Charles L. Murphy

Transmissibility of systemic amyloidosis by a prion-like mechanism.

The generation of amyloid fibrils from an amyloidogenic polypeptide occurs by a nucleation-dependent process initiated in vitro by seeding the protein solution with preformed fibrils. This phenomenon is evidenced in vivo by the fact that amyloid protein A (AA) amyloidosis in mice is markedly accelerated when the animals are given, in addition to an inflammatory stimulus, an i.v. injection of protein extracted from AA amyloid-laden mouse tissue. Heretofore, the chemical nature of this “amyloid enhancing factor” (AEF) has not been definitively identified. Here we report that the active principle of AEF extracted from the spleen of mice with silver nitrate-induced AA amyloidosis was identified unequivocally as the AA fibril itself. Further, we demonstrated that this material was extremely potent, being active in doses <1 ng, and that it retained its biologic activity over a considerable length of time. Notably, the AEF was also effective when administered orally. Our studies have provided evidence that AA and perhaps other forms of amyloidosis are transmissible diseases, akin to the prion-associated disorders.

Antibody-Mediated Resolution of Light Chain-Associated Amyloid Deposits

Primary light-chain-associated (AL) amyloidosis is characterized by the deposition in tissue of monoclonal light chains as fibrils. With rare exception, this process is seemingly irreversible and results in progressive organ dysfunction and eventually death. To determine whether immune factors can effect amyloid removal, we developed an experimental model in which mice were injected with amyloid proteins extracted from the spleens or livers of patients with AL amyloidosis. Notably, the resultant amyloidomas were rapidly resolved, as compared to controls, when animals received injections of an anti-light-chain monoclonal antibody having specificity for an amyloid-related epitope. The reactivity of this monoclonal antibody was not dependent on the V(L) or C(L) isotype of the fibril, but rather seemed to be directed toward a beta-pleated sheet conformational epitope expressed by AL and other amyloid proteins. The amyloidolytic response was associated with a pronounced infiltration of the amyloidoma with neutrophils and putatively involved opsonization of fibrils by the antibody, leading to cellular activation and release of proteolytic factors. The demonstration that AL amyloid resolution can be induced by passive administration of an amyloid-reactive antibody has potential clinical benefit in the treatment of patients with primary amyloidosis and other acquired or inherited amyloid-associated disorders.

Chemical typing of amyloid protein contained in formalin-fixed paraffin-embedded biopsy specimens

The human amyloidoses represent a heterogeneous group of disorders characterized by the deposition of fibrillar protein in vital organs. Given the fact that at least 20 different molecules can form fibrils, the unambiguous identification of the type of amyloid deposited is critical to the correct diagnosis and treatment of patients with these disorders. Heretofore, this information has been inferred from particular clinical features of the disease, ancillary laboratory tests, and results of immunohistochemical analyses. However, to establish unequivocally the kind of protein that is deposited as amyloid, it is necessary to determine its chemical composition through amino acid sequencing or mass spectroscopy of material extracted from fibrillar deposits. We have developed a micromethod whereby such studies can be performed readily using sections of formalin-fixed, paraffin-embedded biopsy specimens. The ability to identify precisely the nature of the tissue deposits has diagnostic, therapeutic, and prognostic implications for patients with amyloid-associated disorders.

Amyloid deposits in transthyretin-derived amyloidosis: cleaved transthyretin is associated with distinct amyloid morphology

The pathological fibrillar deposits found in the heart and other organs of patients with senile systemic amyloidosis (SSA) and Swedish familial amyloidotic polyneuropathy (FAP) contain wild‐type (wt) and a mutant form of transthyretin (TTR), respectively. Previously, it was reported that these two forms of amyloid have different molecular features and it was thus postulated that the mechanism responsible for TTR fibrillogenesis in SSA and FAP may differ. To document further the nature of the amyloid in these entities, detailed morphological, histochemical, immunological, and structural analyses of specimens obtained from 14 individuals with SSA and 11 Swedish FAP patients have been performed. Two distinct patterns of amyloid deposition (designated A and B) were evident. In pattern A, found in all SSA and five of 11 FAP cases, the amyloid had a homogeneous but patchy distribution within the sub‐endocardium, sub‐epicardium, and myocardium; exhibited weak congophilia and green birefringence; and was composed of tightly packed, short, unorientated fibrils. This material contained mainly ∼79‐residue C‐terminal fragments of the amyloidogenic precursor protein. In pattern B, seen in the six other FAP patients, the amyloid appeared as thin streaks throughout the cardiac tissue; often surrounded individual muscle cells; was strongly congophilic and birefringent; had long fibrils arranged in parallel bundles, often penetrating into myocytes; and was composed of virtually intact TTR molecules. These findings provide substantive evidence for the morphological and structural heterogeneity of TTR fibrils and suggest that the two types of deposition may reflect fundamental differences in the pathogenesis of the TTR‐associated amyloidoses. Copyright

Thermodynamic Modulation of Light Chain Amyloid Fibril Formation

To obtain further insight into the pathogenesis of amyloidosis and develop therapeutic strategies to inhibit fibril formation we investigated: 1) the relationship between intrinsic physical properties (thermodynamic stability and hydrogen-deuterium (H-D) exchange rates) and the propensity of human immunoglobulin light chains to form amyloid fibrils in vitro; and 2) the effects of extrinsically modulating these properties on fibril formation. An amyloid-associated protein readily formed amyloid fibrils in vitro and had a lower free energy of unfolding than a homologous nonpathological protein, which did not form fibrils in vitro. H-D exchange was much faster for the pathological protein, suggesting it had a greater fraction of partially folded molecules. The thermodynamic stabilizer sucrose completely inhibited fibril formation by the pathological protein and shifted the values for its physical parameters to those measured for the nonpathological protein in buffer alone. Conversely, urea sufficiently destabilized the nonpathological protein such that its measured physical properties were equivalent to those of the pathological protein in buffer, and it formed fibrils. Thus, fibril formation by light chains is predominantly controlled by thermodynamic stability; and a rational strategy to inhibit amyloidosis is to design high affinity ligands that specifically increase the stability of the native protein.

Characterization of Systemic Amyloid Deposits by Mass Spectrometry

The human systemic (noncerebral) amyloidoses represent a heterogeneous group of disorders characterized by the widespread deposition of proteins as fibrils in organs or tissues throughout the body. The unequivocal identification of the type of amyloid deposited is critical to the correct diagnosis and treatment of patients with these illnesses. Heretofore, this information was inferred from clinical data, ancillary laboratory tests, and results of immunohistochemical, as well as genetic, analyses. However, to establish definitively the type of amyloid present, the chemical composition of the fibrillar components must be determined. For this purpose, we have developed micro-methods, whereby this information can be obtained by tandem mass spectrometry (MS/MS) using material extracted from formalin-fixed, amyloid-containing tissue biopsy specimens or subcutaneous fat aspirates. The ability to identify precisely the protein nature of the pathologic deposits has diagnostic, therapeutic, and prognostic implications for patients with amyloid-associated disease.

Calcifying epithelial odontogenic (Pindborg) tumor-associated amyloid consists of a novel human protein

Calcifying epithelial odontogenic tumors (CEOTs), also known as Pindborg tumors, are characterized by the presence of squamous-cell proliferation, calcification, and, notably, amyloid deposits. On the basis of immunohistochemical analyses, the amyloidogenic component had heretofore been deemed to consist of cytokeratin-related or other molecules; however, its chemical composition had never been elucidated. We have used our microanalytic techniques to characterize the protein nature of CEOT-associated amyloid isolated from specimens obtained from 3 patients. As evidenced by the results of amino-acid sequencing and mass spectrometry, the fibrils were found to be composed of a polypeptide of approximately 46 mer. This component was identical in sequence to the N-terminal portion of a hypothetical 153-residue protein encoded by the FLJ20513 gene cloned from the human KATO III cell line. That the amyloid protein was derived from this larger molecule was demonstrated by reverse transcription-polymerase chain reaction amplification of tumor-cell RNA where a full-length FLJ20513 transcript was found. Furthermore, immunohistochemical analyses revealed that the amyloid within the CEOTs immunostained with antibodies prepared against a synthetic FLJ20513-related dodecapeptide. Our studies provide unequivocal evidence that CEOT-associated amyloid consists of a unique and previously undescribed protein that we provisionally designate APin.

Amyloidogenic potential of foie gras

The human cerebral and systemic amyloidoses and prion-associated spongiform encephalopathies are acquired or inherited protein folding disorders in which normally soluble proteins or peptides are converted into fibrillar aggregates. This is a nucleation-dependent process that can be initiated or accelerated by fibril seeds formed from homologous or heterologous amyloidogenic precursors that serve as an amyloid enhancing factor (AEF) and has pathogenic significance in that disease may be transmitted by oral ingestion or parenteral administration of these conformationally altered components. Except for infected brain tissue, specific dietary sources of AEF have not been identified. Here we report that commercially available duck- or goose-derived foie gras contains birefringent congophilic fibrillar material composed of serum amyloid A-related protein that acted as a potent AEF in a transgenic murine model of secondary (amyloid A protein) amyloidosis. When such mice were injected with or fed amyloid extracted from foie gras, the animals developed extensive systemic pathological deposits. These experimental data provide evidence that an amyloid-containing food product hastened the development of amyloid protein A amyloidosis in a susceptible population. On this basis, we posit that this and perhaps other forms of amyloidosis may be transmissible, akin to the infectious nature of prion-related illnesses.

Microextraction and purification techniques applicable to chemical characterization of amyloid proteins in minute amounts of tissue.

This article described micromethods useful for the extraction, purification, and amino acid sequencing of amyloid proteins contained in minute specimens obtained from patients with systemic forms of amyloidosis. We posit that these procedures can also be applied to the biochemical characterization of cerebral amyloid deposits. The selection of the techniques is dependent on the type of sample to be extracted (fresh or formalin fixed) as well as the amount of congophilic material present. Although amyloid proteins are isolated and purified more easily from fresh tissue, it must be noted that formalin-fixed specimens are available more readily for analysis due to the common diagnostic use of fine needle tissue biopsies and are therefore, important for both current and retrospective studies. Remarkably, despite the expected difficulties associated with formalin treatment we were able to extract and sequence amyloid proteins from fixed tissues presumably due to the resistance of amyloid to formalin cross-linking. Through the continued development of techniques for small-scale protein separation and application of highly sensitive microsequencing and mass spectral methods, exact identification of the protein contained in fibrillar amyloid deposits can be determined. Such information has therapeutic and prognostic relevance and can increase our understanding of the pathogenesis of amyloidosis.

Two different types of amyloid deposits-apolipoprotein A-IV and transthyretin-in a patient with systemic amyloidosis

Certain forms of systemic amyloidosis have been associated with the pathologic deposition as fibrils of three different apolipoprotein-related proteins—apolipoprotein A-I, apolipoprotein A-II, and serum amyloid A. We have previously reported (Bergström et al, Biochem Biophys Res Commun 2001;285:903-908) that amyloid fibrils extracted from the heart of an elderly male with senile systemic amyloidosis contained, in addition to wild-type transthyretin-related molecules, an N-terminal fragment of yet a fourth apolipoprotein—apolipoprotein A-IV (apoA-IV). We now provide the results of our studies that have established the complete amino-acid sequence of this ∼70-residue component and, additionally, have shown this protein to be the product of an unmutated apoA-IV gene. Notably, the apoA-IV and transthyretin fibrils were not codeposited but, rather, had anatomically distinct patterns of distribution within the heart and other organs, as evidenced immunohistochemically, by variation in the ultra structural morphology and by differences in the intensity of Congo red birefringence. These findings provide the first conclusive evidence that two separate forms of amyloid, each derived from a wild-type amyloidogenic precursor protein, were present in a patient with systemic amyloidosis.