The anti-inflammatory sesquiterpene lactone helenalin inhibits the transcription factor NF-kappaB by directly targeting p65.

The sesquiterpene lactone helenalin is a potent anti-inflammatory drug whose molecular mechanism of action remains unclear despite numerous investigations. We have previously shown that helenalin and other sesquiterpene lactones selectively inhibit activation of the transcription factor NF-kappaB, a central mediator of the human immune response. These drugs must target a central step in NF-kappaB pathway, since they inhibit NF-kappaB induction by four different stimuli. It has previously been reported that sesquiterpene lactones exert their effect by inhibiting degradation of IkappaB, the inhibitory subunit of NF-kappaB. These data contradicted our report that IkappaB is not detectable in helenalin-treated, ocadaic acid-stimulated cells. Here we use confocal laser scanning microscopy to demonstrate the presence of IkappaB-released, nuclear NF-kappaB in helenalin-treated, tumor necrosis factor-alpha stimulated cells. These data show that neither IkappaB degradation nor NF-kappaB nuclear translocation are inhibited by helenalin. Rather, we provide evidence that helenalin selectively alkylates the p65 subunit of NF-kappaB. This sesquiterpene lactone is the first anti-inflammatory agent shown to exert its effect by directly modifying NF-kappaB.

Opa binding to cellular CD66 receptors mediates the transcellular traversal of Neisseria gonorrhoeae across polarized T84 epithelial cell monolayers.

We have analysed the capacity of the 11 phase-variable, opacity-associated (Opa) proteins encoded by Neisseria gonorrhoeae MS11 to mediate traversal across polarized monolayers of the human colonic carcinoma T84 cell line. Gonococci expressing either the heparan sulphate proteoglycan (HSPG) binding Opa protein (Opa50) or no Opa protein (Opa-) did not interact with the apical pole of T84 monolayers, whereas the 10 variant Opa proteins previously shown to bind CD66 receptors were found to mediate efficient gonococcal adherence and transepithelial traversal. Consistent with this, T84 cells were shown by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunoblotting to co-express CD66a (BGP), CD66c (NCA) and CD66e (CEA). The recruitment of CD66 receptors by Opa-expressing gonococci indicates their involvement in mediating adherence to the surface of T84 cells, and these bacterial interactions could be inhibited completely using polyclonal antibodies cross-reacting with all of the CD66 proteins co-expressed on T84 cells. Consistent results were obtained when Opa proteins were expressed in Escherichia coli, suggesting that the Opa-CD66 interaction is sufficient to mediate bacterial traversal. Transcytosis of Opa-expressing N. gonorrhoeae or E. coli did not disrupt the barrier function of infected monolayers, as indicated by a sustained transepithelial electrical resistance (TEER) throughout the course of infection, and confocal laser scanning and electron microscopy both suggest a transcellular rather than a paracellular route of traversal across the monolayers. Parallels between the results seen here and previous work done with organ cultures confirm that T84 monolayers provide a valid model for studying neisserial interactions with the mucosal surface, and suggest that CD66 receptors contribute to this process in vivo.