Cholera toxin induced gene expression alterations.

The cholera toxin (CT) is a well-known inducer of cAMP and cAMP regulates gene expression of many genes. However, little is known as to the alterations in gene expression in response to CT. Here the alterations of the expression of 800 selected genes in response to CT were examined using cDNA microarrays. Gene expression alterations in human lymphocytes and monocytes were found after exposure to CT at varying concentrations for different time periods. Over 200 genes showed varying degrees of alterations of expression in CT-treated cells. The CT-induced changes in gene expression were compared by cDNA microarrays under the same conditions to those in response to forskolin, a specific activator of adenylate cyclase, and MDL-12, an irreversible inhibitor of adenylate cyclase. Thirty-five CT-responsive genes were found responded similarly to forskolin but differently to MDL-12. Fourteen CT-responsive genes were affected similarly by MDL-12 but differently by forskolin. Many of these CT responsive genes were involved in immunity, inflammation and oxidative stress. The CT induced responses correlated with those induced by CT subunits. The down regulation of Th1 markers and upregulation of Th2 markers by CT are consistent with the CT induction of Th2 cells.

Cholera toxin induced novel genes in human lymphocytes and monocytes.

Cholera toxin (CT) is well known as an inducer of the accumulation of cellular cAMP through the ADP-ribosylation of the Gs protein by CT. CT is also one of the most powerful mucosal adjuvants. However, the molecular mechanisms of the CT adjuvanticity are not well understood. Here, the transcriptional responses of cultured human lymphocytes and monocytes in response to CT were analyzed using differential display-PCR. The full complement of cellular mRNA was examined by high resolution polyarylamide gel electrophoresis and sequence analyses of the PCR products of 240 primer sets. Over 100 genes with altered expression were initially identified. The expressions of 65 of these genes were further analyzed and confirmed using custom glass cDNA arrays, RT-PCR and real-time PCR. Immunomodulatory genes such as CD2, HIF1, CXCL2, L-plastin, LILR and IFI30 were affected by CT. In addition, 14 novel genes with previously unknown functions were found to be CT induced. These CT induced gene expression alterations provide more insight in the mechanisms of CT actions. The CT induced gene expressions alterations could contribute to the CT adjuvanticity.

Induction of immunomodulator transcriptional responses by cholera toxin.

Cholera toxin (CT) is the causative agent of cholera, binds to GM1 glycosphingolipids, induces the production of cellular cAMP and is also a very powerful mucosal adjuvant. Although the mechanism of the CT induction of cAMP production is well understood, molecular mechanisms of the adjuvanticity of cholera toxin are yet to be delineated. Here, we examined the interaction of CT with human lymphocytes and monocytes by analyzing the host transcriptional profiles using cDNA arrays. The time courses of the transcriptional activations and repressions of affected genes in lymphocytes and monocytes in response to cholera toxin were determined. CT induced the expression of IL-8 and MIP-1 early in the CT exposure. VEGF, TIMP1, HIF-1alpha, MMP11, hek 8, MCP1, IL-6, GCP 2, urokinase plasminogen activator, and TNF-alpha receptor were upregulated after 4h CT treatment. These genes showed increased expression for 48 h. MRP-14, MRP-8A increased expression after 16 h CT treatment. RT-PCR and real-time PCR using cDNA specific primers confirmed the CT induction and repression of selected genes. The results suggest that immunomodulatory genes were among the genes that were affected the most by CT, and induction of these genes may contribute to the CT adjuvanticity.

Deciphering the involvement of innate immune factors in the development of the host response to PRRSV vaccination.

The natural response of pigs to porcine reproductive and respiratory syndrome virus (PRRSV) infections and vaccinations needs to be altered so that better protection is afforded against both homologous and heterologous challenges by this pathogen. To address this problem, real-time gene expression assays were coupled with cytokine Elispot and protein analyses to assess the nature of the anti-PRRSV response of pigs immunized with modified live virus (MLV) vaccine. Although T helper 1 (Th1) immunity was elicited in all vaccinated animals, as evidenced by the genesis of PRRSV-specific interferon-gamma secreting cells (IFNG SC), the overall extent of the memory response was variable and generally weak. Peripheral blood mononuclear cells (PBMC) isolated from these pigs responded to PRRSV exposure with a limited increase in their expression of the Th1 immune markers, IFNG, tumor necrosis factor-alpha and interleukin-15 (IL15), and a reduction in the quantity of mRNAs encoding the innate and inflammatory proteins, IL1B, IL8 and IFNA. Efforts to enhance Th1 immunity, by utilizing an expression plasmid encoding porcine IFNA (pINA) as an adjuvant, resulted in a temporary increase in the frequency of PRRSV-specific IFNG SC but only minor changes overall in the expression of Th1 associated cytokine or innate immune marker mRNA by virus-stimulated PBMC. Administration of pINA, however, did correlate with decreased IL1B secretion by cultured, unstimulated PBMC but had no effect on their ability to release IFNG. Thus, while exogenous addition of IFNA during PRRSV vaccination has an impact on the development of a Th1 immune response, other alterations will be required for substantial boosting of virus-specific protection.

Identification of key immune mediators regulating T helper 1 responses in swine.

This publication describes the cloning of full or partial length sequences for pig TBX21 (T-bet), MYD88, ICSBP1, CD8A (CD8alpha), CD8B (CD8beta), and CD28 cDNAs. Real-time PCR assays have been developed for the relative quantitation of these products as well as previously characterized transcripts that encode exon A-containing CD45, HLX1, IRF1, STAT1 and RPL32. When used for examining temporal immune gene expression in the liver of Toxoplasma gondii infected pigs, the positive regulators of Th1 responses, IRF1, MYD88, and STAT1, were found to be expressed prior to the simultaneous upregulation of interferon gamma (IFNG), HLX1 and TBX21 gene expression. In contrast, in the mesenteric lymph node (MLN), only expression of IRF1 and IFNG was significantly upregulated. Based on their demonstrated utility in establishing an immune response pathway, these PCR assays should be valuable additions to our swine immune toolkit.