PMC:3537549 / 35904-39800
Annnotations
{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/3537549","sourcedb":"PMC","sourceid":"3537549","source_url":"https://www.ncbi.nlm.nih.gov/pmc/3537549","text":"ODN 2216 induce the production of soluble molecules that activate intracellular antiviral mechanisms in feline target cells\nProtective properties of type I IFN against viruses originate from their ability to trigger the production of potent antiviral proteins in nearby cells. The expression of one of these antiviral proteins, the Mx GTPase, is known to be directly stimulated by type I IFN and can be used as marker for the induction of intracellular antiviral mechanisms by these cytokines in cats as well as in other species[19, 64]. Thus, as detection of feline type I IFN on a protein level is rendered difficult by the unavailability of antibodies specifically recognizing these proteins, mRNA levels of Mx were measured in feline PBMCs after stimulation with ODN 2216, as indication for production of type I IFN. Transcription of Mx was significantly enhanced and remained proportional to mRNA expression of type I IFN in stimulated PBMCs of individual cats (Figure4A). Moreover, peak levels of Mx mRNA were measured 24 h after stimulation of the cells, indicating the presence of optimal effects of the type I IFN present in the cell culture medium at this time point (Figure4B). In order to further determine the potential of the type I IFN liberated by ODN 2216 stimulated PBMCs to induce intracellular antiviral mechanisms in non-immune target cells, cell-free supernatants of PBMCs derived from the blood of individual adult cats and stimulated in vitro for 24 h with ODN 2216, ODN 2243 or endotoxin-free PBS (Sup 2216, Sup 2243 and Sup Neg respectively), were incubated with CrFK and fcwf-4 cells. Mx gene transcription was significantly enhanced in cells treated with Sup 2216 compared to Sup 2243 (p = 0.0078) and Sup Neg (p = 0.0078), at levels comparable to those achieved by stimulation of the cells with 100U recombinant feline IFNα (rfeIFNα) (Figure4C and data not shown). The Sup 2216 derived from cells of individual animals (c08, c09, c12) that had shown particularly strong responses to ODN 2216 in previous experiments also induced the highest Mx mRNA expression in both cell lines tested. Although treatment with Sup 2243 systematically increased Mx transcription in target cells, protein levels remained low (Figure4D and data not shown). Furthermore, peak induction of Mx transcription was observed in both cell lines already within 6 hours of incubation with Sup 2216 (Figure4E and data not shown), while protein levels achieved maximum levels within 24 h post stimulation and remained stable thereafter for at least another 24–48 h (Figure4F and data not shown).\nFigure 4 ODN 2216 induces an antiviral state both in stimulated PBMCs directly and in target cells incubated with supernatants from stimulated PBMCs. (A) mRNA expression factors of the indicated genes were measured in PBMCs of the individual cats (c01-c14) from four different age groups after stimulation with ODN 2216 for 24 h. (B) Mx mRNA expression factors were assessed at the indicated time points in PBMCs of one cat after a single stimulation with either ODN 2216 or ODN 2243. (C) Mx mRNA expression factors were measured in fcwf-4 cells incubated for 24 h with supernatants (Sup 2216, Sup 2243, Sup Neg) derived from PBMCs of eight adult cats (groups 2 and 3) or 100U recombinant feline IFNα (rfeIFNα). (D) Mx protein was detected by Western blot in fcwf-4 cells incubated with the indicated supernatants derived from PBMCs of one cat belonging to group 2 or with 100U recombinant feline IFNα (rfeIFNα) for 24 h. (E) Mx mRNA expression factors were measured in fcwf-4 cells at the indicated time points after a single stimulation with Sup 2216, Sup 2243 and Sup Neg respectively. (F) Mx protein was detected by Western blot in fcwf-4 cells at indicated time points after stimulation with Sup 2216 derived from PBMCs of the same cat (right panel). **p \u003c 0.01. rfeIFNα = recombinant feline IFNα.","divisions":[{"label":"Title","span":{"begin":0,"end":123}}],"tracks":[{"project":"2_test","denotations":[{"id":"22906110-17683972-143530981","span":{"begin":529,"end":531},"obj":"17683972"},{"id":"22906110-16740097-143530982","span":{"begin":533,"end":535},"obj":"16740097"}],"attributes":[{"subj":"22906110-17683972-143530981","pred":"source","obj":"2_test"},{"subj":"22906110-16740097-143530982","pred":"source","obj":"2_test"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"2_test","color":"#ecdd93","default":true}]}]}}