We have previously reported that transcriptional induction of cyclooxygenase-2 (COX-2) isoenzyme occurs early after T cell receptor triggering, suggesting functional implications of cyclooxygenase activity in this process. Here, we identify the cis-acting elements responsible for the transcriptional activation of this gene in human T lymphocytes. COX-2 promoter activity was induced upon T cell activation both in primary resting T lymphocytes and in Jurkat cells. This induction was abrogated by inhibition of calcineurin phosphatase with the immunosuppressive drug cyclosporin A, whereas expression of an active calcineurin catalytic subunit enhanced COX-2 transcriptional activation. Moreover, cotransfection of nuclear factor of activated T cells (NFAT) wild type protein transactivated COX-2 promoter activity. Conversely, dominant negative mutants of NFATc or c-Jun proteins inhibited COX-2 induction. Electrophoretic mobility shift assays and site-directed mutagenesis allowed the identification of two regions of DNA located in the positions ؊117 and ؊58 relative to the transcriptional start site that serves as NFAT recognition sequences. These results emphasize the central role that the Ca 2؉ /calcineurin pathway plays in COX-2 transcriptional regulation in T lymphocytes pointing to NFAT/activator protein-1 transcription factors as essential for COX-2 promoter regulation in these cells. Prostaglandin endoperoxide synthase or cyclooxygenase (COX)1 is the enzyme responsible for the conversion of arachidonic acid to prostaglandin H 2 , the main step in the prostaglandin synthesis pathway. Two forms of the enzyme, named COX-1 and COX-2, have been shown to be expressed in mammalian tissues. COX-1 is considered a housekeeping enzyme constitutively expressed in most tissues, whereas the COX-2 isoform is induced by several stimuli including cytokines and mitogens and is thought to be responsible for the increased production of prostaglandins in pathologic processes (reviewed in Refs. 1-3). Promoter regions of the COX-2 gene of mouse (4), rat (5), chicken (6), and humans (7-9) have been cloned. Regardless of the animal species, these promoters contain a classical TATA box, an E-box, and binding sites for transcription factors such as nuclear factor B, nuclear factor-IL6/CCAATenhancer binding protein), and cyclic AMP-response element (CRE) -binding proteins. These sequences have been shown to act as positive regulatory elements for the COX gene transcription in different cell types (5, 10 -14). We have recently shown that COX-2 expression is induced in T cells upon T cell receptor (TCR) activation playing an important role in controlling this process (15). However, no studies about COX-2 promoter regulation in these cells have been reported so far.Activation of T cells triggers a complex regulatory cascade of events that culminates in the induced transcription of a variety of activation-associated genes (16 -18). Many of them are cytokines that in turn regulate cell proliferation, differentiation, and acquisition of effector f...

We have previously described that IFN-γ induces cyclooxygenase 2 and inducible NO synthase expression by a mechanism that involved endogenously produced TNF-α. In this study, we report that TNF-α production is induced by IFN-γ treatment in the murine macrophage cell line RAW 264.7. TNF-α mRNA levels are increased in cells treated with IFN-γ in a time-dependent manner and IFN-γ also increased human TNF-α promoter-dependent transcription. Two regions in the TNF-α promoter seem to be responsible for the IFN-γ response: a distal region between −1311 and −615 bp of the human TNF-α promoter, and a proximal region located between −95 and −36 bp upstream of the transcriptional start. In contrast, IFN-γ stimulation induces the expression of the transcription factors IRF-1 and IRF-8. Overexpression of these transcription factors produces an increase in the transcriptional activity of the human TNF-α promoter. There is a correlation between the regions of the TNF-α promoter responsible of the transcriptional activation elicited by IRF-1 and IRF-8 and those required for IFN-γ response. In addition, IRF-1 and IRF-8 are recruited to the TNF-α promoter in IFN-γ-treated RAW 264.7 cells, as demonstrated by chromatin immunoprecipitation assays. Moreover, overexpression of IRF-1 and IRF-8 induces TNF-α production in unstimulated RAW 264.7 macrophages, comparable to the production of TNF-α elicited by IFN-γ stimulation, and silencing of IRF-1 and/or IRF-8 with specific small interfering RNAs, decreases IFN-γ-elicited TNF-α production. In summary, IFN-γ treatment induces TNF-α expression at transcriptional level requiring the coordinate action of IRF-1 and IRF-8.

In the version of the article originally published, in the top immunoblot (loading control) in Figure 5f, the right half was incorrectly a mirror-image duplication of the left half. The correct immunoblot from a replicate experiment is now presented (along with the corresponding bottom immunoblot). In the version of this article initially published online, the identification of dermal and epidermal γδ T cells in the legend for Figure 3f was reversed; a label was missing above the far left column of Figure 4c; and the red and blue lines were switched in the keys for the far right plots in Figure 6i. The legend for Figure 3f should read "...identified by high expression (top right; epidermal) or low expression (bottom right; dermal) of the γδ TCR. " The far left column in Figure 4c should include the label "CD69-KO" above. The correct keys for Figure 6i are as follows: blue line, FIZC (37 °C), and red line, FICZ + BCH (37 °C); and blue line, CD69-KO (37 °C), and red line, WT (37 °C).

Today, fascioliasis is considered to be an important human disease caused by 2 liver fluke species, Fasciola hepatica and Fasciola gigantica (Fasciolidae), infecting the liver of a wide range of mammals [1] that show a marked variability in their immune response against infection [2]. There are several geographic regions that have been reported to be areas of hypoendemicity, mesoendemicity, and hyperendemicity for fascioliasis in

Recent evidence indicates that PPAR (peroxisome-proliferator-activated receptor) alpha ligands possess anti-inflammatory and antitumoural properties owing to their inhibitory effects on the expression of genes that are involved in the inflammatory response. However, the precise molecular mechanisms underlying these effects are poorly understood. In the present study, we show that tumour promoter PMA-mediated induction of genes that are significantly associated with inflammation, tumour growth and metastasis, such as COX-2 (cyclo-oxygenase 2) and VEGF (vascular endothelial growth factor), is inhibited by PPARalpha ligands in the human colorectal carcinoma cell line SW620. PPARalpha activators LY-171883 and WY-14,643 were able to diminish transcriptional induction of COX-2 and VEGF by inhibiting AP-1 (activator protein-1)-mediated transcriptional activation induced by PMA or by c-Jun overexpression. The actions of these ligands on AP-1 activation and COX-2 and VEGF transcriptional induction were found to be dependent on PPARalpha expression. Our studies demonstrate the existence of a negative cross-talk between the PPARalpha- and AP-1-dependent signalling pathways in these cells. PPARalpha interfered with at least two steps within the pathway leading to AP-1 activation. First, PPARalpha activation impaired AP-1 binding to a consensus DNA sequence. Secondly, PPARalpha ligands inhibited c-Jun transactivating activity. Taken together, these findings provide new insight into the anti-inflammatory and anti-tumoural properties of PPARalpha activation, through the inhibition of the induction of AP-1-dependent genes that are involved in inflammation and tumour progression.

BackgroundVisceral leishmaniasis (VL) is hypoendemic in the Mediterranean region, where it is caused by the protozoan Leishmania infantum. An effective vaccine for humans is not yet available and the severe side-effects of the drugs in clinical use, linked to the parenteral administration route of most of them, are significant concerns of the current leishmanicidal medicines. New drugs are desperately needed to treat VL and phenotype-based High Throughput Screenings (HTS) appear to be suitable to achieve this goal in the coming years.Methodology/Principal findingsWe generated two infrared fluorescent L. infantum strains, which stably overexpress the IFP 1.4 and iRFP reporter genes and performed comparative studies of their biophotonic properties at both promastigote and amastigote stages. To improve the fluorescence emission of the selected reporter in intracellular amastigotes, we engineered distinct constructs by introducing regulatory sequences of differentially-expressed genes (A2, AMASTIN and HSP70 II). The final strain that carries the iRFP gene under the control of the L. infantum HSP70 II downstream region (DSR), was employed to perform a phenotypic screening of a collection of small molecules by using ex vivo splenocytes from infrared-infected BALB/c mice. In order to further investigate the usefulness of this infrared strain, we monitored an in vivo infection by imaging BALB/c mice in a time-course study of 20 weeks.Conclusions/SignificanceThe near-infrared fluorescent L. infantum strain represents an important step forward in bioimaging research of VL, providing a robust model of phenotypic screening suitable for HTS of small molecule collections in the mammalian parasite stage. Additionally, HSP70 II+L. infantum strain permitted for the first time to monitor an in vivo infection of VL. This finding accelerates the possibility of testing new drugs in preclinical in vivo studies, thus supporting the urgent and challenging drug discovery program against this parasitic disease.

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