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1.  Serum Prognostic Biomarkers in Head and Neck Cancer Patients 
The Laryngoscope  2014;124(8):1819-1826.
A reliable estimate of survival is important as it may impact treatment choice. The objective of this study is to identify serum autoantibody biomarkers that can be used to improve prognostication for patients affected with head and neck squamous cell carcinoma (HNSCC).
Study Design
Prospective cohort study.
A panel of 130 serum biomarkers, previously selected for cancer detection using microarray-based serological profiling and specialized bioinformatics, were evaluated for their potential as prognostic biomarkers in a cohort of 119 HNSCC patients followed for up to 12.7 years. A biomarker was considered positive if its reactivity to the particular patient’s serum was greater than one standard deviation above the mean reactivity to sera from the other 118 patients, using a leave-one-out cross-validation model. Survival curves were estimated according to the Kaplan-Meier method, and statistically significant differences in survival were examined using the log rank test. Independent prognostic biomarkers were identified following analysis using multivariate Cox proportional hazards models.
Poor overall survival was associated with African Americans (hazard ratio [HR] for death =2.61; 95% confidence interval [CI]: 1.58–4.33; P =.000), advanced stage (HR =2.79; 95% CI: 1.40–5.57; P =.004), and recurrent disease (HR =6.66; 95% CI: 2.54–17.44; P =.000). On multivariable Cox analysis adjusted for covariates (race and stage), six of the 130 markers evaluated were found to be independent prognosticators of overall survival.
The results shown here are promising and demonstrate the potential use of serum biomarkers for prognostication in HNSCC patients. Further clinical trials to include larger samples of patients across multiple centers may be warranted.
PMCID: PMC4129948  PMID: 24347532
Head and neck squamous cell carcinoma; prognostic biomarkers; prognosis; proteomic
2.  Increased Interaction With Insulin Receptor Substrate 1, a Novel Abnormality in Insulin Resistance and Type 2 Diabetes 
Diabetes  2014;63(6):1933-1947.
Insulin receptor substrate 1 (IRS1) is a key mediator of insulin signal transduction. Perturbations involving IRS1 complexes may lead to the development of insulin resistance and type 2 diabetes (T2D). Surprisingly little is known about the proteins that interact with IRS1 in humans under health and disease conditions. We used a proteomic approach to assess IRS1 interaction partners in skeletal muscle from lean healthy control subjects (LCs), obese insulin-resistant nondiabetic control subjects (OCs), and participants with T2D before and after insulin infusion. We identified 113 novel endogenous IRS1 interaction partners, which represents the largest IRS1 interactome in humans and provides new targets for studies of IRS1 complexes in various diseases. Furthermore, we generated the first global picture of IRS1 interaction partners in LCs, and how they differ in OCs and T2D patients. Interestingly, dozens of proteins in OCs and/or T2D patients exhibited increased associations with IRS1 compared with LCs under the basal and/or insulin-stimulated conditions, revealing multiple new dysfunctional IRS1 pathways in OCs and T2D patients. This novel abnormality, increased interaction of multiple proteins with IRS1 in obesity and T2D in humans, provides new insights into the molecular mechanism of insulin resistance and identifies new targets for T2D drug development.
PMCID: PMC4030113  PMID: 24584551
3.  Intelligent Informatics in Translational Medicine 
BioMed Research International  2015;2015:717210.
PMCID: PMC4438178  PMID: 26064944
4.  Characterization of the myometrial transcriptome in women with an arrest of dilatation during labor 
Journal of perinatal medicine  2013;41(6):665-681.
The molecular basis of failure to progress in labor is poorly understood. This study was undertaken to characterize the myometrial transcriptome of patients with an arrest of dilatation (AODIL).
Study design
Human myometrium was prospectively collected from women in the following groups: 1) spontaneous term labor (TL; n=29); and 2) arrest of dilatation (AODIL; n=14). Gene expression was characterized using Illumina® HumanHT-12 microarrays. A moderated student t-test and false discovery rate adjustment were used for analysis. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) of selected genes was performed in an independent sample set. Pathway analysis was performed on the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database using Pathway Analysis with Down-weighting of Overlapping Genes (PADOG). The Metacore knowledge base was also mined for pathway analysis.
1) 42 genes differentially expressed were identified in women with an AODIL; 2) gene ontology analysis indicated enrichment of biological processes, which included: regulation of angiogenesis, response to hypoxia, inflammatory response, and chemokine-mediated signaling pathway. Enriched molecular functions included: transcription repressor activity, Heat shock protein (Hsp) 90 binding, and nitric oxide synthase (NOS) activity; 3) Metacore analysis identified immune response chemokine (C-C motif) ligand 2 (CCL2) signaling, muscle contraction regulation of eNOS activity in endothelial cells, and Triiodothyronine and Thyroxine signaling as significantly over-represented (FDR<0.05); 4) qRT-PCR confirmed overexpression of Nitric oxide synthase 3 NOS3; hypoxic ischemic factor (HIF1A), Chemokine (C-C motif) ligand 2 (CCL2); angiopoietin-like 4 (ANGPTL4), ADAM metallopeptidase with thrombospondin type 1, motif 9 (ADAMTS9), G protein-coupled receptor 4 (GPR4), metallothionein 1A (MT1A), MT2A, selectin E (SELE) in an AODIL.
The myometrium of women with arrest of dilatation have a stereotypic transcriptome profile. This disorder was associated with a pattern of gene expression involved in muscle contraction, an inflammatory response, and hypoxia. This is the first comprehensive and unbiased examination of the molecular basis of an AODIL.
PMCID: PMC4183453  PMID: 23893668
angiopoietin-like 4 (ANGPTL4); arrest disorders; chemokine (C-C motif) ligand 2 (CCL2); metallothionein (MT); myometrium; nitric oxide synthase (NOS); pregnancy; parturition; systems biology; TGF signaling pathway; transcriptomics
5.  Differences and Similarities in the Transcriptional Profile of Peripheral Whole Blood in Early and Late-Onset Preeclampsia: Insights into the Molecular Basis of the Phenotype of Preeclampsia 
Journal of perinatal medicine  2013;41(5):485-504.
Preeclampsia (PE) has been sub-divided into early- and late-onset phenotypes. The pathogenesis of these two phenotypes has not been elucidated. To gain insight into the mechanisms of disease, the transcriptional profiles of whole blood from women with early- and late-onset PE were examined.
A cross-sectional study was conducted to include women with: 1) early-onset PE (diagnosed prior to 34 weeks, n=25); 2) late-onset PE (after 34 weeks, n=47); and 3) uncomplicated pregnancy (n=61). Microarray analysis of mRNA expression in peripheral whole blood was undertaken using Affymetrix microarrays. Differential gene expression was evaluated using a moderated t-test (false discovery rate <0.1 and fold change >1.5), adjusting for maternal WBC count and gestational age. Validation by real-time qRT-PCR was performed in a larger sample size [early PE (n=31), late PE (n=72) and controls (n=99)] in all differentially expressed genes. Gene Ontology analysis and pathway analysis were performed.
1) 43 and 28 genes were differentially expressed in early- and late-onset PE compared to the control group respectively; 2) qRT-PCR confirmed the microarray results for early and late-onset PE in 77% (33/43) and 71% (20/28) of genes, respectively; 3) 20 genes that are involved in coagulation (SERPINI2), immune regulation (VSIG4, CD24), developmental process (H19) and inflammation (S100A10) were differentially expressed in early-onset PE alone. In contrast, only seven genes that encoded proteins involved in innate immunity (LTF, ELANE) and cell-to-cell recognition in the nervous system (CNTNAP3) were differentially expressed in late-onset PE alone. Thirteen genes that encode proteins involved in host defense (DEFA4, BPI, CTSG, LCN2), tight junctions in blood-brain barrier (EMP1) and liver regeneration (ECT2) were differentially expressed in both early- and late-onset PE.
Early- and late-onset PE are characterized by a common signature in the transcriptional profile of whole blood. A small set of genes were differentially regulated in early- and late-onset PE. Future studies of the biological function, expression timetable and protein expression of these genes may provide insight into the pathophysiology of PE.
PMCID: PMC4164302  PMID: 23793063
Gene expression; transcriptomic; pregnancy; microarray; PAX gene; H19; white blood cell count; Affymetrix; PCR
6.  Expression Patterns of MicroRNAs in the Chorioamniotic Membranes: a Role for MicroRNAs in Human Pregnancy and Parturition 
The Journal of pathology  2009;217(1):113-121.
MicroRNAs (miRNAs) are involved in the post-transcriptional regulation of gene expression during development. This study was performed to determine gestational age-dependent changes in miRNA expression in the chorioamniotic membranes and to assess the significance of miRNAs in human pregnancy and parturition. The expression profile of 455 miRNAs was compared between patients at term without labor (TNL: n=10), in labor (TL: n=10), and preterm labor (PTL: n=10) using microarrays. A total of 39 miRNAs were differentially expressed between term and preterm cases, of which 31 (79.5%) were down-regulated at term. Expression of 10 miRNAs, including miR-338, differentially expressed between PTL and TL groups was decreased at term. Computational analyses using miRBase Targets have identified PLA2G4B, a phospholipase implicated in parturition, as a putative target of miR-338. Inhibition of endogenous miR-338 with anti-miR-338 increased the mRNA and protein expression of PLA2G4B in decidual cells. Luciferase assay with reporter constructs confirmed that the suppression of PLA2G4B occurs through binding of miR-338 to the 3’UTR of PLA2G4B. Interestingly, the expression of Dicer, a key miRNA-processing enzyme, was markedly decreased at term, particularly with labor in the chorioamniotic membranes. Collectively, the novel findings reported herein strongly suggest that post-transcriptional regulation of genes by miRNAs, coupled with the changes of miRNA processing machinery in the chorioamniotic membranes, plays a role in pregnancy and parturition. Furthermore, the expression level of Dicer in the chorioamniotic membranes dichotomizes pathological preterm labor and physiological spontaneous labor at term.
PMCID: PMC4160233  PMID: 18991333
miR-338; group IVB phospholipase A2; Dicer; development; decidua; labor; preterm birth
7.  Developing Classifiers for the Detection of Cancer Using Multianalytes 
The development of a successful classifier from multiple predictors (analytes) is a multistage process complicated typically by the paucity of the data samples when compared to the number of available predictors. Choosing an adequate validation strategy is key for drawing sound conclusions about the usefulness of the classifier. Other important decisions have to be made regarding the type of prediction model to be used and training algorithm, as well as the way in which the markers are selected. This chapter described the principles of the classifier development and underlines the most common pitfalls. A simulated dataset is used to illustrate the main concepts involved in supervised classification.
PMCID: PMC3748835  PMID: 19381961
Multiple predictors; Supervised classification; Feature/marker selection
8.  Predicting Novel Human Gene Ontology Annotations Using Semantic Analysis 
The correct interpretation of many molecular biology experiments depends in an essential way on the accuracy and consistency of the existing annotation databases. Such databases are meant to act as repositories for our biological knowledge as we acquire and refine it. Hence, by definition, they are incomplete at any given time. In this paper, we describe a technique that improves our previous method for predicting novel GO annotations by extracting implicit semantic relationships between genes and functions. In this work, we use a vector space model and a number of weighting schemes in addition to our previous latent semantic indexing approach. The technique described here is able to take into consideration the hierarchical structure of the Gene Ontology (GO) and can weight differently GO terms situated at different depths. The prediction abilities of 15 different weighting schemes are compared and evaluated. Nine such schemes were previously used in other problem domains, while six of them are introduced in this paper. The best weighting scheme was a novel scheme, n2tn. Out of the top 50 functional annotations predicted using this weighting scheme, we found support in the literature for 84 percent of them, while 6 percent of the predictions were contradicted by the existing literature. For the remaining 10 percent, we did not find any relevant publications to confirm or contradict the predictions. The n2tn weighting scheme also outperformed the simple binary scheme used in our previous approach.
PMCID: PMC3712327  PMID: 20150671
Gene function prediction; gene annotation; Gene Ontology; vector space model; latent semantic indexing; weighting schemes
9.  Intelligent Informatics in Biomedicine 
BioMed Research International  2013;2013:185839.
PMCID: PMC3654645  PMID: 23710437
10.  Identification of Novel Type 2 Diabetes Candidate Genes Involved in the Crosstalk between the Mitochondrial and the Insulin Signaling Systems 
PLoS Genetics  2012;8(12):e1003046.
Type 2 Diabetes (T2D) is a highly prevalent chronic metabolic disease with strong co-morbidity with obesity and cardiovascular diseases. There is growing evidence supporting the notion that a crosstalk between mitochondria and the insulin signaling cascade could be involved in the etiology of T2D and insulin resistance. In this study we investigated the molecular basis of this crosstalk by using systems biology approaches. We combined, filtered, and interrogated different types of functional interaction data, such as direct protein–protein interactions, co-expression analyses, and metabolic and signaling dependencies. As a result, we constructed the mitochondria-insulin (MITIN) network, which highlights 286 genes as candidate functional linkers between these two systems. The results of internal gene expression analysis of three independent experimental models of mitochondria and insulin signaling perturbations further support the connecting roles of these genes. In addition, we further assessed whether these genes are involved in the etiology of T2D using the genome-wide association study meta-analysis from the DIAGRAM consortium, involving 8,130 T2D cases and 38,987 controls. We found modest enrichment of genes associated with T2D amongst our linker genes (p = 0.0549), including three already validated T2D SNPs and 15 additional SNPs, which, when combined, were collectively associated to increased fasting glucose levels according to MAGIC genome wide meta-analysis (p = 8.12×10−5). This study highlights the potential of combining systems biology, experimental, and genome-wide association data mining for identifying novel genes and related variants that increase vulnerability to complex diseases.
Author Summary
It has been shown that the crosstalk between insulin signaling and the mitochondria may be involved in the etiology of type 2 diabetes. In order to characterize the molecular basis of this crosstalk, we mined and filtered several interaction databases of different natures, including protein–protein interactions, gene co-expression, signaling, and metabolic pathway interactions, to identify reliable direct and indirect interactions between insulin signaling cascade and mitochondria genes. This allowed us to identify 286 genes that are associated simultaneously with insulin signaling and mitochondrial genes and therefore could act as a molecular bridge between both systems. We performed in vitro and in vivo experiments where the insulin signaling or the mitochondrial function were disrupted, and we found deregulation of these connecting genes. Finally, we found that common variants in genomic regions where these genes lie are enriched for genetic associations with type 2 diabetes and glycemic traits according to large genome-wide association meta-analyses. In summary, we reconstructed the network implicated in the crosstalk between the mitochondria and the insulin signaling and provide a list of genes connecting both systems. We also propose new potential type 2 diabetes candidate genes.
PMCID: PMC3516534  PMID: 23236286
11.  A survey of small RNAs in human sperm 
Human Reproduction (Oxford, England)  2011;26(12):3401-3412.
There has been substantial interest in assessing whether RNAs (mRNAs and sncRNAs, i.e. small non-coding) delivered from mammalian spermatozoa play a functional role in early embryo development. While the cadre of spermatozoal mRNAs has been characterized, comparatively little is known about the distribution or function of the estimated 24 000 sncRNAs within each normal human spermatozoon.
RNAs of <200 bases in length were isolated from the ejaculates from three donors of proved fertility. RNAs of 18–30 nucleotides in length were then used to construct small RNA Digital Gene Expression libraries for Next Generation Sequencing. Known sncRNAs that uniquely mapped to a single location in the human genome were identified.
Bioinformatic analysis revealed the presence of multiple classes of small RNAs in human spermatozoa. The primary classes resolved included microRNA (miRNAs) (≈7%), Piwi-interacting piRNAs (≈17%), repeat-associated small RNAs (≈65%). A minor subset of short RNAs within the transcription start site/promoter fraction (≈11%) frames the histone promoter-associated regions enriched in genes of early embryonic development. These have been termed quiescent RNAs.
A complex population of male derived sncRNAs that are available for delivery upon fertilization was revealed. Sperm miRNA-targeted enrichment in the human oocyte is consistent with their role as modifiers of early post-fertilization. The relative abundance of piRNAs and repeat-associated RNAs suggests that they may assume a role in confrontation and consolidation. This may ensure the compatibility of the genomes at fertilization.
PMCID: PMC3212879  PMID: 21989093
microRNA; Piwi-interacting RNAs RNA; spermatozoa; small non-coding RNA
12.  The Transcriptome of the Fetal Inflammatory Response Syndrome 
The fetal inflammatory response syndrome (FIRS) is considered the counterpart of the systemic inflammatory response syndrome (SIRS), but similarities in their regulatory mechanisms are unclear. This study characterizes the fetal mRNA transcriptome of peripheral leukocytes to identify key biological processes and pathways involved in FIRS.
Method of Study
Umbilical cord blood from preterm neonates with FIRS (funisitis, plasma IL-6>11 pg/ml; n=10) and neonates with no evidence of inflammation (n=10) was collected at birth.
Microarray analysis of leukocyte RNA revealed differential expression of 541 unique genes, changes confirmed by qRT-PCR for 41 or of 44 genes tested. Similar to SIRS and sepsis, ontological and pathway analyses yielded significant enrichment of biological processes including antigen processing and presentation, immune response, and processes critical to cellular metabolism. Results are comparable with microarray studies of endotoxin challenge models and pediatric sepsis, identifying 25 genes across all studies.
This study is the first to profile genome-wide expression in FIRS, which demonstrates a substantial degree of similarity with SIRS despite differences in fetal and adult immune systems.
PMCID: PMC3437779  PMID: 20059468
prematurity; preterm birth; chorioamnionitis; FIRS; microarray; transcriptomics
13.  Down-weighting overlapping genes improves gene set analysis 
BMC Bioinformatics  2012;13:136.
The identification of gene sets that are significantly impacted in a given condition based on microarray data is a crucial step in current life science research. Most gene set analysis methods treat genes equally, regardless how specific they are to a given gene set.
In this work we propose a new gene set analysis method that computes a gene set score as the mean of absolute values of weighted moderated gene t-scores. The gene weights are designed to emphasize the genes appearing in few gene sets, versus genes that appear in many gene sets. We demonstrate the usefulness of the method when analyzing gene sets that correspond to the KEGG pathways, and hence we called our method Pathway Analysis with Down-weighting of Overlapping Genes (PADOG). Unlike most gene set analysis methods which are validated through the analysis of 2-3 data sets followed by a human interpretation of the results, the validation employed here uses 24 different data sets and a completely objective assessment scheme that makes minimal assumptions and eliminates the need for possibly biased human assessments of the analysis results.
PADOG significantly improves gene set ranking and boosts sensitivity of analysis using information already available in the gene expression profiles and the collection of gene sets to be analyzed. The advantages of PADOG over other existing approaches are shown to be stable to changes in the database of gene sets to be analyzed. PADOG was implemented as an R package available at:
PMCID: PMC3443069  PMID: 22713124
Gene expression; Gene set analysis; Pathway analysis; Overlapping gene sets
14.  A molecular signature of an arrest of descent in human parturition 
American journal of obstetrics and gynecology  2011;204(2):177.e15-177.e33.
This study was undertaken to identify the molecular basis of an arrest of descent.
Study Design
Human myometrium was obtained from women in term labor (TL; n=29) and arrest of descent (AODes, n=21). Gene expression was characterized using Illumina® HumanHT-12 microarrays. A moderated t-test and false discovery rate adjustment were applied for analysis. Confirmatory qRT-PCR and immunoblot was performed in an independent sample set.
400 genes were differentially expressed between women with an AODes compared to those with TL. Gene Ontology analysis indicated enrichment of biological processes and molecular functions related to inflammation and muscle function. Impacted pathways included inflammation and the actin cytoskeleton. Overexpression of HIF1A, IL-6, and PTGS2 in AODES was confirmed.
We have identified a stereotypic pattern of gene expression in the myometrium of women with an arrest of descent. This represents the first study examining the molecular basis of an arrest of descent using a genome-wide approach.
PMCID: PMC3053040  PMID: 21284969
pregnancy; parturition; myometrium; spontaneous term labor; systems biology; inflammation; transcriptomics; interleukin-6; IL6; prostaglandin-endoperoxide synthase 2; COX2; PTGS2; hypoxia inducible factor-1a; HIF1A; ATPase, Na+/K+ transporting, alpha 1 polypeptide; ATP1A1
15.  Characterization of the myometrial transcriptome and biological pathways of spontaneous human labor at term 
Journal of perinatal medicine  2010;38(6):617-643.
To characterize the transcriptome of human myometrium during spontaneous labor at term.
Myometrium was obtained from women with (n=19) and without labor (n=20). Illumina® HumanHT-12 microarrays were utilized. Moderated t-tests and False Discovery Rate adjustment of p-values were applied. qRT-PCR was performed for a select set of differentially expressed genes in a separate set of samples. ELISA and Western Blot were utilized to confirm differential protein production in a third sample set.
1) 471 genes were differentially expressed; 2) Gene Ontology analysis indicated enrichment of 103 biological processes and 18 molecular functions including: a) inflammatory response; b) cytokine activity; and c) chemokine activity; 3) systems biology pathway analysis using Signaling Pathway Impact Analysis indicated 6 significant pathways: a) cytokine-cytokine receptor interaction; b) Jak-Stat signaling; and c) complement and coagulation cascades; d) NOD-like receptor signaling pathway; e) Systemic Lupus Erythematosus; and f) Chemokine signaling pathway; 3) qRT-PCR confirmed over-expression of prostaglandin-endoperoxide synthase-2 (PTGS2/COX2), heparin binding EGF-like growth factor (HBEGF), chemokine C-C motif ligand 2 (CCL2/MCP1), leukocyte immunoglobulin-like receptor, subfamily A member 5 (LILRA5/LIR9), IL-8, IL-6, chemokine C-X-C motif ligand 6 (CXCL6/GCP2), nuclear factor of kappa light chain gene enhancer in B-cells inhibitor zeta (NFKBIZ), suppressor of cytokine signaling 3 (SOCS3) and decreased expression of FK506 binding-protein 5 (FKBP5) and aldehyde dehydrogenase (ALDH2) in labor; 4) IL-6, CXCL6, CCL2 and SOCS3 protein expression was significantly higher in the term labor group compared to the term not in labor group.
Myometrium of women in spontaneous labor at term is characterized by a stereotypic gene expression pattern consistent with over-expression of the inflammatory response and leukocyte chemotaxis. Differential gene expression identified with microarray was confirmed with qRT-PCR using an independent set of samples. This study represents an unbiased description of the biological processes involved in spontaneous labor at term based on transcriptomics.
PMCID: PMC3097097  PMID: 20629487
inflammation; microarray; pregnancy; parturition; systems biology; aldehyde dehydrogenase; ALDH2; CCL2/MCP-1; CXCL6/GCP2; FK506 binding-protein 5; FKBP5; heparin binding EGF-like growth factor; HBEGF; IL-6; IL-8; leukocyte immunoglobulin-like receptor; subfamily A member 5; LILRA5/LIR9; nuclear factor of kappa light chain gene enhancer in B-cells inhibitor zeta; NFKBIZ; PTGS2/COX2; suppressor of cytokine signaling 3; SOCS3; progesterone; inflammasome
16.  The Biological Connection Markup Language: a SBGN-compliant format for visualization, filtering and analysis of biological pathways 
Bioinformatics  2011;27(15):2127-2133.
Motivation: Many models and analysis of signaling pathways have been proposed. However, neither of them takes into account that a biological pathway is not a fixed system, but instead it depends on the organism, tissue and cell type as well as on physiological, pathological and experimental conditions.
Results: The Biological Connection Markup Language (BCML) is a format to describe, annotate and visualize pathways. BCML is able to store multiple information, permitting a selective view of the pathway as it exists and/or behave in specific organisms, tissues and cells. Furthermore, BCML can be automatically converted into data formats suitable for analysis and into a fully SBGN-compliant graphical representation, making it an important tool that can be used by both computational biologists and ‘wet lab’ scientists.
Availability and implementation: The XML schema and the BCML software suite are freely available under the LGPL for download at They are implemented in Java and supported on MS Windows, Linux and OS X.
Supplementary information: Supplementary data are available at Bioinformatics online.
PMCID: PMC3137220  PMID: 21653523
17.  Surfactant Protein-A as an Anti-Inflammatory Component in the Amnion: Implications for Human Pregnancy1 
The mechanism of mouse parturition is thought to involve myometrial infiltration by amniotic fluid (AF) macrophages, activated by surfactant protein-A (SP-A). In humans, the concentration of AF SP-A decreases during labor, and no fetal macrophages are found in the myometrium after labor. Therefore, it appears that the mechanisms of labor in mice and humans are different. We investigated a potential role for SP-A in human pregnancy and parturition by examining SP-A expression patterns in AF and amnion. High molecular weight (HMW; >250 kD) oligomeric SP-A was increased in AF with advancing gestation. Interestingly, these oligomers were more abundant in placental amnion before labor at term, while they increased primarily in reflected amnion during labor (p<0.05). Immunoblotting showed a binding of HMW SP-A in AF to amnion. In C57BL/6 mice, oligomeric SP-A was also readily detected in AF from E15 onwards, but not in amnion. Macrophage density in mice myometrium did not change with advancing gestational age. Microarray analysis of human amnion explants incubated with SP-A revealed a molecular signature of inhibited cytokine-cytokine receptor interaction with down-regulation of IL-1β, CXCL2, and CXCL5 mRNA expression. The findings herein strongly suggest that SP-A signals amniotic anti-inflammatory response via amniotic fluid during pregnancy. We propose that a SP-A interaction among amniotic fluid, placental amnion, and reflected amnion is a unique mechanism for immunoregulation in human pregnancy akin to that established in lung biology. However, amniotic fluid SP-A and fetal macrophages by themselves do not seem to be exclusive effectors of parturition in humans.
This is an author-produced version of a manuscript accepted for publication in The Journal of Immunology (The JI). The American Association of Immunologists, Inc. (AAI), publisher of The JI, holds the copyright to this manuscript. This version of the manuscript has not yet been copyedited or subjected to editorial proofreading by The JI; hence, it may differ from the final version published in The JI (online and in print). AAI (The JI) is not liable for errors or omissions in this author-produced version of the manuscript or in any version derived from it by the U.S. National Institutes of Health or any other third party. The final, citable version of record can be found at
PMCID: PMC3103775  PMID: 20439915
human; inflammation; mucosa; macrophages; reproductive immunology
18.  DC-ATLAS: a systems biology resource to dissect receptor specific signal transduction in dendritic cells 
Immunome Research  2010;6:10.
The advent of Systems Biology has been accompanied by the blooming of pathway databases. Currently pathways are defined generically with respect to the organ or cell type where a reaction takes place. The cell type specificity of the reactions is the foundation of immunological research, and capturing this specificity is of paramount importance when using pathway-based analyses to decipher complex immunological datasets. Here, we present DC-ATLAS, a novel and versatile resource for the interpretation of high-throughput data generated perturbing the signaling network of dendritic cells (DCs).
Pathways are annotated using a novel data model, the Biological Connection Markup Language (BCML), a SBGN-compliant data format developed to store the large amount of information collected. The application of DC-ATLAS to pathway-based analysis of the transcriptional program of DCs stimulated with agonists of the toll-like receptor family allows an integrated description of the flow of information from the cellular sensors to the functional outcome, capturing the temporal series of activation events by grouping sets of reactions that occur at different time points in well-defined functional modules.
The initiative significantly improves our understanding of DC biology and regulatory networks. Developing a systems biology approach for immune system holds the promise of translating knowledge on the immune system into more successful immunotherapy strategies.
PMCID: PMC3000836  PMID: 21092113
19.  Villitis of Unknown Etiology is Associated with a Distinct Pattern of Chemokine Up-regulation in the Feto-maternal and Placental Compartments: Implications for Conjoint Maternal Allograft Rejection and Maternal Anti-fetal Graft-versus-Host Disease1 
The co-presence of histoincompatible fetal and maternal cells is a characteristic of human placental inflammation. Villitis of unknown etiology (VUE), a destructive inflammatory lesion of villous placenta, is characterized by participation of Hofbauer cells (placental macrophages) and maternal T cells. In contrast to acute chorioamnionitis of infection-related origin, the fundamental immunopathology of VUE is unknown. This study was performed to investigate the placental transcriptome of VUE and to determine whether VUE is associated with systemic maternal and/or fetal inflammatory response(s). Comparison of the transcriptome between term placentas without and with VUE revealed differential expression of 206 genes associated with pathways related to immune response. The mRNA expression of a subset of chemokines and their receptors (CXCL9, CXCL10, CXCL11, CXCL13, CCL4, CCL5, CXCR3, CCR5) was higher in VUE placentas than in normal placentas (p < 0.05). Analysis of blood cell mRNA showed a higher expression of CXCL9 and CXCL13 in the mother, and CXCL11 and CXCL13 in the fetus of VUE cases (p < 0.05). The median concentrations of CXCL9, CXCL10, and CXCL11 in maternal and fetal plasma were higher in VUE (p < 0.05). Comparison of preterm cases without and with acute chorioamnionitis revealed elevated CXCL9, CXCL10, CXCL11, and CXCL13 concentrations in fetal plasma (p < 0.05), but not in maternal plasma with chorioamnionitis. We report for the first time the placental transcriptome of VUE. A systemic derangement of CXC chemokines in maternal and fetal circulation distinguishes VUE from acute chorioamnionitis. We propose that VUE be a unique state combining maternal allograft rejection and maternal antifetal graft-vs-host disease mechanisms.
PMCID: PMC2754231  PMID: 19265171
human; inflammation; chemokines; graft versus host disease; transplantation
20.  Gene expression profiling demonstrates a novel role for foetal fibrocytes and the umbilical vessels in human fetoplacental development 
There is a difference in the susceptibility to inflammation between the umbilical vein (UV) and the umbilical arteries (UAs). This led us to hypothesize that there is an intrinsic difference in the pro-inflammatory response between the UA and UV. Real-time quantitative RT-PCR and microarray analysis revealed higher expression of interleukin (IL)-1β and IL-8 mRNA in the UV and differential expression of 567 genes between the UA and UV associated with distinct biological processes, including the immune response. Differential expression of human leukocyte antigen (HLA)-DRA mRNA between the UA and UV was due to unexpected HLA-DR+ cells migrating via the umbilical vessels into Wharton’s jelly, more frequently in the UV. A significant proportion of these cells co-expressed CD45 and type I pro-collagen, and acquired CD163 or α-smooth muscle actin immunoreactivity in Wharton’s jelly. Migrating cells were also found in the chorionic and stem villous vessels. Furthermore, the extent of migration increased with progression of gestation, but diminished in intrauterine growth restriction (IUGR). The observations herein strongly suggest that circulating foetal fibrocytes, routing via umbilical and placental vessels, are a reservoir for key cellular subsets in the placenta. This study reports fibrocytes in the human umbilical cord and placenta for the first time, and a novel role for both circulating foetal cells and the umbilical vessels in placental development, which is deranged in IUGR.
PMCID: PMC2837362  PMID: 18298660
umbilical vein; umbilical artery; placenta; funisitis; chorioamnionitis; intrauterine growth restriction; transcriptome; microarray
21.  Mitochondrial MnSOD mRNA expression in human chorioamniotic membranes and its association with labor, inflammation and infection 
Human parturition is characterized by the activation of genes involved in acute inflammatory in the fetal membranes. Manganese superoxide dismutase (MnSOD) is a mitochondrial enzyme that scavenges reactive oxygen species (ROS). MnSOD is up-regulated in sites of inflammation and has an important role in the down-regulation of acute inflammatory processes. Therefore, the aim of this study was to determine the differences in MnSOD mRNA expression in the fetal membranes in patients with term and preterm labor as well as in acute chorioamnionitis.
Study design
Fetal membranes were obtained from patients in the following groups: 1) term not in labor (n=29); 2) term in labor (n=29); 3) spontaneous preterm labor with intact mebranes (n=16); 4) PTL with histological chorioamnionitis (n=12); 5) preterm prelabor rupture of membranes (PPROM; n=17); and 6) PPROM with histological chorioamnionitis (n=21). MnSOD mRNA expression in the membranes was determined by quantitative real-time RT-PCR.
1) MnSOD mRNA expression was higher in the fetal membranes of patients at term in labor than those not in labor (2.4-fold; p=0.02); 2) the amount of MnSOD mRNA in the fetal membranes was higher in PTL than in term labor or in PPROM (7.2-fold, p=0.03; 3.2-fold, p=0.03, respectively); 3) MnSOD mRNA expression was higher when histological chorioamnionitis was present both among patients with PPROM (3.8-fold, p=0.02) and with PTL (5.4-fold, p=0.02) than in patients with these conditions without histological chorioamnionitis; 4) expression of MnSOD mRNA was higher in PTL with chorioamnionitis than in PPROM with chorioamnionitis (4.3-fold, p=0.03);
The increase in MnSOD mRNA expression by fetal membranes in term labor and in histological chorioamnionitis in PTL and PPROM suggests that the fetus deploys anti-oxidant mechanisms to constrain the inflammatory processes in the chorioamniotic membranes.
PMCID: PMC2823267  PMID: 19900038
fetal gender; gene expression; preterm delivery; preterm labor; preterm prelabor rupture of the membranes; reactive oxygen species; scavenger
22.  Differential expression of microRNAs with progression of gestation and inflammation in the human chorioamniotic membranes 
The aim of this study was to identify differential expression of microRNAs (miRNAs) in chorioamniotic membranes with advancing gestation, labor, and inflammation.
Study design
Expression profiles of 157 miRNAs in the chorioamniotic membranes were obtained from patients in the following groups: 1) term not in labor (n=10); 2) term in labor (n=10); 3) preterm labor with histologic chorioamnionitis (n=9); and 4) without histologic chorioamnionitis (n=10).
More than 95% of the miRNAs screened were expressed. Gestational age-dependent changes in expression were observed for 13 miRNAs. No differences in miRNA expression were observed between women without labor and women in labor. Membranes with chorioamnionitis displayed increased expression of miR-223 and miR-338. Gene Ontology analysis of genes targeted by differentially expressed miRNAs revealed enrichment for specific biological process categories.
Chorioamniotic membranes with advancing gestational age and chorioamnionitis are associated with the differential expression of a subset of miRNAs.
PMCID: PMC2810125  PMID: 17826424
chorioamniotic membranes; gestation; inflammation; labor; microRNA; real-time qRT-PCR
23.  NetPath: a public resource of curated signal transduction pathways 
Genome Biology  2010;11(1):R3.
NetPath, a novel community resource of curated human signaling pathways is presented and its utility demonstrated using immune signaling data.
We have developed NetPath as a resource of curated human signaling pathways. As an initial step, NetPath provides detailed maps of a number of immune signaling pathways, which include approximately 1,600 reactions annotated from the literature and more than 2,800 instances of transcriptionally regulated genes - all linked to over 5,500 published articles. We anticipate NetPath to become a consolidated resource for human signaling pathways that should enable systems biology approaches.
PMCID: PMC2847715  PMID: 20067622
24.  Region-Specific Gene Expression Profiling: Novel Evidence for Biological Heterogeneity of the Human Amnion1 
Biology of reproduction  2008;79(5):954-961.
The amnion plays an important role during pregnancy and parturition. Though referred to as a single structure, this fetal tissue is regionally divided into placental amnion, reflected amnion, and umbilical amnion. Histological differences between placental amnion and reflected amnion led us to hypothesize that the amnion is biologically heterogeneous. The gene expression profiles of placental amnion and reflected amnion were compared in patients at term with no labor (TNL; n = 10) and in labor (TIL; n = 10). Real-time quantitative RT-PCR revealed a higher expression of IL1B mRNA in reflected amnion than in placental amnion in TNL cases but not in TIL cases. Extended screening using microarrays showed differential expression of 17 genes in labor, regardless of the region. Interestingly, 839 genes were differentially expressed between placental amnion and reflected amnion. Pathway analysis identified 19 signaling pathways, such as mitogen-activated protein kinase and transforming growth factor beta pathways, associated with region. Lipopolysaccharide (LPS) treatment of the amnion explants showed more robust activation of mitogen-activated protein kinase 3/1 (extracellular signal-regulated kinase 1/2) in placental amnion of TNL but not in TIL cases. Placental amnion from TNL and TIL cases showed a significant difference in the amplitude of IL1B mRNA induction by LPS. We report that the anatomical region has a substantial impact on the transcriptional program and the biological properties of the amnion. Labor-associated switching to a proinflammatory signature is a feature particular to placental amnion. The novel observations herein strongly suggest that the seemingly homogeneous amnion is biologically heterogeneous and compartmentalized, with implications for the physiology of pregnancy and parturition.
PMCID: PMC2714995  PMID: 18685129
IL1B; lipopolysaccharide; microarray; parturition; placenta; pregnancy
25.  A novel signaling pathway impact analysis 
Bioinformatics  2008;25(1):75-82.
Motivation: Gene expression class comparison studies may identify hundreds or thousands of genes as differentially expressed (DE) between sample groups. Gaining biological insight from the result of such experiments can be approached, for instance, by identifying the signaling pathways impacted by the observed changes. Most of the existing pathway analysis methods focus on either the number of DE genes observed in a given pathway (enrichment analysis methods), or on the correlation between the pathway genes and the class of the samples (functional class scoring methods). Both approaches treat the pathways as simple sets of genes, disregarding the complex gene interactions that these pathways are built to describe.
Results: We describe a novel signaling pathway impact analysis (SPIA) that combines the evidence obtained from the classical enrichment analysis with a novel type of evidence, which measures the actual perturbation on a given pathway under a given condition. A bootstrap procedure is used to assess the significance of the observed total pathway perturbation. Using simulations we show that the evidence derived from perturbations is independent of the pathway enrichment evidence. This allows us to calculate a global pathway significance P-value, which combines the enrichment and perturbation P-values. We illustrate the capabilities of the novel method on four real datasets. The results obtained on these data show that SPIA has better specificity and more sensitivity than several widely used pathway analysis methods.
Availability: SPIA was implemented as an R package available at
Supplementary information: Supplementary data are available at Bioinformatics online.
PMCID: PMC2732297  PMID: 18990722

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