Loss of cone cyclic nucleotide-gated channel leads to alterations in light response modulating system and cellular stress response pathways: a gene expression profiling study.

The cone photoreceptor cyclic nucleotide-gated (CNG) channel is essential for central and color vision and visual acuity. Mutations in the channel subunits CNGA3 and CNGB3 are associated with achromatopsia and cone dystrophy. We investigated the gene expression profiles in mouse retina with CNG channel deficiency using whole genome expression microarrays. As cones comprise only 2 to 3% of the total photoreceptor population in the wild-type mouse retina, the mouse lines with CNG channel deficiency on a cone-dominant background, i.e. Cnga3-/-/Nrl-/- and Cngb3-/-/Nrl-/- mice, were used in our study. Comparative data analysis revealed a total of 105 genes altered in Cnga3-/-/Nrl-/- and 92 in Cngb3-/-/Nrl-/- retinas, relative to Nrl-/- retinas, with 27 genes changed in both genotypes. The differentially expressed genes primarily encode proteins associated with cell signaling, cellular function maintenance and gene expression. Ingenuity pathway analysis (IPA) identified 26 and 9 canonical pathways in Cnga3-/-/Nrl-/- and Cngb3-/-/Nrl-/- retinas, respectively, with 6 pathways being shared. The shared pathways include phototransduction, cAMP/PKA-mediated signaling, endothelin signaling, and EIF2/endoplasmic reticulum (ER) stress, whereas the IL-1, CREB, and purine metabolism signaling were found to specifically associate with Cnga3 deficiency. Thus, CNG channel deficiency differentially regulates genes that affect cell processes such as phototransduction, cellular survival and gene expression, and such regulations play a crucial role(s) in the retinal adaptation to impaired cone phototransduction. Though lack of Cnga3 and Cngb3 shares many common pathways, deficiency of Cnga3 causes more significant alterations in gene expression. This work provides insights into how cones respond to impaired phototransduction at the gene expression levels.

The canonical Wnt pathway shapes niches supportive of hematopoietic stem/progenitor cells.

Considerable information has accumulated about components of BM that regulate the survival, self-renewal, and differentiation of hematopoietic cells. In the present study, we investigated Wnt signaling and assessed its influence on human and murine hematopoiesis. Hematopoietic stem/progenitor cells (HSPCs) were placed on Wnt3a-transduced OP9 stromal cells. The proliferation and production of B cells, natural killer cells, and plasmacytoid dendritic cells were blocked. In addition, some HSPC characteristics were maintained or re-acquired along with different lineage generation potentials. These responses did not result from direct effects of Wnt3a on HSPCs, but also required alterations in the OP9 cells. Microarray, PCR, and flow cytometric experiments revealed that OP9 cells acquired osteoblastic characteristics while down-regulating some features associated with mesenchymal stem cells, including the expression of angiopoietin 1, the c-Kit ligand, and VCAM-1. In contrast, the production of decorin, tenascins, and fibromodulin markedly increased. We found that at least 1 of these extracellular matrix components, decorin, is a regulator of hematopoiesis: upon addition of this proteoglycan to OP9 cocultures, decorin caused changes similar to those caused by Wnt3a. Furthermore, hematopoietic stem cell numbers in the BM and spleen were elevated in decorin-knockout mice. These findings define one mechanism through which canonical Wnt signaling could shape niches supportive of hematopoiesis.

Functional characterization of the MECP2/IRAK1 lupus risk haplotype in human T cells and a human MECP2 transgenic mouse.

Genetic polymorphism in MECP2/IRAK1 on chromosome Xq28 is a confirmed and replicated susceptibility locus for lupus. High linkage disequilibrium in this locus suggests that both MECP2 and IRAK1 are candidate genes for the disease. DNA methylation changes in lupus T cells play a central role in the pathogenesis of lupus, and MeCp-2 (encoded by MECP2) is a master regulator of gene expression and is also known to recruit DNA methyltransferase 1 (DNMT1) during DNA synthesis. Using human T cells from normal individuals with either the lupus risk or the lupus protective haplotype in MECP2/IRAK1, we demonstrate that polymorphism in this locus increases MECP2 isoform 2 mRNA expression in stimulated but not unstimulated T cells. By assessing DNA methylation levels across over 485,000 methylation sites across the entire genome, we further demonstrate that the lupus risk variant in this locus is associated with significant DNA methylation changes, including in the HLA-DR and HLA-DQ loci, as well as interferon-related genes such as IFI6, IRF6, and BST2. Further, using a human MECP2 transgenic mouse, we show that overexpression of MECP2 alters gene expression in stimulated T cells. This includes overexpression of Eif2c2 that regulates the expression of multiple microRNAs (such as miR-21), and the histone demethylase Jhdm1d. In addition, we show that MECP2 transgenic mice develop antinuclear antibodies. Our data suggest that the lupus-associated variant in the MECP2/IRAK1 locus has the potential to affect all 3 epigenetic mechanisms: DNA methylation, microRNA expression, and histone modification. Importantly, these data support the notion that variants within the MECP2 gene can alter DNA methylation in other genetic loci including the HLA and interferon-regulated genes, thereby providing evidence for genetic-epigenetic interaction in lupus.

Inflammation and neurological disease-related genes are differentially expressed in depressed patients with mood disorders and correlate with morphometric and functional imaging abnormalities.

Depressed patients show evidence of both proinflammatory changes and neurophysiological abnormalities such as increased amygdala reactivity and volumetric decreases of the hippocampus and ventromedial prefrontal cortex (vmPFC). However, very little is known about the relationship between inflammation and neuroimaging abnormalities in mood disorders. A whole genome expression analysis of peripheral blood mononuclear cells yielded 12 protein-coding genes (ADM, APBB3, CD160, CFD, CITED2, CTSZ, IER5, NFKBIZ, NR4A2, NUCKS1, SERTAD1, TNF) that were differentially expressed between 29 unmedicated depressed patients with a mood disorder (8 bipolar disorder, 21 major depressive disorder) and 24 healthy controls (HCs). Several of these genes have been implicated in neurological disorders and/or apoptosis. Ingenuity Pathway Analysis yielded two genes networks, one centered around TNF with NFKß, TGFß, and ERK as connecting hubs, and the second network indicating cell cycle and/or kinase signaling anomalies. fMRI scanning was conducted using a backward-masking task in which subjects were presented with emotionally-valenced faces. Compared with HCs, the depressed subjects displayed a greater hemodynamic response in the right amygdala, left hippocampus, and the ventromedial prefrontal cortex to masked sad versus happy faces. The mRNA levels of several genes were significantly correlated with the hemodynamic response of the amygdala, vmPFC and hippocampus to masked sad versus happy faces. Differentially-expressed transcripts were significantly correlated with thickness of the left subgenual ACC, and volume of the hippocampus and caudate. Our results raise the possibility that molecular-level immune dysfunction can be mapped onto macro-level neuroimaging abnormalities, potentially elucidating a mechanism by which inflammation leads to depression.

Estrogen receptor signaling promotes dendritic cell differentiation by increasing expression of the transcription factor IRF4.

During inflammation, elevated granulocyte macrophage-colony-stimulating factor (GM-CSF) directs the development of new dendritic cells (DCs). This pathway is influenced by environmental factors, and we previously showed that physiologic levels of estradiol, acting through estrogen receptor alpha (ERalpha), promote the GM-CSF-mediated differentiation of a CD11b(+) DC subset from myeloid progenitors (MPs). We now have identified interferon regulatory factor 4 (IRF4), a transcription factor induced by GM-CSF and critical for CD11b(+) DC development in vivo, as a target of ERalpha signaling during this process. In MPs, ERalpha potentiates and sustains GM-CSF induction of IRF4. Furthermore, retroviral delivery of the Irf4 cDNA to undifferentiated ERalpha(-/-) bone marrow cells restored the development of the estradiol/ERalpha-dependent DC population, indicating that an elevated amount of IRF4 protein substitutes for ERalpha signaling. Thus at an early stage in the MP response to GM-CSF, ERalpha signaling induces an elevated amount of IRF4, which leads to a developmental program underlying CD11b(+) DC differentiation.

External Qi of Yan Xin Qigong induces cell death and gene expression alterations promoting apoptosis and inhibiting proliferation, migration and glucose metabolism in small-cell lung cancer cells.

Small-cell lung cancer (SCLC) is a highly malignant carcinoma with poor long-term survival. Effective treatment remains highly demanded. In the present study, we demonstrated that External Qi of Yan Xin Qigong (YXQ-EQ) exerted potent cytotoxic effect towards SCLC cell line NCI-H82 via induction of apoptosis. Global gene expression profiling identified 39 genes whose expression was altered by YXQ-EQ in NCI-82 cells. Among them, semi-quantitative RT-PCR and real-time qPCR analyses confirmed that the gene expression levels of apoptotic proteins death-associated protein kinase 2 and cell death-inducing DFFA-like effector b were upregulated, whereas that of oncoproteins DEK and MYCL1, cell migration-promoting proteins CD24 and integrin-alpha 9, and glycolytic enzyme aldolase A were downregulated. These findings suggest that YXQ-EQ may exert anticancer effect through modulating gene expression in a way that facilitates cancer cell apoptosis while represses proliferation, metastasis, and glucose metabolism.

Erroneous augmentation of multiplex assay measurements in patients with rheumatoid arthritis due to heterophilic binding by serum rheumatoid factor.

OBJECTIVE: Serum rheumatoid factor (RF) and other heterophilic antibodies potentially interfere with antibody-based immunoassays by nonspecifically binding detection reagents. The purpose of this study was to assess whether these factors confound multiplex-based immunoassays, which are used with increasing frequency to measure cytokine and chemokine analytes in patients with rheumatoid arthritis (RA). METHODS: We performed multiplex immunoassays using different platforms to measure analyte concentrations in RA patient samples. Samples were depleted of RF by column-based affinity absorption or were exposed to agents that block heterophilic binding activity. RESULTS: In RA patients with high-titer RF, 69% of analytes demonstrated at least a 2-fold stronger multiplex signal in non-RF-depleted samples as compared to RF-depleted samples. This degree of erroneous signal amplification was less frequent in low-titer RF samples (17% of analytes; P < 0.0000001). Signal amplification by heterophilic antibodies was blocked effectively by HeteroBlock (≥ 150 µg/ml). In 35 RA patients, multiplex signals for 14 of 22 analytes were amplified erroneously in unblocked samples as compared to blocked samples (some >100-fold), but only in patients with high-titer RF (P < 0.002). Two other blocking agents, heterophilic blocking reagent and immunoglobulin-inhibiting reagent, also blocked heterophilic activity. CONCLUSION: All multiplex protein detection platforms we tested exhibited significant confounding by RF or other heterophilic antibodies. These findings have broad-reaching implications in the acquisition and interpretation of data derived from multiplex immunoassay testing of RA patient serum and possibly also in other conditions in which RF or other heterophilic antibodies may be present. Several available blocking agents effectively suppressed this erroneous signal amplification in the multiplex platforms tested.

Unique patterns of molecular profiling between human prostate cancer LNCaP and PC-3 cells.

BACKGROUND: Human prostate cancer LNCaP and PC-3 cell lines have been extensively used to study prostate cancer progression and to develop therapeutic agents. Although LNCaP and PC-3 cells are generally assumed to represent early and late stages of prostate cancer, respectively, there is limited information regarding gene expression patterns between these two cell lines and its relationship to prostate cancer. METHODS: Comprehensive gene expression analysis was performed. Total RNA was isolated from cultured cells and hybridized to Illumina human BeadChips representing 24,526 transcripts. Bioinformatics analysis was applied to identify cell line specific genes as well as biological mechanisms, pathways, and functions related to the genes. RESULTS: A total of 2,198 genes were differentially expressed between LNCaP and PC-3 cells. Using a robust statistical analysis and high significance criteria, 115 and 188 genes were identified to be unique to LNCaP and PC-3 cells, respectively. LNCaP cells maintained various metabolic pathways including a gene cluster that encodes UDP-glucuronosyltransferases. Several transcription factors including Tal alpha/beta, GATA-1, and c-Myc/Max may be responsible for regulating LNCaP cell specific genes. By contrast, PC-3 cells were characterized by their unique expression of cytoskeleton-related genes and other genes including VEGFC, IL8, and TGF beta 2. CONCLUSIONS: This study showed that LNCaP and PC-3 cells represent two distinct prostate cancer cell lineages. LNCaP cells retain many prostate cell specific properties, whereas PC-3 cells have acquired a more aggressive phenotype. Future studies for prostate cancer research need to consider similarities and differences between these two cells and their relationship to prostate cancer.

Frankincense oil derived from Boswellia carteri induces tumor cell specific cytotoxicity.

BACKGROUND: Originating from Africa, India, and the Middle East, frankincense oil has been important both socially and economically as an ingredient in incense and perfumes for thousands of years. Frankincense oil is prepared from aromatic hardened gum resins obtained by tapping Boswellia trees. One of the main components of frankincense oil is boswellic acid, a component known to have anti-neoplastic properties. The goal of this study was to evaluate frankincense oil for its anti-tumor activity and signaling pathways in bladder cancer cells. METHODS: Frankincense oil-induced cell viability was investigated in human bladder cancer J82 cells and immortalized normal bladder urothelial UROtsa cells. Temporal regulation of frankincense oil-activated gene expression in bladder cancer cells was identified by microarray and bioinformatics analysis. RESULTS: Within a range of concentration, frankincense oil suppressed cell viability in bladder transitional carcinoma J82 cells but not in UROtsa cells. Comprehensive gene expression analysis confirmed that frankincense oil activates genes that are responsible for cell cycle arrest, cell growth suppression, and apoptosis in J82 cells. However, frankincense oil-induced cell death in J82 cells did not result in DNA fragmentation, a hallmark of apoptosis. CONCLUSION: Frankincense oil appears to distinguish cancerous from normal bladder cells and suppress cancer cell viability. Microarray and bioinformatics analysis proposed multiple pathways that can be activated by frankincense oil to induce bladder cancer cell death. Frankincense oil might represent an alternative intravesical agent for bladder cancer treatment.

Modeling Transcriptional Rewiring in Neutrophils Through the Course of Treated Juvenile Idiopathic Arthritis.

Neutrophils in children with the polyarticular form of juvenile idiopathic arthritis (JIA) display abnormal transcriptional patterns linked to fundamental metabolic derangements. In this study, we sought to determine the effects of therapy on mRNA and miRNA expression networks in polyarticular JIA. Using exon and miRNA microarrays, we studied children with untreated active JIA (ADU, n = 35), children with active disease on therapy with methotrexate ± etanercept (ADT, n = 26), and children with inactive disease also on therapy (ID, n = 14). We compared the results to findings from healthy control children (HC, n = 35). We found substantial re-ordering of mRNA and miRNA expression networks after the initiation of therapy. Each disease state was associated with a distinct transcriptional profile, with the ADT state differing the most from HC, and ID more strongly resembling HC. Changes at the mRNA level were mirrored in changes in miRNA expression patterns. The analysis of the expression dynamics from differentially expressed genes across three disease states indicated that therapeutic response is a complex process. This process does not simply involve genes slowly correcting in a linear fashion over time. Computational modeling of miRNA and transcription factor (TF) co-regulatory networks demonstrated that combinational regulation of miRNA and TF might play an important role in dynamic transcriptome changes.

Discriminators of mouse bladder response to intravesical Bacillus Calmette-Guerin (BCG).

BACKGROUND: Intravesical Bacillus Calmette-Guerin (BCG) is an effective treatment for bladder superficial carcinoma and it is being tested in interstitial cystitis patients, but its precise mechanism of action remains poorly understood. It is not clear whether BCG induces the release of a unique set of cytokines apart from its pro-inflammatory effects. Therefore, we quantified bladder inflammatory responses and alterations in urinary cytokine protein induced by intravesical BCG and compared the results to non-specific pro-inflammatory stimuli (LPS and TNF-alpha). We went further to determine whether BCG treatment alters cytokine gene expression in the urinary bladder. METHODS: C57BL/6 female mice received four weekly instillations of BCG, LPS, or TNF-alpha. Morphometric analyses were conducted in bladders isolated from all groups and urine was collected for multiplex analysis of 18 cytokines. In addition, chromatin immune precipitation combined with real-time polymerase chain reaction assay (CHIP/Q-PCR) was used to test whether intravesical BCG would alter bladder cytokine gene expression. RESULTS: Acute BCG instillation induced edema which was progressively replaced by an inflammatory infiltrate, composed primarily of neutrophils, in response to weekly administrations. Our morphological analysis suggests that these polymorphonuclear neutrophils are of prime importance for the bladder responses to BCG. Overall, the inflammation induced by BCG was higher than LPS or TNF-alpha treatment but the major difference observed was the unique granuloma formation in response to BCG. Among the cytokines measured, this study highlighted the importance of IL-1beta, IL-2, IL-3, IL-4, IL-6, IL-10, IL-17, GM-CSF, KC, and Rantes as discriminators between generalized inflammation and BCG-specific inflammatory responses. CHIP/Q-PCR indicates that acute BCG instillation induced an up-regulation of IL-17A, IL-17B, and IL-17RA, whereas chronic BCG induced IL-17B, IL-17RA, and IL-17RB. CONCLUSION: To the best of our knowledge, the present work is the first to report that BCG induces an increase in the IL-17 family genes. In addition, BCG induces a unique type of persisting bladder inflammation different from TNF-alpha, LPS, and, most likely, other classical pro-inflammatory stimuli.

Complexity and Specificity of the Neutrophil Transcriptomes in Juvenile Idiopathic Arthritis.

NIH projects such as ENCODE and Roadmap Epigenomics have revealed surprising complexity in the transcriptomes of mammalian cells. In this study, we explored transcriptional complexity in human neutrophils, cells generally regarded as nonspecific in their functions and responses. We studied distinct human disease phenotypes and found that, at the gene, gene isoform, and miRNA level, neutrophils exhibit considerable specificity in their transcriptomes. Thus, even cells whose responses are considered non-specific show tailoring of their transcriptional repertoire toward specific physiologic or pathologic contexts. We also found that miRNAs had a global impact on neutrophil transcriptome and are associated with innate immunity in juvenile idiopathic arthritis (JIA). These findings have important implications for our understanding of the link between genes, non-coding transcripts and disease phenotypes.

Protection against hydrogen peroxide-induced cell death in cultured human retinal pigment epithelial cells by 17beta-estradiol: a differential gene expression profile.

It has been demonstrated that estrogen receptors are present in the retinal pigment epithelium (RPE)-choroids complex regardless of sex. This suggests that estrogen could play a functional role in the outer retina, especially the RPE. To gain further insights on the molecular mechanisms differentially activated by 17beta-estradiol (betaE2) in RPE cells, we investigated gene expression changes in response to betaE2 in cultured RPE cells using cDNA microarray technology. A total of 47 genes among 21,329 human genes are significantly altered in response to betaE2 treatment in RPE cells. Among these 47 altered genes, 34 are up-regulated and 13 are down-regulated by betaE2. The products of 34 genes have a known or suspected function. These functions belong to various categories, including caspases; extracellular matrix proteins; metabolism pathway components; GTP/GDP exchangers and G-protein GTPase activity modulators; transcription activators and repressors. Six genes which may contribute to the unique functions of the RPE cells have been validated by both quantitative real-time reverse transcription (RT)-PCR and semi-quantitative RT-PCR. In addition, we also demonstrated that betaE2 quenches H2O2-induced up-regulation of apoptosis-related protein, and protects RPE cell degeneration. These results indicate that estrogen regulates functions of RPE cells and is involved in the maintaining and survival of RPE cells during oxidative stress, and its deficiency during menopause period may be a factor contributing to the development of age-related macular degeneration in elderly women.

Structural analysis of IgG2A monoclonal antibodies in relation to complement deposition and renal immune complex deposition.

This study explores the structural features of murine monoclonal IgG2a anti-dinitrophenyl (DNP) antibodies that were previously shown to form immune complexes (IC) differing in their capacity to bind complement, their clearance from the circulation and their deposition in the kidney. Interestingly, the sequence of one of these antibodies has a missing stretch of 14 amino acids within FR3. Molecular modeling suggests that this sequence deletion corresponds to the loss of beta-pleated sheet structure for two beta-strands (designated 4-3 and 4-4) on the external surface of the V(H) domain. Despite this sequence and conformational abnormality, the antibody retains affinity for DNP comparable to other IgG2a antibodies. Data presented here identify monoclonal IgG2a antibodies that form IC with varying propensity for both complement binding and renal deposition and yet have similar V(H) domain sequences. In fact, in the case of two IgG2a antibodies that form IC with very different renal tropisms and complement binding capacity, sequence variation within V(H) was observed only at three clustered residues within FR2, a single residue within FR3 and nine clustered residues spanning CDR3 and FR4. Sequence and modeling analysis also yielded the paradoxical finding that an antibody forming IC with a relatively high capacity to serve as a target for complement binding displays a relatively low number of solvent exposed acceptor residues for C4b and C3b. These data underscore the complex relationship between V domain structure, complement activation and renal deposition of model IC.

Dynamic tracking of functional gene modules in treated juvenile idiopathic arthritis.

BACKGROUND: We have previously shown that childhood-onset rheumatic diseases show aberrant patterns of gene expression that reflect pathology-associated co-expression networks. In this study, we used novel computational approaches to examine how disease-associated networks are altered in one of the most common rheumatic diseases of childhood, juvenile idiopathic arthritis (JIA). METHODS: Using whole blood gene expression profiles derived from children in a pediatric rheumatology clinical trial, we used a network approach to understanding the impact of therapy and the underlying biology of response/non-response to therapy. RESULTS: We demonstrate that therapy for JIA is associated with extensive re-ordering of gene expression networks, even in children who respond inadequately to therapy. Furthermore, we observe distinct differences in the evolution of specific network properties when we compare children who have been treated successfully with those who have inadequate treatment response. CONCLUSIONS: Despite the inherent noisiness of whole blood gene expression data, our findings demonstrate how therapeutic response might be mapped and understood in pathologically informative cells in a broad range of human inflammatory diseases.

Ribosomal and immune transcripts associate with relapse in acquired ADAMTS13-deficient thrombotic thrombocytopenic purpura.

Approximately 40% of patients who survive acute episodes of thrombotic thrombocytopenic purpura (TTP) associated with severe acquired ADAMTS13 deficiency experience one or more relapses. Risk factors for relapse other than severe ADAMTS13 deficiency and ADAMTS13 autoantibodies are unknown. ADAMTS13 autoantibodies, TTP episodes following infection or type I interferon treatment and reported ensuing systemic lupus erythematosus in some patients suggest immune dysregulation. This cross-sectional study asked whether autoantibodies against RNA-binding proteins or peripheral blood gene expression profiles measured during remission are associated with history of prior relapse in acquired ADAMTS13-deficient TTP. Peripheral blood from 38 well-characterized patients with autoimmune ADAMTS13-deficient TTP in remission was examined for autoantibodies and global gene expression. A subset of TTP patients (9 patients, 24%) exhibited a peripheral blood gene signature composed of elevated ribosomal transcripts that associated with prior relapse. A non-overlapping subset of TTP patients (9 patients, 24%) displayed a peripheral blood type I interferon gene signature that associated with autoantibodies to RNA-binding proteins but not with history of relapse. Patients who had relapsed bimodally expressed higher HLA transcript levels independently of ribosomal transcripts. Presence of any one potential risk factor (ribosomal gene signature, elevated HLA-DRB1, elevated HLA-DRB5) associated with relapse (OR = 38.4; p = 0.0002) more closely than any factor alone or all factors together. Levels of immune transcripts typical of natural killer (NK) and T lymphocytes positively correlated with ribosomal gene expression and number of prior episodes but not with time since the most recent episode. Flow cytometry confirmed elevated expression of cell surface markers encoded by these transcripts on T and/or NK cell subsets of patients who had relapsed. These data associate elevated ribosomal and immune transcripts with relapse history in acquired, ADAMTS13-deficient TTP.

Gene expression arrays reveal a rapid return to normal homeostasis in immunologically-challenged trophoblast-like JAR cells.

The immunologic adaptations of pregnancy have come under increasing scrutiny in the past 15 years. Existing experimental evidence clearly demonstrates that placental trophoblasts play an important role in regulating immunologic/inflammatory responses at the maternal-fetal interface. We used a well-developed gene expression array to examine in greater detail the physiologic response of trophoblast-like choriocarcinoma cells to a model immunologic 'challenge.' We co-cultured PHA-activated or resting peripheral blood mononuclear cells (PBMC) with the human choriocarcinoma cell line JAR for time periods ranging from 2 to 18 h. Messenger RNA expression in the JAR cells was then assessed using a 21,329-gene microarray and novel biostatistical analyses that we have previously published. Patterns of differential gene expression were assessed using a commercial pathway analysis software program. Differences in gene expression between JAR cells cultured with activated PBMC (experimental samples) and JAR cells cultured with resting PBMC (control samples) were seen only at the 2h time point. That is, multiple genes were transcribed in JAR cells in response to activated PBMC, but expression levels of the genes had all returned to baseline by 6h. Molecular modeling demonstrated that the differentially expressed genes were largely associated with cell growth and differentiation. This model was confirmed by noting a two-fold increase in CD10/neutral endopeptidase expression (a marker for cell differentiation) in JAR cells incubated with media from activated PBMC compared with JAR cells incubated with resting PBMC. These findings support the hypothesis that there is a delicate immunologic milieu at the maternal-fetal interface that must be maintained. Immunologic/inflammatory challenge at the maternal-fetal interface is compensated by cellular mechanisms that work to reduce inflammation and rapidly restore immunologic balance.

Early effects of Staphylococcus aureus biofilm secreted products on inflammatory responses of human epithelial keratinocytes.

BACKGROUND: Chronic wounds such as diabetic foot ulcers, pressure ulcers, and venous leg ulcers contribute to a considerable amount of mortality in the U.S. annually. The inability of these wounds to heal has now been associated with the presence of microbial biofilms. The aim of this study was to determine if products secreted by S. aureus biofilms play an active role in chronic wounds by promoting inflammation, which is a hallmark of chronic wounds. METHODS: In vitro experiments were conducted to examine changes in gene expression profiles and inflammatory response of human epithelial keratinocytes (HEKa) exposed to products secreted by S. aureus grown in biofilms or products secreted by S. aureus grown planktonically. RESULTS: After only two hours of exposure, gene expression microarray data showed marked differences in inflammatory, apoptotic, and nitric oxide responses between HEKa cells exposed to S. aureus biofilm conditioned media (BCM) and HEKa cells exposed to S. aureus planktonic conditioned media (PCM). As early as 4 hours post exposure, ELISA results showed significant increases in IL-6, IL-8, TNFα, and CXCL2 production by HEKa cells exposed to BCM compared to HEKa cells exposed to PCM or controls. Nitric oxide assay data also showed significant increases in nitric oxide production by HEKa cells treated with BCM compared to HEKa cells treated with PCM, or controls. CONCLUSIONS: Taken together, these results support and extend previous findings that indicate products secreted by S. aureus biofilms directly contribute to the chronic inflammation associated with chronic wounds.

Anthrax lethal toxin-induced gene expression changes in mouse lung.

A major virulence factor of Bacillus anthracis is the anthrax Lethal Toxin (LeTx), a bipartite toxin composed of Protective Antigen and Lethal Factor. Systemic administration of LeTx to laboratory animals leads to death associated with vascular leakage and pulmonary edema. In this study, we investigated whether systemic exposure of mice to LeTx would induce gene expression changes associated with vascular/capillary leakage in lung tissue. We observed enhanced susceptibility of A/J mice to death by systemic LeTx administration compared to the C57BL/6 strain. LeTx-induced groups of both up- and down-regulated genes were observed in mouse lungs 6 h after systemic administration of wild type toxin compared to lungs of mice exposed to an inactive mutant form of the toxin. Lungs of the less susceptible C57BL/6 strain showed 80% fewer differentially expressed genes compared to lungs of the more sensitive A/J strain. Expression of genes known to regulate vascular permeability was modulated by LeTx in the lungs of the more susceptible A/J strain. Unexpectedly, the largest set of genes with altered expression was immune specific, characterized by the up-regulation of lymphoid genes and the down-regulation of myeloid genes. Transcripts encoding neutrophil chemoattractants, modulators of tumor regulation and angiogenesis were also differentially expressed in both mouse strains. These studies provide new directions for the investigation of vascular leakage and pulmonary edema induced by anthrax LeTx.

Functional genomics and rheumatoid arthritis: where have we been and where should we go?

Studies in model organisms and humans have begun to reveal the complexity of the transcriptome. In addition to serving as passive templates from which genes are translated, RNA molecules are active, functional elements of the cell whose products can detect, interact with, and modify other transcripts. Gene expression profiling is the method most commonly used thus far to enrich our understanding of the molecular basis of rheumatoid arthritis in adults and juvenile idiopathic arthritis in children. The feasibility of this approach for patient classification (for example, active versus inactive disease, disease subsets) and improving prognosis (for example, response to therapy) has been demonstrated over the past 7 years. Mechanistic understanding of disease-related differences in gene expression must be interpreted in the context of interactions with transcriptional regulatory molecules and epigenetic alterations of the genome. Ongoing work regarding such functional complexities in the human genome will likely bring both insight and surprise to our understanding of rheumatoid arthritis.