Today I encountered the following error after changing a value in a web.config file of a published webservice on two nodes of an NLB cluster
"Attempted to access an unloaded AppDomain"
After the web.config change all web service requests failed. The solution was to reset the IIS and restart the corresponding BizTalk Hostinstance. (although I think restarting IIS should have been enough) The interesting thing was that another application, running under the same AppPool also had the same error.
Update: See this Link http://technet.microsoft.com/en-us/library/aa577833.aspx for a describtion and resoltution of the problem.