1 ± 2.9 19.3 ± 1.5 Percentage of ADAM8+/HPIV2- cells 15 ± 6.7 37.9 ± 3.6 78.9 ± 1.9 Figure 2 Immunofluorescence double staining of ADAM8 and HPIV2 marker of HPIV2 HDAC inhibitors cancer stimulated HSY cell cultures on culture day 0 (panel B), 1 (panel C), 3 (panel D). ADAM8 staining is

shown in red and HPIV2 shown in green (arrowheads), together with the blue nuclear counterstain of the same field. Panel A shows the staining of HSY cells that HPIV2 did not infect as negative control, therefore (A) only ADAM8 weak staining with nuclear counterstain, (B, C, D) overlay of double staining of ADAM8 and HPIV2 marker with blue nuclear counterstain of beta-catenin activation the same field on culture days 0, 1 and 3, respectively. Bar = 10 μm. Figure 3 The proportion of mononuclear (black square), binuclear (black upwards pointing triangle) and multinuclear positive cells (downwards pointing triangle) of all ADAM8 positive cells in the immunofluorescence staining of ADAM8 in HPIV2 stimulated human salivary adenocarcinoma cell cultures on culture days 0, 1 and 3 as a function of time. Expression of ADAM8 HPIV2 infected cell cultures was studied using the rabbit anti-human ADAM8 carboxy-terminal antibody as it was reasoned that the antibody recognizing the intracytoplasmic carboxy-terminal end of the molecule would provide an idea of the amount of the full-length ADAM8 molecule, find more with the amino-terminal propeptide and metalloproteinase domains,

as well as its amino-terminal end trimmed counterparts. Indeed, in non-infected HSY cells the proportion

of ADAM8-positive cells was relatively low and stable over time. In contrast, HPIV2 clearly and dramatically up-regulated ADAM8 expression, which in only 3 days increased from 7.9 to 99.2% (p < 0.001). Apart from this dramatic up-regulation of host cell encoded Interleukin-2 receptor ADAM8, two other interesting observations were made in these experiments. First, this increase in ADAM8 expression was accompanied by the formation of binuclear cells and very soon also of multinuclear syncytia. By kinetic association between the increased ADAM8 expression and cell-to-cell fusion it was concluded to indicate that HPIV2 induces this tentative host fusion molecule for enhancement of host-host cell fusion. This conclusion is in part based on the general role of ADAM8 in such fusion processes in the formation of osteoclasts [10] and foreign body giant cells [12]. It can also be asked whether this host-host cell fusion could provide some survival advantages to the HPIV2 virus. Interestingly, it was noticed that at the beginning of the culture period most of the ADAM8-positive host cells were negative for HPIV2 hemagglutinin-neuraminidase antigen indicating that they were non-infected. However, it is also conceivable that the detection of nucleocapsid protein, the most abundant viral protein, would have raised the number of cells identified as HPIV2-positive.