I'm not sure if you're talking about the ACORN output or AM26C31 input.
There are only pull up resistors on older ACORN revisions. If your ACORN is 170918 or newer, it has push-pull drivers for the DB25.
The AM26C31 has TTL compatible input levels, but no pull up resistors.
That would be a start, but there is still some signal degradation that can be avoided by using the DB25. This would only be an issue if running higher output frequencies is part of the goal when switching to line drivers.
Yes, that is no problem. All the I/Os are always active. ACORN does not "know" which headers you are using.