A multi-target antisense approach against PDE4 and PDE7 reduces smoke-induced lung inflammation in mice

<p>Abstract</p> <p>Background</p> <p>Recent development in the field of COPD has focused on strategies aimed at reducing the underlying inflammation through selective inhibition of the phosphodiesterase type IV (PDE4) isoform. Although the anti-inflammatory and bronchodilator activity of selective PDE4 inhibitors has been well documented, their low therapeutic ratio and dose-dependent systemic side effects have limited their clinical utility. This study examined the effect of 2'-deoxy-2'-Fluoro-β-D-Arabinonucleic Acid (FANA)-containing antisense oligonucleotides (AON) targeting the mRNA for the PDE4B/4D and 7A subtypes on lung inflammatory markers, both <it>in vitro </it>and <it>in vivo</it>.</p> <p>Methods</p> <p>Normal human bronchial epithelial (NHBE) cells were transfected with FANA AON against PDE4B/4D and 7A alone or in combination. mRNA levels for target PDE subtypes, as well as secretion of pro-inflammatory chemokines were then measured following cell stimulation. Mice were treated with combined PDE4B/4D and 7A AON via endo-tracheal delivery, or with roflumilast via oral delivery, and exposed to cigarette smoke for one week. Target mRNA inhibition, as well as influx of inflammatory cells and mediators were measured in lung lavages. A two-week smoke exposure protocol was also used to test the longer term potency of PDE4B/4D and 7A AONs.</p> <p>Results</p> <p>In NHBE cells, PDE4B/4D and 7A AONs dose-dependently and specifically inhibited expression of their respective target mRNA. When used in combination, PDE4B/4D and 7A AONs significantly abrogated the cytokine-induced secretion of IL-8 and MCP-1 to near baseline levels. In mice treated with combined PDE4B/4D and 7A AONs and exposed to cigarette smoke, significant protection against the smoke-induced recruitment of neutrophils and production of KC and pro-MMP-9 was obtained, which was correlated with inhibition of target mRNA in cells from lung lavages. In this model, PDE AONs exerted more potent and broader anti-inflammatory effects against smoke-induced lung inflammation than roflumilast. Moreover, the protective effect of PDE4B/4D and 7A AON was maintained when a once-weekly treatment schedule was used.</p> <p>Conclusion</p> <p>These results indicate that inhaled AON against PDE4B/4D and 7A have unique effects on biomarkers that are believed to be important in the pathophysiology of COPD, which supports further development as a potential therapy in this disease.</p

Modulation of macrophage functions by components of Entamoeba histolytica

Entamoeba histolytica is a protozoan parasite and the causative agent of amebiasis. Activated macrophages are the main host effector cells in host defence against E. histolytica, through the production of nitric oxide (NO) which is cytotoxic for the parasite. NO is upregulated by tumor necrosis factor-alpha (TNF-$alpha$) produced by macrophages. The objective of this study was to determine the effect of amebic components on TNF-$alpha$ and NO production by macrophages. Soluble E. histolytica proteins stimulated naive macrophages for enhanced TNF-$alpha$ mRNA expression through PKC signal transduction. E. histolytica-induced TNF-$alpha$ mRNA expression was unstable, and macrophages pretreated with E. histolytica proteins expressed reduced levels of TNF-$alpha$ mRNA in response to LPS or IFN-$gamma$ + LPS. In contrast, the purified galactose-adherence lectin (Gal-lectin) of E. histolytica stimulated naive macrophages for stable TNF-$alpha$ mRNA expression and protein production. Furthermore, IFN-$gamma$ primed macrophages produced TNF-$alpha$ and NO in response to the Gal-lectin. Naive macrophages exposed to Gal-lectin + IFN-$gamma$ were activated to kill E. histolytica trophozoites in vitro by NO. Anti-lectin monoclonal antibodies that recognize non-overlapping epitopes of the kDa heavy subunit of the Gal-lectin identified amino acids 596-1082 as important in mediating amebic adherence to target cells and TNF-$alpha$ mRNA induction in macrophages. Likewise, a region between amino acids 596-818 of the 170 kDa Gal-lectin, in conjunction with IFN-$gamma$, activated macrophages for TNF-$alpha$ and NO production and amebicidal activity. This research demonstrates the immunogenic potential of the E. histolytica Gal-lectin and the critical regions that could be used as a subunit vaccine candidate against amebiasis