In vitro identification and verification of inflammatory biomarkers in swine.

Currently there are no non-steroidal anti-inflammatory drugs (NSAIDs) approved for the control of inflammation in swine due to a lack of validated animal models and suitable biomarkers to assess drug efficacy. This study investigates the differential expression of genes altered in response to Escherichia coli lipopolysaccharide (LPS) induced inflammation which may serve as indicators of NSAID efficacy. Unstimulated whole blood from swine was mixed with tissue culture media, stimulated with LPS, and RNA extracted at the following time points 0h, 1h, 3h, 24h and 48h. Total RNA was extracted and analyzed using a commercial swine DNA microarray. The DNA microarray was utilized as a screen to determine potential biomarkers, focusing on the genes that exhibited the greatest degree of differential expression. A master list of 57 genes was formed based on the differential expression as a result of the stimulation. Following analysis, 12 genes whose expressions were significantly altered (8 up- and 4 down-regulated) were chosen for verification via quantitative RT-PCR (qRT-PCR). The qRT-PCR analysis confirmed the differential expression of 11 of the 12 genes chosen via the microarray analyses. Specifically, traditional genes such as SAA, G-CSF, and IL-10 were up-regulated, while CD4 was down-regulated; all of the genes were altered by 24h or 48h post-stimulation. We demonstrate here that expression of these 11 genes is altered as a direct result of LPS stimulation and consequently inflammation.

Intragraft gene expression profile associated with the induction of tolerance by allochimeric MHC I in the rat heart transplantation model.

The MHC class I allochimeric protein containing donor-type epitopes on recipient-type heavy chains induces indefinite survival of heterotopic cardiac allografts in rats. We analyzed gene expression profile of heart allograft tissue. Mutated peptide [alpha1h1/u]-RT1.Aa that contains donor-type (Wistar Furth, WF; RT1u) immunogenic epitopes displayed on recipient-type (ACI, RT1a) was delivered into ACI recipients of WF hearts at the time of transplantation in addition to a 3 days course of oral cyclosporine. Microarray analysis was performed using Affymetrix Rat 230 2.0 Microarray. Allochimeric molecule treatment caused upregulation of genes involved in structural integrity of heart muscle, downregulation of IL-1beta a key modulator of the immune response, and downregulation of partitioning defective six homolog gamma PAR6, which is involved in T cell polarity, motility, and ability to scan dendritic cells (DC). These indicate that the immunosuppressive function of allochimeric molecule and/or the establishment of allograft tolerance depend on the induction of genes responsible for the heart tissue integrity, the suppression of cytokine pathway(s), and possibly the impairment of T cells mobility and their DC scanning ability. These novel findings may have important clinical implications for inhibition of chronic rejection in transplant recipients.

Down regulation of genes involved in T cell polarity and motility during the induction of heart allograft tolerance by allochimeric MHC I.

BACKGROUND: The allochimeric MHC class I molecule [alpha1h1/u]-RT1.Aa that contains donor-type (Wistar Furth, WF; RT1u) epitopes displayed on recipient-type (ACI, RT1a) administered in conjunction with sub-therapeutic dose of cyclosporine (CsA) induces indefinite survival of heterotopic cardiac allografts in rat model. In vascularized transplantation models, the spleen contributes to graft rejection by generating alloantigen reactive T cells. The immune response in allograft rejection involves a cascade of molecular events leading to the formation of immunological synapses between T cells and the antigen-presenting cells. METHODOLOGY/PRINCIPAL FINDINGS: To elucidate the molecular pathways involved in the immunosuppressive function of allochimeric molecule we performed microarray and quantitative RTPCR analyses of gene expression profile of splenic T cells from untreated, CsA treated, and allochimeric molecule + subtherapeutic dose of CsA treated animals at day 1, 3 and 7 of post transplantation. Allochimeric molecule treatment caused down regulation of genes involved in actin filament polymerization (RhoA and Rac1), cell adhesion (Catna1, Vcam and CD9), vacuolar transport (RhoB, Cln8 and ATP6v1b2), and MAPK pathway (Spred1 and Dusp6) involved in tubulin cytoskeleton reorganization and interaction between actin and microtubule cytoskeleton. All these genes are involved in T cell polarity and motility, i.e., their ability to move, scan and to form functional immunological synapse with antigen presenting cells (APCs). CONCLUSIONS: These results indicate that the immunosuppressive function of allochimeric molecule may depend on the impairment of T cells' movement and scanning ability, and possibly also the formation of immunological synapse. We believe that these novel findings may have important clinical implications for organ transplantation.

Text mining of full-text journal articles combined with gene expression analysis reveals a relationship between sphingosine-1-phosphate and invasiveness of a glioblastoma cell line.

BACKGROUND: Sphingosine 1-phosphate (S1P), a lysophospholipid, is involved in various cellular processes such as migration, proliferation, and survival. To date, the impact of S1P on human glioblastoma is not fully understood. Particularly, the concerted role played by matrix metalloproteinases (MMP) and S1P in aggressive tumor behavior and angiogenesis remains to be elucidated. RESULTS: To gain new insights in the effect of S1P on angiogenesis and invasion of this type of malignant tumor, we used microarrays to investigate the gene expression in glioblastoma as a response to S1P administration in vitro. We compared the expression profiles for the same cell lines under the influence of epidermal growth factor (EGF), an important growth factor. We found a set of 72 genes that are significantly differentially expressed as a unique response to S1P. Based on the result of mining full-text articles from 20 scientific journals in the field of cancer research published over a period of five years, we inferred gene-gene interaction networks for these 72 differentially expressed genes. Among the generated networks, we identified a particularly interesting one. It describes a cascading event, triggered by S1P, leading to the transactivation of MMP-9 via neuregulin-1 (NRG-1), vascular endothelial growth factor (VEGF), and the urokinase-type plasminogen activator (uPA). This interaction network has the potential to shed new light on our understanding of the role played by MMP-9 in invasive glioblastomas. CONCLUSION: Automated extraction of information from biological literature promises to play an increasingly important role in biological knowledge discovery. This is particularly true for high-throughput approaches, such as microarrays, and for combining and integrating data from different sources. Text mining may hold the key to unraveling previously unknown relationships between biological entities and could develop into an indispensable instrument in the process of formulating novel and potentially promising hypotheses.

Regulation of murine TGFbeta2 by Pax3 during early embryonic development.

Previously our laboratory identified TGFbeta2 as a potential downstream target of Pax3 by utilizing microarray analysis and promoter data base mining (Mayanil, C. S. K., George, D., Freilich, L., Miljan, E. J., Mania-Farnell, B. J., McLone, D. G., and Bremer, E. G. (2001) J. Biol. Chem. 276, 49299-49309). Here we report that Pax3 directly regulates TGFbeta2 transcription by binding to cis-regulatory elements within its promoter. Chromatin immunoprecipitation revealed that Pax3 bound to the cis-regulatory elements on the TGFbeta2 promoter (GenBanktrade mark accession number AF118263). Both TGFbeta2 promoter-luciferase activity measurements in transient cotransfection experiments and electromobility shift assays supported the idea that Pax3 regulates TGFbeta2 by directly binding to its cis-regulatory regions. Additionally, by using a combination of co-immunoprecipitation and chromatin immunoprecipitation, we show that the TGFbeta2 cis-regulatory elements between bp 741-940 and bp 1012-1212 bind acetylated Pax3 and are associated with p300/CBP and histone deacetylases. The cis-regulatory elements between bp 741 and 940 in addition to associating with acetylated Pax3 and HDAC1 also associated with SIRT1. Whole mount in situ hybridization and quantitative real time reverse transcription-PCR showed diminished levels of TGFbeta2 transcripts in Pax3(-/-) mouse embryos (whose phenotype is characterized by neural tube defects) as compared with Pax3(+/+) littermates (embryonic day 10.0; 30 somite stage), suggesting that Pax3 regulation of TGFbeta2 may play a pivotal role during early embryonic development.

Proteasome inhibition induces both pro- and anti-cell death pathways in prostate cancer cells.

The proteasome-mediated protein degradation is critical for regulation of a variety of cellular processes, including cell cycle, cell death, differentiation and immune response. Proteasome inhibitors have recently been shown to be potent anti-cancer agents against a variety of cancer cells. Our study demonstrated that proteasome inhibitor MG132 (carbobenzoxy-L-leucyle-L-leucyl-L-leucinal) was a potent death-inducing agent for PC3 prostate cancer cells. MG132-induced cell death was partially inhibited by pan-caspase inhibitor zAVD-fmk and translational inhibitor cycloheximide. To understand the signaling pathways of proteasome inhibitor-induced cell death, we performed gene profiling study using Affymetrix human DNA microarrays to identify the genes whose expression was affected by proteasome inhibitor MG132 in PC3 cells. The genes with more than threefold increased expression induced by MG132 were functionally categorized into the following groups: heat shock and chaperone proteins, ubiquitination and protein degradation, transcription/translation factors, cell death and cell cycle arrest, signaling molecules and enzymes, and secreted cytokines. Among them, heat shock proteins and anti-oxidant enzymes may promote cell survival, while pro-death proteins such as GADD45B and STK17a may promote cell death. Interestingly, expression of a few autophagic genes was elevated by MG132 treatment. Furthermore, autophagy inhibitor 3-methyladenine partially inhibited MG132-induced cell death, indicating that autophagic cell death may contribute to MG132-induced cell death. Taken together, our results demonstrated that proteasome inhibition elicits activation of multiple signaling pathways in prostate cancer cells.

Microarray analysis of postictal transcriptional regulation of neuropeptides.

Unlike adults, kainic acid (KA)-induced status epilepticus (SE) in immature rats causes neither cell death nor recurrent spontaneous seizures. To elucidate the mechanisms of these distinct responses, transcriptional changes in neuropeptides were examined following KA-induced SE. We aimed to determine whether neuropeptides with anticonvulsant/neuroprotective properties were preferentially increased in immature rats while those with a proconvulsant/neurotoxic role were elevated to a greater extent in mature rats. We used high-density oligonucleotide gene arrays and directly compared transcriptional regulation of seven select neuropeptides at P15 and P30 over five time points. Total RNAs were isolated from hippocampi of 12 animals and pooled to hybridize to triplicate Affymetrix Genechips. Microarray results were validated by real-time quantitative RT-PCR (qRT-PCR). Independent individual RNA samples were purified for triplicate runs of qRT-PCR. Neuropeptides are significantly regulated by seizures in both immature and mature hippocampus. The magnitude of increase is significantly higher at P30 compared with that at P15, not only for neuropeptides with neurotoxic/proconvulsant properties but also for those with neuroprotective/ anticonvulsant properties. Galanin is induced at 24 h only in P30 rats. CST shows high expression in immature hippocampus and is further increased after KA-induced SE only in P15. The expression trends seen in the microarray data are confirmed by qRT-PCR for all six neuropeptides analyzed. CST might play a neuroprotective role in immature rats, and its overexpression might prevent neuronal loss after seizure in adults. Also, suppression of tachykinin and corticotropin-releasing hormone might be effective in alleviating seizure-induced neuronal damage.

The effect of phosphogypsum on greenhouse gas emissions during cattle manure composting.

Phosphogypsum (PG), a by-product of the phosphate fertilizer industry, reduces N losses when added to composting livestock manure, but its impact on greenhouse gas emissions is unclear. The objective of this research was to assess the effects of PG addition on greenhouse gas emissions during cattle feedlot manure composting. Sand was used as a filler material for comparison. The seven treatments were PG10, PG20, PG30, S10, S20, and S30, representing the rate of PG or sand addition at 10, 20, or 30% of manure dry weight and a check treatment (no PG or sand) with three replications. The manure treatments were composted in open windrows and turned five times during a 134-d period. Addition of PG significantly increased electrical conductivity (EC) and decreased pH in the final compost. Total carbon (TC), total nitrogen (TN), and mineral nitrogen contents in the final composted product were not affected by the addition of PG or sand. From 40 to 54% of initial TC was lost during composting, mostly as CO(2), with CH(4) accounting for <14%. The addition of PG significantly reduced CH(4) emissions, which decreased exponentially with the compost total sulfur (TS) content. The emission of N(2)O accounted for <0.2% of initial TN in the manure, increasing as compost pH decreased from alkaline to near neutral. Based on the total greenhouse gas budget, PG addition reduced greenhouse gas emissions (CO(2)-C equivalent) during composting of livestock manure by at least 58%, primarily due to reduced CH(4) emission.

Gene expression profiling in murine autoimmune arthritis during the initiation and progression of joint inflammation.

We present here an extensive study of differential gene expression in the initiation, acute and chronic phases of murine autoimmune arthritis with the use of high-density oligonucleotide arrays interrogating the entire mouse genome. Arthritis was induced in severe combined immunodeficient mice by using adoptive transfer of lymphocytes from proteoglycan-immunized arthritic BALB/c mice. In this unique system only proteoglycan-specific lymphocytes are transferred from arthritic mice into syngeneic immunodeficient recipients that lack adaptive immunity but have intact innate immunity on an identical (BALB/c) genetic background.Differential gene expression in response to donor lymphocytes that migrated into the joint can therefore be monitored in a precisely timed manner, even before the onset of inflammation. The initiation phase of adoptively transferred disease (several days before the onset of joint swelling) was characterized by differential expression of 37 genes, mostly related to chemokines, interferon-gamma and tumor necrosis factor-alpha signaling, and T cell functions. These were designated early arthritis 'signature' genes because they could distinguish between the naive and the pre-arthritic state. Acute joint inflammation was characterized by at least twofold overexpression of 256 genes and the downregulation of 21 genes, whereas in chronic arthritis a total of 418 genes with an equal proportion of upregulated and downregulated transcripts were expressed differentially. Hierarchical clustering and functional classification of inflammation-related and arthritis-related genes indicated that the most common biological activities were represented by genes encoding interleukins, chemokine receptors and ligands, and by those involved in antigen recognition and processing.

Modulation of Growth Factor response in brain tumors by complex carbohydrates.

It is well established that growth factors and their receptors are overexpressed in brain tumors and play a key role in tumor cell proliferation. Glycoconjugate molecules expressed at the plasma membrane of mammalian cells have been also reported to be associated with tumor progression. Growth factor receptors and glycoconjugate molecules are able to interact with each other and this interaction usually results in modulation of growth factor receptor mediated signaling and the biological function of the cell. This review addresses the expression of both growth factor receptors and glycoconjugates molecules in the brain and brain tumors. The mechanism by which these two entities interact with each other and the consequences of their interaction on the biological function of tumor cell are also discussed. Glycoconjugate molecules seems to act more specifically on growth factor receptor signaling pathways than most of tyrosine kinase inhibitors. The use of glycoconjugates or their derivatives may represent a new approach to modulate the proliferative behavior of tumors that overexpress growth factor receptors such as brain tumors.

Gene expression profiling reveals a highly specialized genetic program of plasma cells.

The formation of terminally differentiated plasma cells represents the critical final step in B-cell differentiation. In this study, utilizing oligonucleotide microarray analysis, we describe the highly specialized genetic profile exhibited by terminally differentiated plasma cells. A total of 1476 known genes were differentially expressed by plasma cells compared with B cells. Plasma cells displayed an up-regulation, induction, or a selective retention of a unique constellation of transcription factors, including members of the AP-1, nuclear factor-kappaB (NF-kappaB), nuclear factor of activated T cells (NFAT), and octamer binding factor families. Interestingly, plasma cells also displayed a down-regulation of several RNA polymerase I- related factors, consistent with terminal differentiation, and exhibited a down-regulation of the TATA box binding protein. Furthermore, plasma cells displayed alterations in multiple components of the Wnt and Notch signaling pathways and showed a unique pattern of apoptosis and proliferation-associated genes. Unexpectedly, plasma cells displayed an up-regulation of 2 factors normally associated with microenvironmental positioning of neuronal cells, reelin and neuropilin-1. These results supply insight into the developmental genetics of plasma cell differentiation and provide a foundation for further analysis of plasma cell biology.

High mannose glycans and sialic acid on gp120 regulate binding of mannose-binding lectin (MBL) to HIV type 1.

Mannose-binding lectin (MBL) is a C-type lectin of the innate immune system that binds to carbohydrates on the surface of certain microorganisms. Previous studies showed that MBL binds to gp120, the envelope glycoprotein of HIV-1. gp120 is extensively glycosylated, with N-linked complex and high mannose carbohydrates accounting for about half of the molecular weight. The objectives of this study were to determine the types of glycans on gp120 important for MBL binding and to determine if alteration of complex glycans with neuraminidase (NA) could enhance the interaction of MBL with virus. Lectin blot analyses revealed that MBL interacted with recombinant gp120 (rgp120) from both T cell-tropic and M-tropic virus strains. Treatment of rgp120 with endoglycosidase H (eH) or endoglycosidase F1 (eF1) abrogated binding of MBL, but did not decrease binding of wheat germ agglutinin indicating that high mannose and/or hybrid N-linked glycans were required for MBL binding. Removal of sialic acids from rgp120 with NA enhanced MBL binding. Treatment of intact virus from T cell lines or primary isolates with eF1 also significantly decreased HIV binding to MBL, while treatment with NA substantially increased binding. Treatment of virus with both eF1 and NA did not decrease binding compared to NA alone suggesting that NA treatment exposed binding sites on gp120 that are not high mannose glycans. These studies provide evidence that MBL binds to HIV via high mannose carbohydrates on gp120 and shows that the interaction of MBL with virus is regulated by sialylation.

Regulation of growth factor receptors by gangliosides.

Since their discovery in the 1940s, gangliosides have been associated with a number of biological processes, such as growth, differentiation, and toxin uptake. Hypotheses about regulation of these processes by gangliosides are based on indirect observations and lack a clear definition of their mechanisms within the cell. The first insights were provided when a reduction in cell proliferation in the presence of gangliosides was attributed to inhibition of the epidermal growth factor receptor (EGFR). Since that initial finding, most, if not all, growth factor receptors have been described as regulated by gangliosides. In this review, we describe the effects of gangliosides on growth factor receptors, beginning with a list of known effects of gangliosides on growth factor receptors; we then present three models based on fibroblast growth factor (FGFR), platelet-derived growth factor receptor (PDGFR), and EGFR. We focus first on ganglioside modulation of ligand binding; second, we discuss ganglioside regulation of receptor dimerization; and third, we describe a model that implicates gangliosides with receptor activation state and subcellular localization. The methodology used to develop the three models may be extended to all growth factor receptors, bearing in mind that the three models may not be mutually exclusive. We believe that gangliosides do not act independently of many well-established mechanisms of receptor regulation, such as clathrin-coated pit internalization and ubiquitination, but that gangliosides contribute to these functions and to signal transduction pathways. We hypothesize a role for the diverse structures of gangliosides in biology through the organization of the plasma membrane into lipid raft microdomains of unique ganglioside composition, which directly affect the signal duration and membrane localization of the growth factor receptor.

Engraftment assessment in human and mouse liver tissue after sex-mismatched liver cell transplantation by real-time quantitative PCR for Y chromosome sequences.

Transplanted hepatocytes can engraft, proliferate, and function permanently in host animals. After one cell infusion, however, engrafted hepatocytes constitute only between 1 in 200 to 1 in 3,000 host liver cells. Although transplanted cells can be identified using biochemical and molecular techniques, more accurate methods are needed to evaluate interventions that could improve cell engraftment rates. Real-time polymerase chain reaction (PCR) was done using primers and probes complementary to human testis determining gene (SRY) and mouse testis-specific Y-encoded protein (TSPY) pseudogene. Tissue samples from human or mouse recipients of liver cell transplantation were used to determine the test ability to detect transplanted cell DNA. Real-time PCR for the human SRY and mouse TSPY were species- and sex-specific. These two tests were sensitive in the detection of male DNA. Test sensitivity was consistently found at minimum 1:10,000 of male and female DNA mixing curve in both human SRY and mouse TSPY assays. The optimal amount of sample DNA per reaction to produce the highest sensitivity was 300 ng to 1 microg. Real-time PCR gave similar results whether standard male-female mixtures were prepared from liver cells or mononuclear cells. Engraftment of male liver cells in female liver tissues in mice and humans ranging from 0.125% to 0.257% was successfully measured using this method. Real-time PCR for SRY and TSPY affords a specific, sensitive, and reproducible tool for chimerism analysis in transplanted human and mouse liver tissues. This method could be used to optimize current models of cell transplantation.

Gene expression profiling in DQA1*0501+ children with untreated dermatomyositis: a novel model of pathogenesis.

Juvenile dermatomyositis (JDM), the most common pediatric inflammatory myopathy, is a systemic vasculopathy affecting young children. Epidemiology studies documenting an antecedent illness in the 3 mo before the first definite symptom (rash and/or weakness) of JDM are supported by immunologic data that suggest that the disease pathophysiology is Ag driven. The purpose of this study was to compare the gene expression profiles in muscle biopsies of four untreated DQA1*0501(+) JDM children with profiles from children with a known necrotizing myopathy (Duchenne muscular dystrophy), as well as an in vitro antiviral model (NF90), and healthy pediatric controls. Nearly half (47%) of the dysregulated genes in JDM were associated with the immune response. In particular, increased expression of IFN-alphabeta-inducible genes 6-16, myxovirus resistance protein p78, latent cytosolic transcription factor, LMP2, and TAP1 was observed. This profile is consistent with an IFN-alphabeta transcription cascade seen in the in vitro viral resistance model. The IFN-alphabeta-inducible profile was superimposed on transcription profiles reflective of myofiber necrosis and regeneration shared with Duchenne muscular dystrophy. Expressed genes were confirmed by quantitative real-time PCR (6-16), immunofluorescence (thrombospondin 4), and immunolocalization (IFN-gamma, p21). We hypothesize that these data support a model of Ag (?viral) induction of an apparent autoimmune disease based on dynamic interaction between the muscle, vascular, and immune systems in the genetically susceptible (DQA1*0501(+)) child.

Quantification of human immunodeficiency virus type 1 proviral DNA by using TaqMan technology.

A protocol for quantification of human immunodeficiency virus type 1 (HIV-1) proviral DNA with the TaqMan technology was developed and validated. The assay was specific for HIV-1, with an analytic sensitivity of 10 copies and a linear dynamic range of >6 logs. Viral RNA levels, when at a stable state, were highly correlated with proviral DNA levels in 80 specimens of 18 HIV-infected children.

Interaction of the extracellular domain of the epidermal growth factor receptor with gangliosides.

Ganglioside GM3 inhibits epidermal growth factor (EGF)-dependent cell proliferation in a variety of cell lines. Both in vitro and in vivo, this glycosphingolipid inhibits the kinase activity of the EGF receptor (EGFR). Furthermore, membrane preparations containing EGFR can bind to GM3-coated surfaces. These data suggest that GM3 may interact directly with the EGFR. In this study, the interaction of gangliosides with the extracellular domain (ECD) of the EGFR was investigated. The purified human recombinant ECD from insect cells bound directly to ganglioside GM3. The ganglioside interaction site appears to be distinct from the EGF-binding site. In agreement with previous reports on the effects of specific gangliosides on EGFR kinase activity, the ECD preferentially interacted with GM3. The order of relative binding of other gangliosides investigated was as follows: GM3 GM2, GD3, GM4 > GM1, GD1a, GD1b, GT1b, GD2, GQ1b > lactosylceramide. These data suggest that NeuAc-lactose is essential for binding and that any sugar substitution reduces binding. In agreement with the specificity of soluble ECD binding to gangliosides, GM3 specifically inhibited EGFR autophosphorylation. Identification of a ganglioside interaction site on the ECD of the EGFR is consistent with the hypothesis that endogenous GM3 may function as a direct modulator of EGFR activity.