS administration in dfy−/− and dfy+/+ mice reconstituted with dfy−/− or dfy+/+ marrow. Mice lacking endothelial dfy expression had higher MIP-2 and keratinocyte chemoattractant concentrations in the airspaces. Mice lacking erythrocyte dfy had higher MIP-2 and keratinocyte chemoattractant concentrations in the lung tissue vascular space, but lower plasma chemokine concentrations associated with attenuated neutrophil recruitment into the airspaces. These data indicate that dfy alters soluble chemokine concentrations in blood and local tissue compartments and enhances systemic bioavailability of chemokines produced during local tissue inflammation.
PMCID: PMC2665269  PMID: 17114483
19.  Essential Role of MMP-12 in Fas-Induced Lung Fibrosis 
Acute lung injury (ALI) is characterized by an early inflammatory response followed by a late fibroproliferative phase, and by an increase in the bronchoalveolar lavage fluid (BALF) concentrations of bioactive soluble FasL (sFasL). Activation of Fas (CD95) has been associated with the development of lung fibrosis in mice. The goal of this study was to determine the mechanisms that link Fas activation with the development of fibrosis in the lungs. We treated mice with three daily intratracheal instillations of a Fas-activating monoclonal antibody (Jo2) or a control IgG, and studied the animals at sequential times. Mice treated with Jo2 had increased caspase-3 activation in alveolar wall cells on Days 2, 4, and 7; an inflammatory response peaking on Day 7, and increased total lung collagen on Day 21. Gene expression profiling performed on Days 2, 4, and 7 showed sequential activation of co-regulated profibrotic genes, including marked up-regulation of matrix metalloproteinase 12 (MMP-12). Targeted deletion of MMP-12 protected mice from Fas-induced pulmonary fibrosis, even though the inflammatory responses in the lungs were similar to those of wild-type mice. Compared with wild-type mice, the mmp12−/− mice showed decreased expression of the profibrotic genes egr1 and cyr61. We conclude that Fas activation in the lungs induces a complex response that includes apoptosis, inflammation, and eventually fibrosis, and that MMP-12 is essential for the fibrotic phenotype. We speculate that MMP-12 activity is required for activation of the profibrotic genes egr1 and cyr61.
doi:10.1165/rcmb.2006-0471OC
PMCID: PMC1976544  PMID: 17446527
apoptosis; inflammation; MMP-12; Fas; CYR61/CCN1
20.  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.
doi:10.1164/rccm.200412-1667OC
PMCID: PMC2041799  PMID: 15947280
asthma; eicosanoid; epithelial cell; exercise-induced bronchoconstriction; mast cell
21.  Genetic pleiotropy between asthma and obesity in a community-based sample of twins 
Background
Asthma and obesity are common conditions that are strongly associated. This association might be due to shared genetic or environmental causes.
Objective
We sought to determine whether a shared genetic cause is responsible for the association between asthma and obesity and to estimate the magnitude of shared genetic cause.
Methods
The analyses were performed with 1001 monozygotic and 383 dizygotic same-sex twin pairs within the University of Washington Twin Registry. The presence of asthma was determined by self-report of a physician diagnosis of asthma, and body mass index (BMI) was calculated by using self-reported height and weight. Obesity was defined as a BMI of 30 or greater. The association between asthma and BMI was assessed by means of mixed-effects ordinal regression. Twin correlations examined the association of asthma and obesity. Univariate and bivariate structural equation models estimated the components of variance attributable to genetic and environmental effects.
Results
A strong association between asthma and BMI was identified in the sample population (P < .001). Substantial heritability was detected for asthma (53%) and obesity (77%), which is indicative of additive genetic influences on each disorder. The best-fitting model of shared components of variance indicated that 8% of the genetic component of obesity is shared with asthma.
Conclusion
The covariation between obesity and asthma is predominantly caused by shared genetic risk factors for both conditions.
doi:10.1016/j.jaci.2005.09.016
PMCID: PMC2014783  PMID: 16337451
Asthma; obesity; genetic; twin
22.  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.
doi:10.1164/rccm.200412-1667OC
PMCID: PMC2041799  PMID: 15947280
asthma; eicosanoid; epithelial cell; exercise-induced bronchoconstriction; mast cell
23.  Targeted Deletion of the Lipopolysaccharide (LPS)-binding Protein Gene Leads to Profound Suppression of LPS Responses Ex Vivo, whereas In Vivo Responses Remain Intact  
The Journal of Experimental Medicine  1997;186(12):2051-2056.
Gram-negative bacterial lipopolysaccharide (LPS) stimulates phagocytic leukocytes by interacting with the cell surface protein CD14. Cellular responses to LPS are markedly potentiated by the LPS-binding protein (LBP), a lipid-transfer protein that binds LPS aggregates and transfers LPS monomers to CD14. LBP also transfers LPS to lipoproteins, thereby promoting the neutralization of LPS. LBP present in normal plasma has been shown to enhance the LPS responsiveness of cells in vitro. The role of LBP in promoting LPS responsiveness in vivo was tested in LBP-deficient mice produced by gene targeting in embryonic stem cells. Whole blood from LBP-deficient animals was 1,000-fold less responsive to LPS as assessed by the release of tumor necrosis factor (TNF)-α. Blood from gene-targeted mice was devoid of immunoreactive LBP, essentially incapable of transferring LPS to CD14 in vitro, and failed to support cellular responses to LPS. These activities were restored by the addition of exogenous recombinant murine LBP to the plasma. Despite these striking in vitro findings, no significant differences in TNF-α levels were observed in plasma from wild-type and LBP-deficient mice injected with LPS. These data suggest the presence of an LBP-independent mechanism for responding to LPS. These LBP knockout mice may provide a tool for discovering the nature of the presumed second mechanism for transferring LPS to responsive cells.
PMCID: PMC2199164  PMID: 9396775
24.  Inflammation and Immune-Related Candidate Gene Associations with Acute Lung Injury Susceptibility and Severity: A Validation Study 
PLoS ONE  2012;7(12):e51104.
Introduction
Common variants in genes related to inflammation, innate immunity, epithelial cell function, and angiogenesis have been reported to be associated with risks for Acute Lung Injury (ALI) and related outcomes. We tested whether previously-reported associations can be validated in an independent cohort at risk for ALI.
Methods
We identified 37 genetic variants in 27 genes previously associated with ALI and related outcomes. We prepared allelic discrimination assays for 12 SNPs from 11 genes with MAF>0.05 and genotyped these SNPs in Caucasian subjects from a cohort of critically ill patients meeting criteria for the systemic inflammatory response syndrome (SIRS) followed for development of ALI, duration of mechanical ventilation, and in-hospital death. We tested for associations using additive and recessive genetic models.
Results
Among Caucasian subjects with SIRS (n = 750), we identified a nominal association between rs2069832 in IL6 and ALI susceptibility (ORadj 1.61; 95% confidence interval [CI], 1.04–2.48, P = 0.03). In a sensitivity analysis limiting ALI cases to those who qualified for the Acute Respiratory Distress Syndrome (ARDS), rs61330082 in NAMPT was nominally associated with risk for ARDS. In terms of ALI outcomes, SNPs in MBL2 (rs1800450) and IL8 (rs4073) were nominally associated with fewer ventilator-free days (VFDs), and SNPs in NFE2L2 (rs6721961) and NAMPT (rs61330082) were nominally associated with 28-day mortality. The directions of effect for these nominal associations were in the same direction as previously reported but none of the associations survived correction for multiple hypothesis testing.
Conclusion
Although our primary analyses failed to statistically validate prior associations, our results provide some support for associations between SNPs in IL6 and NAMPT and risk for development of lung injury and for SNPs in IL8, MBL2, NFE2L2 and NAMPT with severity in ALI outcomes. These associations provide further evidence that genetic factors in genes related to immunity and inflammation contribute to ALI pathogenesis.
doi:10.1371/journal.pone.0051104
PMCID: PMC3522667  PMID: 23251429
25.  Genetic Variation in the TNF Gene Is Associated with Susceptibility to Severe Sepsis, but Not with Mortality 
PLoS ONE  2012;7(9):e46113.
Background
Tumor necrosis factor (TNF) and TNF receptor superfamily (TNFR)-mediated immune response play an essential role in the pathogenesis of severe sepsis. Studies examining associations of TNF and lymphotoxin-α (LTA) single nucleotide polymorphisms (SNPs) with severe sepsis have produced conflicting results. The objective of this study was to investigate whether genetic variation in TNF, LTA, TNFRSF1A and TNFRSF1B was associated with susceptibility to or death from severe sepsis in Chinese Han population.
Methodology/Principal Findings
Ten SNPs in TNF, LTA, TNFRSF1A and TNFRSF1B were genotyped in samples of patients with severe sepsis (n = 432), sepsis (n = 384) and healthy controls (n = 624). Our results showed that rs1800629, a SNP in the promoter region of TNF, was significantly associated with risk for severe sepsis. The minor allele frequency of rs1800629 was significantly higher in severe sepsis patients than that in both healthy controls (Padj = 0.00046, odds ratio (OR)adj = 1.92) and sepsis patients (Padj = 0.002, ORadj = 1.56). Further, we investigated the correlation between rs1800629 genotypes and TNF-α concentrations in peripheral blood mononuclear cells (PBMCs) of healthy volunteers exposed to lipopolysaccharides (LPS) ex vivo, and the association between rs1800629 and TNF-α serum levels in severe sepsis patients. After exposure to LPS, the TNF-α concentration in culture supernatants of PBMCs was significantly higher in the subjects with AA+AG genotypes than that with GG genotype (P = 0.007). Moreover, in patients with severe sepsis, individuals with AA+AG genotypes had significantly higher TNF-α serum concentrations than those with GG genotype (Padj = 0.02). However, there were no significant associations between SNPs in the four candidate genes and 30 day mortality for patients with severe sepsis.
Conclusions/Significance
Our findings suggested that the functional TNF gene SNP rs1800629 was strongly associated with susceptibility to severe sepsis, but not with lethality in Chinese Han population.
doi:10.1371/journal.pone.0046113
PMCID: PMC3459853  PMID: 23029405

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