Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Effect of human TNF-alpha on schistosomulum stage of Schistosoma mansoni gene expression profile

ABSTRACT: Schistosoma mansoni is the major causative agent of schistosomiasis in the Americas. This parasite takes advantage from host signaling molecules such as cytokines and hormones to complete its development inside the host. TNF-Î± is the most important cytokine involved in the inflammatory response when cercaria, the infective stage, penetrates the human skin and a severe inflammatory response is started. In this work the authors describe the complete sequence of a possible TNF-Î± receptor in S. mansoni and detect that the receptor is most highly expressed in cercaria among all life cycle stages. In an attempt to mimic the situation at the site of skin penetration cercariae have been mechanically transformed in vitro into schistosomula and immediately exposed to human TNF-Î± . Exposure of these early schistosomula to the human hormone caused a large-scale change in the expression of parasite genes. Exposure of adult worms to human TNF-Î± caused gene expression changes as well, although the set of parasite altered genes was entirely different from that of schistosomula. This work increases the number of known signaling pathways of the parasite, and opens new perspectives into understanding the molecular components of TNF-Î± response as well as possibly interfering with parasite-host interaction. A 4 x 44k microarray platform was designed with the same probe set described by our group in a previous publication (PMID: 17517391). 230 ng of RNA from schistosomula treated with TNF and its control were used for the hybridization. RNA amplification and labeling kit (Agilent Technologies) were used according to manufacturerÂ´s instructions. Each sample was separately labeled with either Cy3 or Cy5 and 825 ng cRNA from each amplification were used for hybridization a control sample was combined with a treated sample and hybridized. Washing and scanning procedures were according to the manufacturerÂ´s instructions using GenePix 4000B scanner (Molecular Devices, Sunnyvale, CA, USA). Data was extracted using Feature Extraction software (Agilent Technologies). Low intensity data points were filtered out according to Feature Extraction software criteria, which essentially determine those points that are significantly below the average background signal of the array. Total intensity data from each experiment were normalized by Quantiles Normalization Method, excluding positive and negative external controls. Significance Analysis of Microarray (SAM) was used as to find differentially expressed genes. z-score transformation of normalized data was performed and SAM two class tests were applied; genes were considered as significantly differentially expressed at q-value â¤ 0.05. Hierarchical clustering of selected genes was generated using Spotfire Decision Site software (TIBCO Software Inc., Palo Alto, CA, USA). For a gene that was represented in the array by multiple probes, we picked a single representative probe by selecting the probe with the highest absolute value of the Log2 ratio between intensities of TNF-Î± Treated/Control.

ORGANISM(S): Schistosoma mansoni

SUBMITTER: Sergio Verjovski-Almeida

PROVIDER: E-GEOD-16260 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Identification of the Schistosoma mansoni TNF-alpha receptor gene and the effect of human TNF-alpha on the parasite gene expression profile.

Oliveira Katia C KC Carvalho Mariana L P ML Venancio Thiago M TM Miyasato Patricia A PA Kawano Toshie T DeMarco Ricardo R Verjovski-Almeida Sergio S

PLoS neglected tropical diseases 20091201 12

<h4>Background</h4>Schistosoma mansoni is the major causative agent of schistosomiasis. The parasite takes advantage of host signals to complete its development in the human body. Tumor necrosis factor-alpha (TNF-alpha) is a human cytokine involved in skin inflammatory responses, and although its effect on the adult parasite's metabolism and egg-laying process has been previously described, a comprehensive assessment of the TNF-alpha pathway and its downstream molecular effects is lacking.<h4>Me ...[more]

PMID: 19956564

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Project description:Schistosoma mansoni is the major causative agent of schistosomiasis in the Americas. This parasite takes advantage from host signaling molecules such as cytokines and hormones to complete its development inside the host. TNF-Î± is the most important cytokine involved in the inflammatory response when cercaria, the infective stage, penetrates the human skin and a severe inflammatory response is started. In this work the authors describe the complete sequence of a possible TNF-Î± receptor in S. mansoni and detect that the receptor is most highly expressed in cercaria among all life cycle stages. In an attempt to mimic the situation at the site of skin penetration cercariae have been mechanically transformed in vitro into schistosomula and immediately exposed to human TNF-Î± . Exposure of these early schistosomula to the human hormone caused a large-scale change in the expression of parasite genes. Exposure of adult worms to human TNF-Î± caused gene expression changes as well, although the set of parasite altered genes was entirely different from that of schistosomula. This work increases the number of known signaling pathways of the parasite, and opens new perspectives into understanding the molecular components of TNF-Î± response as well as possibly interfering with parasite-host interaction. A platform previously designed by our group (PMID: 17517391) was used. 300 ng of RNA from adult schistosomes treated with TNF and its control were used for the hybridization. RNA amplification and labeling kit (Agilent Technologies) were used according to manufacturerÂ´s instructions. Each sample was separately labeled with either Cy3 or Cy5. 825 ng cRNA from each amplification were used for hybridization; self-self experiments were performed. Washing and scanning procedures were according to the manufacturerÂ´s instructions using GenePix 4000B scanner (Molecular Devices, Sunnyvale, CA, USA). Data was extracted using Feature Extraction software (Agilent Technologies). We calculated a virtual Log2 ratio between intensities of TNF-Î± Treated/Control, for each gene. With these ratios, we used two different approaches for SAM statistical analyses. SAM one-class approach was used to identify genes with sustained changes in their expression levels along the entire time period of observation (1 and 24 h); SAM two-class approach was used to identify genes with transient changes in their expression levels. In both cases, genes were considered differentially expressed at q-value â?¤ 0.05. Hierarchical clustering of selected genes was generated using Spotfire Decision Site software (TIBCO Software Inc., Palo Alto, CA, USA). For a gene that was represented in the array by multiple probes, we picked a single representative probe by selecting the probe with the highest absolute value of the Log2 ratio between intensities of TNF-Î± Treated/Control.

Project description:Schistosomiasis continues to be a significant public health problem1. Although vaccine development against this disease has experienced more failures than successes, encouraging results have recently been obtained using membrane-spanning protein antigens from the tegument of S. mansoni. Our group recently identified Sm29, an antigen that is predominantly recognized by IgG1 and IgG3 antibodies of resistant patients2. In the present study, we show that Sm29 is located on the surface of adult worms and lung-stage schistosomula. Immunization of mice with recombinant (r) Sm29 engendered 51% reduction in adult worm burdens, 60% reduction in intestinal eggs and 55% reduction in liver granulomas. Protective immunity in mice was associated with high titers of specific IgG1 and IgG2a and elevated production of IFN-Î³, TNF-Î± and IL-12. Further, cellular responses of infected schistosomiasis patients to rSm29 consisted of elevated IFN-Î³ and an absence of IL-5. Gene expression analysis of worms recovered from rSm29 vaccinated mice relative to control mice revealed a significant (q< 0.01) down-regulation of 498 genes, while no up-regulation was detected. Among down-regulated genes, many of them encode surface antigens and proteins associated with immune signals suggesting that under immune attack schistosomes reduce the expression of critical surface proteins. This study demonstrates that the membrane-bound Sm29 protein is a new molecule that has great potential as a vaccine candidate against schistosomiasis. Keywords: Schistosoma mansoni gene expression in vaccinated mice Two biological samples were used in the study, each used in two arrays (technical replicates with dye swap) and each array containing two replicates of each spot. Overall, there are eight data points data points for each spot. Only cases with more than 4 of 8 points are valid ratios were considered. Value and its respective dye swap value [after changing the log(ratio) signal] were averaged, reducing the data point to four. The average values were than used in the significance testing using SAM (Tusher et al., 2001). The samples were used to get average values using the following combination: 3000139790L with 3000139792L; 3000139790R with 3000139792R; 3000139791L with 3000139795L; 3000139791R with 3000139795R The normalized, averaged dyeswap values are provided in a supplementary file at the foot of the record.

Dataset Information

Effect of human TNF-alpha on schistosomulum stage of Schistosoma mansoni gene expression profile

Publications

Identification of the Schistosoma mansoni TNF-alpha receptor gene and the effect of human TNF-alpha on the parasite gene expression profile.

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