AP-1 negatively regulates the IP-10 promoter in response to poly I:C, with transfection of FADD augmenting AP-1 luciferase activity.

<p>HEK-293/TLR3 cells were transfected with full length IP-10 luciferase plasmids or IP-10 luciferase plasmids containing point deletions in binding sites for NF-κB (κB1, κB2), proximal ISRE or AP-1. A schematic of these putative binding sites is illustrated <b>(A)</b>. 24 hrs post transfection, cells were stimulated with 20 μg/ml poly I:C for 6 hours with IP-10 luciferase activity expressed as fold-change over TK-renilla activity <b>(B)</b>. Cells were transfected with an AP-1 luciferase reporter plasmid and the indicated doses of FADD plasmid. 24 hrs after transfection cells were stimulated with 20 μg/ml poly I:C or 10 ng/ml TNFα for a further 6 hrs. AP-1 activity were measured and expressed as fold-change over TK-renilla activity <b>(C)</b>. Data shown are a combination of three independent experiments, with values shown as Mean ± SEM.</p

Fas activation in THP-1-derived macrophages does not induce caspase 3/7 activation.

<p>THP-1-derived macrophages and Jurkat T cells were treated with 100 ng/ml CH11 and/or 20 μg/ml poly I:C for 24 hrs, or with 5 μM staurosporine as indicated. Both caspase 3/7 activation (<b>A</b>) and cell viability (<b>B</b>) were determined after 24 hrs by fluorescence and trypan blue, respectively. Data shown are representative of three independent experiments, with values shown as Mean ± SEM.</p

The augmentation of poly-I:C-induced IP-10 production following ligation of Fas is mediated by both TLR3 and RIG-I.

<p>THP-1-derived macrophages were treated with 100 ng/ml CH11 for 1 hr followed by infection with Sendai virus (<b>A,B</b>) or stimulation with poly A:U (<b>C</b>) for a further 8 hrs. Changes in IP-10 production were determined by qRT-PCR (<b>A,C</b>) or ELISA (<b>B</b>). Immortalised wild type and TRIF-^/- BMDMs were stimulated with 10 ng/ml murine agonistic Fas antibody (Jo2) for 1 hr followed by stimulation with 20 μg/ml poly I:C for a further 8 hrs, with changes in cytokine expression were detected by qRT-PCR (<b>D</b>). Data shown are a combination of three independent experiments, with values shown as Mean ± SEM. * p<0.05, ** p<0.01 and *** p<0.001.</p

Schematic of mechanism whereby Fas activation may enhance poly I:C-induced IP-10.

<p>In the absence of Fas engagement, poly I:C, acting through both TLR3 and RIG-I, cause the phosphorylation of JNK and p38 MAPK in a FADD dependant manner. This results in AP-1 activation and translocation of AP-1 to the nucleus, where it acts to repress IP-10 production. Dashed lines indicate that the precise position of FADD in this pathway is unknown <b>(A)</b>. Upon Fas activation, FADD is recruited to Fas, reducing poly I:C-induced JNK and p38 MAPK activation. This reduces the levels of AP-1, thereby alleviating AP-1-mediated repression of the IP-10 promoter, resulting in enhanced IP-10 production. <b>(B)</b></p

Stimulation of macrophages with poly I:C increases expression of Fas and FasL.

<p>THP-1 cells were differentiated with 100 μg/ml PMA for 72 hrs. Cells were treated with increasing concentrations of poly I:C, and Fas and FasL expression detected by qRT-PCR after 8 hrs (<b>A, B</b>) and Western blotting after 48 hrs (<b>C</b>). Human monocyte-derived macrophages from three separate donors were treated with poly I:C (20 μg/ml) and Fas and FasL expression detected by qRT-PCR after 8 hrs (<b>D</b>). Results shown are representative of three separate experiments. Values are shown as Mean ± SEM, (n = 3). ** p<0.01 and *** p<0.001.</p

Fas activation suppresses poly I:C-induced activation of MAP Kinases p38 and JNK.

<p>THP-1 macrophages were treated with CH11 1 hr prior to poly I:C stimulation and Western blotting for phosphorylated p38 MAPK, JNK, IκBα and p42/p44 MAPK was performed. Levels of β-actin were assessed to ensure equal loading of protein. Data shown are representative of three independent experiments.</p

Pregnancy-specific glycoprotein expression in normal gastrointestinal tract and in tumors detected with novel monoclonal antibodies

Pregnancy-specific glycoproteins (PSGs) are immunoglobulin superfamily members related to the carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family and are encoded by 10 genes in the human. They are secreted at high levels by placental syncytiotrophoblast into maternal blood during pregnancy, and are implicated in immunoregulation, thromboregulation, and angiogenesis. To determine whether PSGs are expressed in tumors, we characterized 16 novel monoclonal antibodies to human PSG1 and used 2 that do not cross-react with CEACAMs to study PSG expression in tumors and in the gastrointestinal (GI) tract using tissue arrays and immunohistochemistry. Staining was frequently observed in primary squamous cell carcinomas and colonic adenocarcinomas and was correlated with the degree of tumor differentiation, being largely absent from metastatic samples. Staining was also observed in normal oesophageal and colonic epithelium. PSG expression in the human and mouse GI tract was confirmed using quantitative RT-PCR. However, mRNA expression was several orders of magnitude lower in the GI tract compared to placenta. Our results identify a non-placental site of PSG expression in the gut and associated tumors, with implications for determining whether PSGs have a role in tumor progression, and utility as tumor biomarkers