Amyloid enhancing factor (AEF) is an activity that appears naturally during the course of persistent
inflammation and precedes, by 24-48 h, AA
amyloid deposition in appropriate murine models.
AEF is defined by its
biological properties, namely, when administered intravenously or intraperitoneally to a mouse, it primes the recipient for the rapid induction of AA
amyloid when they are given an inflammatory stimulus. Available evidence indicates that
AEF is
protein in nature, but a specific molecular species (if a singular species exits) has not been identified. Past work (Ganowiak et al., Biochem. Biophys. Res. Commun. 199:306-312, 1994) has shown that
AEF activity may be imparted to two different
proteins (IAPP and beta-
protein) provided each is organized in the form of an
amyloid fibril. Since a characteristic property of
proteins in
amyloid fibrils is their beta-sheet organization, one possibility is that
AEF activity, in part, depends on such organization, and other
proteins with such properties may also have
AEF activity. To investigate this possibility,
silk, a
protein which contains substantial beta-sheet content, was denatured in LiSCN and allowed to renature slowly under reducing conditions to form a gel. The denatured
silk preparation was then sonicated thoroughly to permit
intravenous injection and assessed for
AEF activity. The modified
silk, presented as small fibrils in a beta-sheet conformation as assessed by electron microscopy and circular dichroism, respectively. This
silk at 0-50 micrograms/animal was administered intravenously as "
AEF" followed immediately by subcutaneous AgNO3 as the inflammatory stimulus. Six days later the spleens were examined for the presence of AA
amyloid and following
Congo red staining, the amount of
amyloid quantified by image analysis. Modified
silk without an inflammatory stimulus, and non-sonicated modified
silk, failed to induce AA
amyloid. Sonicated modified
silk followed by AgNO3 induced large quantities of splenic AA
amyloid in a dose dependent fashion. Modified
silk in quantities as small as 1-5 micrograms/animal can function as
AEF. The
AEF properties of the modified
silk were stable at 4 degrees C for at least 4 weeks (the longest period tested). This procedure may provide a means of standardizing
AEF preparations.