Induction of tissue factor expression and release as microparticles in ECV304 cell line by Chlamydia pneumoniae infection.

The association between Chlamydia pneumoniae (C. pneumoniae) infection and the onset and progression of atherosclerosis has become apparent recently. Moreover, increased expression of tissue factor (TF) as a result of C. pneumoniae infection has been previously demonstrated. We have examined the expression of TF on the surface of endothelial cells and the release of TF-containing cell-derived microparticles, over seven days. Additionally, using cells expressing a procoagulantly active EGFP-TF hybrid protein, we examined the kinetics of TF trafficking on the cells and incorporation into shed microparticles. Finally, in an attempt to associate this with the activation of NFkappaB, we used a luciferase reporter to measure the duration of the activation of this transcription factor. TF-containing microparticles were released within 24h of infection and continued for up to 7 days. Moreover, the initial release of TF containing microparticles was associated with NFkappaB activation and was suppressed on inclusion of an NFkappaB inhibitor, pyrrolidinedithiocarbamate ammonium. Moreover, persistent dissemination of TF-containing microparticles at later stages of infection was associated with the release of the infective C. pneumoniae elementary bodies. The released procoagulant, cellular microparticles are known to be strongly atherogenic and therefore we suggest a mechanism for the involvement of C. pneumoniae in the onset and progression of vascular disease.

Flow cytometric detection of adenoviruses and intracellular adenovirus proteins.

Precise and simple assay of purified and crude preparations of human adenoviruses is essential for basic and gene therapy research. Previous bioassays used to quantitate adenoviruses (such as the plaque assay or fluorescent focus assay) are time-consuming and subjective in their interpretation. Here we describe a flow cytometric method that eliminates these disadvantages and provides a quantitative and reliable method of focus-forming unit (FFU) assay.

Sexually transmitted infections in teenagers.

Prevention of sexually transmitted infection in teenagers is a high priority. The introduction of non-invasive methods for diagnostic testing of Chlamydia trachomatis and Neisseria gonorrhoeae has enabled screening for these sexually transmitted micro-organisms in a range of community-based settings among populations at high risk, such as teenagers, who do not readily access existing sexual health services.

The role of Cajal bodies in the expression of late phase adenovirus proteins.

Cajal bodies (CBs) are subnuclear structures involved in RNA metabolism. Here we show that, following infection of HeLa cells by adenovirus type 5 (Ad5), CBs fragment and form ordered structures, which we have termed "rosettes". Formation of CB rosettes was prevented by inhibition of viral DNA synthesis and preceded expression of the L4-33K protein. CB rosettes localised to the periphery of E2A-72K-containing replication centers and to the edges of ASF/SF2 and hnRNP A1 ring structures that demarcate sites of viral transcription and splicing. At later times of infection, CB rosettes were undetectable. Furthermore, knock-down of p80-coilin (the major structural protein of CBs) by RNA interference reduced the yield of infectious Ad5 and expression of the late proteins IIIa (from L1), hexon (from L3) and fiber (from L5), whereas the E2A-72K protein was unaffected. We conclude that CBs have an important role in the expression of adenovirus major late gene products.

Differential functions of tissue factor in the trans-activation of cellular signalling pathways.

In this study we examined the ability of tissue factor (TF) alone, or in conjunction with factor VIIa, factor Xa and TFPI in activating a number of key signalling pathways associated with cellular growth, stress and differentiation responses in human endothelial cells. We used luciferase reporter systems to demonstrate the activation of p42/44 MAPK by the TF-FVIIa complex, mediated via the PAR1 receptor. TF alone was capable of interacting with the cell surface and was sufficient to activate the JNK-SAPK pathway and subsequently AP-1, but the level of activation was enhanced by the activity of FXa on PAR1 and 2. Furthermore, the phosphorylated form of the transmembrane-cytoplasmic domain of TF was directly responsible for activation of these pathways. CREB activation occurred in response to TF-FVIIa in a non-protease dependent manner but was lowered on addition of FXa. Finally, NFkappaB activation occurred in response to FVIIa or FXa, with the latter exhibiting higher levels of activation. In conclusion, we have shown that TF is capable of activating differing signalling pathways, via more than one mechanism. The differential influence of TF is modified depending on the presence of other coagulation factors and ultimately acts as a deciding factor in the determination of cellular fate.