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1.  Identification of new biomarkers for Acute Respiratory Distress Syndrome by expression-based genome-wide association study 
Accumulated to-date gene microarray data on Acute Respiratory Distress Syndrome (ARDS) in the Gene Expression Omnibus (GEO) represent a rich source for identifying new unsuspected targets and mechanisms of ARDS. The recently developed expression-based genome-wide association study (eGWAS) for analysis of GEO data was successfully used for analysis of gene expression of comparatively noncomplex adipose tissue, 75 % of which is represented by adipocytes. Although lung tissue is more heterogenic and does not possess a prevalent cell type for driving gene expression patterns, we hypothesized that eGWAS of ARDS samples will generate biologically meaningful results.
The eGWAS was conducted according to (Proc Natl Acad Sci U S A 109:7049-7054, 2012) and genes were ranked according to p values of chi-square test.
The search of GEO retrieved 487 ARDS related entries. These entries were filtered for multiple qualitative and quantitative conditions and 219 samples were selected: mouse nsham/ARDS = 67/92, rat n = 13/13, human cells n = 11/11, canine n = 6/6 with the following ARDS model distributions: mechanical ventilation (MV)/cyclic stretch n = 11; endotoxin (LPS) treatment n = 8; MV + LPS n = 3; distant organ injury induced ARDS n = 3; chemically induced ARDS n = 2; Staphylococcus aureus induced ARDS n = 2; and one experiment each for radiation and shock induced ARDS. The eGWAS of this dataset identified 42 significant (Bonferroni threshold P < 1.55 × 10−6) genes. 66.6 % of these genes, were associated previously with lung injury and include the well known ARDS genes such as IL1R2 (P = 4.42 × 10−19), IL1β (P = 3.38 × 10−17), PAI1 (P = 9.59 × 10−14), IL6 (P = 3.57 × 10−12), SOCS3 (P = 1.05 × 10−10), and THBS1 (P = 2.01 × 10−9). The remaining genes were new ARDS candidates. Expression of the most prominently upregulated genes, CLEC4E (P = 4.46 × 10−14) and CD300LF (P = 2.31 × 10−16), was confirmed by real time PCR. The former was also validated by in silico pathway analysis and the latter by Western blot analysis.
Our first in the field application of eGWAS in ARDS and utilization of more than 120 publicly available microarray samples of ARDS not only justified applicability of eGWAS to complex lung tissue, but also discovered 14 new candidate genes which associated with ARDS. Detailed studies of these new candidates might lead to identification of unsuspected evolutionarily conserved mechanisms triggered by ARDS.
Electronic supplementary material
The online version of this article (doi:10.1186/s12890-015-0088-x) contains supplementary material, which is available to authorized users.
PMCID: PMC4545360  PMID: 26285698
Expression-based genome-wide association studies; Acute respiratory distress syndrome; Gene expression; Biomarkers; Microarray; Genomics
2.  Identification of Novel Single Nucleotide Polymorphisms Associated with Acute Respiratory Distress Syndrome by Exome-Seq 
PLoS ONE  2014;9(11):e111953.
Acute respiratory distress syndrome (ARDS) is a lung condition characterized by impaired gas exchange with systemic release of inflammatory mediators, causing pulmonary inflammation, vascular leak and hypoxemia. Existing biomarkers have limited effectiveness as diagnostic and therapeutic targets. To identify disease-associating variants in ARDS patients, whole-exome sequencing was performed on 96 ARDS patients, detecting 1,382,399 SNPs. By comparing these exome data to those of the 1000 Genomes Project, we identified a number of single nucleotide polymorphisms (SNP) which are potentially associated with ARDS. 50,190SNPs were found in all case subgroups and controls, of which89 SNPs were associated with susceptibility. We validated three SNPs (rs78142040, rs9605146 and rs3848719) in additional ARDS patients to substantiate their associations with susceptibility, severity and outcome of ARDS. rs78142040 (C>T) occurs within a histone mark (intron 6) of the Arylsulfatase D gene. rs9605146 (G>A) causes a deleterious coding change (proline to leucine) in the XK, Kell blood group complex subunit-related family, member 3 gene. rs3848719 (G>A) is a synonymous SNP in the Zinc-Finger/Leucine-Zipper Co-Transducer NIF1 gene. rs78142040, rs9605146, and rs3848719 are associated significantly with susceptibility to ARDS. rs3848719 is associated with APACHE II score quartile. rs78142040 is associated with 60-day mortality in the overall ARDS patient population. Exome-seq is a powerful tool to identify potential new biomarkers for ARDS. We selectively validated three SNPs which have not been previously associated with ARDS and represent potential new genetic biomarkers for ARDS. Additional validation in larger patient populations and further exploration of underlying molecular mechanisms are warranted.
PMCID: PMC4221189  PMID: 25372662
3.  Combined meta-analysis of systemic effects of allogeneic stem cell transplantation and systemic sclerosis 
BMC Hematology  2014;14:7.
Chronic graft-versus-host disease (cGVHD) is a major factor of morbidity and mortality for allogeneic stem cell transplantation (aSCT). The skin and internal organ involvement is the most common systemic complication of cGVHD and closely resembles systemic sclerosis (SSc). Circulating lymphocytes characterize the autoimmune nature of both conditions. Therefore we hypothesized that the common clinical manifestation (systemic organ and skin injury) and the common underlying players (lymphocytes) justify the combined meta-analysis of these diseases.
The aSCT and SSc datasets were uploaded from Gene Expression Omnibus (GEO), a public functional genomics data repository. The available microarray studies of peripheral blood mononuclear cells (PBMCs) and isolated lymphocytes were limited to well established microarray platforms (Affymetrix, Agilent, Canvac, and Illumina) and experimental settings with ≥10 patients per group. The resulting pools of data were merged by unique gene identifier and analyzed by the expression genome-wide association studies (eGWAS) coupled with the subtraction of the cGVHD+ and cGVHD− molecular signatures. The eGWAS was applied to 47 and 50 lymphocyte profiles from aSCT and SSc patients, respectively. The identified 35 candidates were represented by 8 known cGVHD genes (including CXCR4, LTBR and PML) and 28 new candidate genes (including SEPX1 and DNJGB1). The further mutual subtraction of cGVHD+ and cGVHD− candidates and pathway analysis identified a list of 25 genes. Seven of these genes belong to the fibroblast development and function pathway, consisting of the well known cGVHD genes CCND1, JUN, and FOS, and the new molecular targets MMP2, FOSB, TNFAIP8, and DUSP1. These genes become primary candidates for a potential link of systemic effects of cGVHD and SSc.
We designed a new approach for meta-analysis by combining data from different diseases using common clinical manifestation as a linker. This allowed us to power up the insufficient standalone meta-analysis of aSCT microarray studies, by adding SSc samples to the data pool. This new method has successfully identified novel molecular targets for systemic effects of both aSCT and SSc. We believe that this approach is generalizable and can be applied to an array of diseases with common clinical manifestations.
PMCID: PMC4021691  PMID: 24656173
Meta-analysis; Allogeneic stem cell transplantation; Systemic sclerosis; Microarray; Molecular signature; Gene expression; Public data repository; Peripheral mononuclear cells; Circulating lymphocytes
4.  Meta-analysis of molecular response of kidney to ischemia reperfusion injury for the identification of new candidate genes 
BMC Nephrology  2013;14:231.
Accumulated to-date microarray data on ischemia reperfusion injury (IRI) of kidney represent a powerful source for identifying new targets and mechanisms of kidney IRI. In this study, we conducted a meta-analysis of gene expression profiles of kidney IRI in human, pig, rat, and mouse models, using a new scoring method to correct for the bias of overrepresented species. The gene expression profiles were obtained from the public repositories for 24 different models. After filtering against inclusion criteria 21 experimental settings were selected for meta-analysis and were represented by 11 rat models, 6 mouse models, and 2 models each for pig and human, with a total of 150 samples. Meta-analysis was conducted using expression-based genome-wide association study (eGWAS). The eGWAS results were corrected for a rodent species bias using a new weighted scoring algorithm, which favors genes with unidirectional change in expression in all tested species.
Our meta-analysis corrected for a species bias, identified 46 upregulated and 1 downregulated genes, of which 26 (55%) were known to be associated with kidney IRI or kidney transplantation, including LCN2, CCL2, CXCL1, HMOX1, ICAM1, ANXA1, and TIMP1, which justified our approach. Pathway analysis of our candidates identified “Acute renal failure panel” as the most implicated pathway, which further validates our new method. Among new IRI candidates were 10 novel (<5 published reports related to kidney IRI) and 11 new candidates (0 reports related to kidney IRI) including the most prominent candidates ANXA2, CLDN4, and TYROBP. The cross-species expression pattern of these genes allowed us to generate three workable hypotheses of kidney IRI, one of which was confirmed by an additional study.
Our first in the field kidney IRI meta-analysis of 150 microarray samples, corrected for a species bias, identified 10 novel and 11 new candidate genes. Moreover, our new meta-analysis correction method improved gene candidate selection by identifying genes that are model and species independent, as a result, function of these genes can be directly extrapolated to the disease state in human and facilitate translation of potential diagnostic or therapeutic properties of these candidates to the bedside.
PMCID: PMC4016589  PMID: 24152794
Kidney; Ischemia reperfusion injury; Bioinformatics; Meta-analysis
5.  Use of consomic rats for genomic insights into ventilator-associated lung injury 
Increasing evidence supports the contribution of genetic influences on susceptibility/severity in acute lung injury (ALI), a devastating syndrome requiring mechanical ventilation with subsequent risk for ventilator-associated lung injury (VALI). To identify VALI candidate genes, we determined that Brown Norway (BN) and Dahl salt-sensitive (SS) rat strains were differentially sensitive to VALI (tidal volume of 20 ml/kg, 85 breaths/min, 2 h) defined by bronchoalveolar lavage (BAL) protein and leukocytes. We next exploited differential sensitivities and phenotyped both the VALI-sensitive BN and the VALI-resistant SS rat strains by expression profiling coupled to a bioinformatic-intense candidate gene approach (Significance Analysis of Microarrays, i.e., SAM). We identified 106 differentially expressed VALI genes representing gene ontologies such as “transcription” and “chemotaxis/cell motility.” We mapped the chromosomal location of the differentially expressed probe sets and selected consomic SS rats with single BN introgressions of chromosomes 2, 13, and 16 (based on the highest density of probe sets) while also choosing chromosome 20 (low probe sets density). VALI exposure of consomic rats with introgressions of BN chromosomes 13 and 16 resulted in significant increases in both BAL cells and protein (compared to parental SS strain), whereas introgression of BN chromosome 2 displayed a large increase only in BAL protein. Introgression of BN chromosome 20 had a minimal effect. These results suggest that genes residing on BN chromosomes 2, 13, and 16 confer increased sensitivity to high tidal volume ventilation. We speculate that the consomic-microarray-SAM approach is a time- and resource-efficient tool for the genetic dissection of complex diseases including VALI.
PMCID: PMC3616407  PMID: 17468131
rodent mechanical ventilation; consomics; bioinformatics; microarrays; candidate gene approach
6.  The HECT-Type E3 Ubiquitin Ligase AIP2 Inhibits Activation-Induced T-Cell Death by Catalyzing EGR2 Ubiquitination▿  
Molecular and Cellular Biology  2009;29(19):5348-5356.
E3 ubiquitin ligases, which target specific molecules for proteolytic destruction, have emerged as key regulators of immune functions. Several E3 ubiquitin ligases, including c-Cbl, Cbl-b, GRAIL, Itch, and Nedd4, have been shown to negatively regulate T-cell activation. Here, we report that the HECT-type E3 ligase AIP2 positively regulates T-cell activation. Ectopic expression of AIP2 in mouse primary T cells enhances their proliferation and interleukin-2 production by suppressing the apoptosis of T cells. AIP2 interacts with and promotes ubiquitin-mediated degradation of EGR2, a zinc finger transcription factor that has been found to regulate Fas ligand (FasL) expression during activation-induced T-cell death. Suppression of AIP2 expression by small RNA interference upregulates EGR2, inhibits EGR2 ubiquitination and FasL expression, and enhances the apoptosis of T cells. Therefore, AIP2 regulates activation-induced T-cell death by suppressing EGR2-mediated FasL expression via the ubiquitin pathway.
PMCID: PMC2747983  PMID: 19651900
7.  Essential Role of Pre–B-Cell Colony Enhancing Factor in Ventilator-induced Lung Injury 
Rationale: We previously demonstrated pre–B-cell colony enhancing factor (PBEF) as a biomarker in sepsis and sepsis-induced acute lung injury (ALI) with genetic variants conferring ALI susceptibility.
Objectives: To explore mechanistic participation of PBEF in ALI and ventilator-induced lung injury (VILI).
Methods: Two models of VILI were utilized to explore the role of PBEF using either recombinant PBEF or PBEF+/− mice.
Measurements and Main Results: Initial in vitro studies demonstrated recombinant human PBEF (rhPBEF) as a direct rat neutrophil chemotactic factor with in vivo studies demonstrating marked increases in bronchoalveolar lavage (BAL) leukocytes (PMNs) after intratracheal injection in C57BL/6J mice. These changes were accompanied by increased BAL levels of PMN chemoattractants (KC and MIP-2) and modest increases in lung vascular and alveolar permeability. We next explored the potential synergism between rhPBEF challenge (intratracheal) and a model of limited VILI (4 h, 30 ml/kg tidal volume) and observed dramatic increases in BAL PMNs, BAL protein, and cytokine levels (IL-6, TNF-α, KC) compared with either challenge alone. Gene expression profiling identified induction of ALI- and VILI-associated gene modules (nuclear factor-κB, leukocyte extravasation, apoptosis, Toll receptor pathways). Heterozygous PBEF+/− mice were significantly protected (reduced BAL protein, BAL IL-6 levels, peak inspiratory pressures) when exposed to a model of severe VILI (4 h, 40 ml/kg tidal volume) and exhibited significantly reduced expression of VILI-associated gene expression modules. Finally, strategies to reduce PBEF availability (neutralizing antibody) resulted in significant protection from VILI.
Conclusions: These studies implicate PBEF as a key inflammatory mediator intimately involved in both the development and severity of ventilator-induced ALI.
PMCID: PMC2542434  PMID: 18658108
visfatin; acute lung injury; chemotaxis; apoptosis; mechanical ventilation
8.  Macrophage Migration Inhibitory Factor in Acute Lung Injury: Expression, Biomarker and Associations 
Macrophage migration inhibitory factor (MIF), a proinflammatory cytokine central to the response to endotoxemia, is a putative biomarker in acute lung injury (ALI). To explore MIF as a molecular target and candidate gene in ALI, we examined MIF gene and protein expression in murine and canine models of ALI (high tidal volume mechanical ventilation, endotoxin exposure) and in patients with either sepsis or sepsis-induced ALI. MIF gene expression and protein levels were significantly increased in each ALI model, with serum MIF levels significantly higher in patients with either sepsis or ALI compared to healthy controls (African- and European- descent). We next studied the association of 8 MIF gene polymorphisms (SNPs) (within a 9.7 kb interval on chromosome 22q11.23) with the development of sepsis and ALI in European- and African- descent populations. Genotyping in 506 DNA samples (sepsis patients, sepsis-associated ALI patients, and healthy controls) revealed haplotypes located in the 3′ end of the MIF gene, but not individual SNPs, associated with sepsis and ALI in both populations. These data, generated via functional genomic and genetic approaches, suggest that MIF is a relevant molecular target in ALI.
PMCID: PMC1989118  PMID: 17585860
mechanical ventilation; MIF; gene expression profiling; polymorphism
9.  Novel Polymorphisms in the Myosin Light Chain Kinase Gene Confer Risk for Acute Lung Injury 
The genetic basis of acute lung injury (ALI) is poorly understood. The myosin light chain kinase (MYLK) gene encodes the nonmuscle myosin light chain kinase isoform, a multifunctional protein involved in the inflammatory response (apoptosis, vascular permeability, leukocyte diapedesis). To examine MYLK as a novel candidate gene in sepsis-associated ALI, we sequenced exons, exon–intron boundaries, and 2 kb of 5′ UTR of the MYLK, which revealed 51 single-nucleotide polymorphisms (SNPs). Potential association of 28 MYLK SNPs with sepsis-associated ALI were evaluated in a case-control sample of 288 European American subjects (EAs) with sepsis alone, subjects with sepsis-associated ALI, or healthy control subjects, and a sample population of 158 African American subjects (AAs) with sepsis and ALI. Significant single locus associations in EAs were observed between four MYLK SNPs and the sepsis phenotype (P < 0.001), with an additional SNP associated with the ALI phenotype (P = 0.03). A significant association of a single SNP (identical to the SNP identified in EAs) was observed in AAs with sepsis (P = 0.002) and with ALI (P = 0.01). Three sepsis risk-conferring haplotypes in EAs were defined downstream of start codon of smooth muscle MYLK isoform, a region containing putative regulatory elements (P < 0.001). In contrast, multiple haplotypic analyses revealed an ALI-specific, risk-conferring haplotype at 5′ of the MYLK gene in both European and African Americans and an additional 3′ region haplotype only in African Americans. These data strongly implicate MYLK genetic variants to confer increased risk of sepsis and sepsis-associated ALI.
PMCID: PMC2644210  PMID: 16399953
MYLK/MLCK; genetic association; SNP; ALI; sepsis
10.  Pluripotent Allospecific CD8+ Effector T Cells Traffic to Lung in Murine Obliterative Airway Disease 
Long-term success in lung transplantation is limited by obliterative bronchiolitis, whereas T cell effector mechanisms in this process remain incompletely understood. Using the mouse heterotopic allogeneic airway transplant model, we studied T cell effector responses during obliterative airways disease (OAD). Allospecific CD8+IFN-γ+ T cells were detected in airway allografts, with significant coexpression of TNF-α and granzyme B. Therefore, using IFN-γ as a surrogate marker, we assessed the distribution and kinetics of extragraft allo-specific T cells during OAD. Robust allospecific IFN-γ was produced by draining the lymph nodes, spleen, and lung mononuclear cells from allograft, but not isograft recipients by Day 14, and significantly decreased by Day 28. Although the majority of allospecific T cells were CD8+, allospecific CD4+ T cells were also detected in these compartments, with each employing distinct allorecognition pathways. An influx of pluripotent CD8+ effector cells with a memory phenotype were detected in the lung during OAD similar to those seen in the allografts and secondary lymphoid tissue. Antibody depletion of CD8+ T cells markedly reduced airway lumen obliteration and fibrosis at Day 28. Together, these data demonstrate that allospecific CD8+ effector T cells play an important role in OAD and traffic to the lung after heterotopic airway transplant, suggesting that the lung is an important immunologic site, and perhaps a reservoir, for effector cells during the rejection process.
PMCID: PMC2644186  PMID: 16195540
effector T cells; lung allograft rejection; lung transplantation; obliterative airways disease
11.  In vitro identification and in silico utilization of interspecies sequence similarities using GeneChip® technology 
BMC Genomics  2005;6:62.
Genomic approaches in large animal models (canine, ovine etc) are challenging due to insufficient genomic information for these species and the lack of availability of corresponding microarray platforms. To address this problem, we speculated that conserved interspecies genetic sequences can be experimentally detected by cross-species hybridization. The Affymetrix platform probe redundancy offers flexibility in selecting individual probes with high sequence similarities between related species for gene expression analysis.
Gene expression profiles of 40 canine samples were generated using the human HG-U133A GeneChip (U133A). Due to interspecies genetic differences, only 14 ± 2% of canine transcripts were detected by U133A probe sets whereas profiling of 40 human samples detected 49 ± 6% of human transcripts. However, when these probe sets were deconstructed into individual probes and examined performance of each probe, we found that 47% of human probes were able to find their targets in canine tissues and generate a detectable hybridization signal. Therefore, we restricted gene expression analysis to these probes and observed the 60% increase in the number of identified canine transcripts. These results were validated by comparison of transcripts identified by our restricted analysis of cross-species hybridization with transcripts identified by hybridization of total lung canine mRNA to new Affymetrix Canine GeneChip®.
The experimental identification and restriction of gene expression analysis to probes with detectable hybridization signal drastically increases transcript detection of canine-human hybridization suggesting the possibility of broad utilization of cross-hybridizations of related species using GeneChip technology.
PMCID: PMC1156887  PMID: 15871745

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