New clothes for amyloid enhancing factor (AEF): silk as AEF.

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.