A. S. Cohen

A new form of amyloid protein associated with chronic hemodialysis was identified as β2-microglobulin

Amyloid fibrils were isolated from amyloid-laden tissue obtained from a chronic hemodialysis patient with carpal tunnel syndrome. After solubilization in guanidine HCl, a significant amount of the protein was located in a homogeneous low molecular weight fraction. The protein was found to be identical to beta 2-microglobulin, with regard to its molecular weight of 11,000, amino acid composition and 16 amino-terminal amino acids: Ile-Gln-Arg-Thr-Pro-Lys-Ile-Gln-Val-Tyr-Ser-Arg-His-Pro-Ala-Glu-. These results demonstrate that the amyloid associated with chronic hemodialysis contains as major component a new form of amyloid fibril protein that is homologous to beta 2-microglobulin.

ISOLATION OF AMYLOID P COMPONENT (PROTEIN AP) FROM NORMAL SERUM AS A CALCIUM-DEPENDENT BINDING PROTEIN

The P component of amyloid (protein AP, pentagonal unit) has been isolated from normal serum by using its hitherto undescribed calcium-dependent affinity for agarose. The presence of P component in all forms of amyloid may be due to this calcium-dependent binding to certain polyanions.

In vitro formation of amyloid fibrils from intact β2-microglobulin

Prompted by the identification of hemodialysis-associated amyloid protein as beta 2-microglobulin, we attempted to create in vitro amyloid fibrils from the native protein. Beta 2-microglobulin in PBS was slowly dialyzed free of salt and then concentrated. The residue showed Congophilia with green birefringence by light microscopy and polarization, and non-branching fibrils of indeterminate length, measuring 8 to 10 nm in diameter by electron microscopy, thus meeting the morphologic criteria for amyloid. The present study demonstrates the first successful in vitro creation of amyloid fibrils with intact precursor protein molecules and provides supporting evidence that hemodialysis-associated amyloid is constituted from beta 2-microglobulin.

Immunocytochemical identification of amyloid in formalin-fixed paraffin sections

SummaryFormalin-fixed paraffin sections of livers, spleens and kidneys from patients with primary, secondary and familial amyloidosis as well as from a casein-induced murine amyloid model were analysed by an immunocy-tochemical (unlabeled antibody enzyme) method utilizing antisera to amyloid-related proteins. All amyloid deposits of all amyloid types showed positive reactions with anti-AP of the respective species. Positive reaction of anti-human AA to human secondary amyloid deposits and of anti-mouse AA to the deposits of casein-induced murine amyloid was also observed, but there was no species cross reactivity. No significant deposition of the reaction products was produced by anti-immunoglobulin light chains on deposits of any amyloid type, or by anti-AA in the tissues from primary or familial amyloidosis. The results indicate that amyloid proteins AA and AP can survive as antigens through routine histologic preparation, that anti-AP can be a universal marker for deposits of any amyloid type within the same species, and that AA-type amyloid can be identified by this method while there may as yet be no feasible universal marker for the AL-type at present.

RENAL TRANSPLANTATION IN TWO CASES OF AMYLOIDOSIS

Abstract Bilateral nephrectomy and renal transplantation were performed in two patients with severe renal amyloidosis and progressive azotaemia. One patient, whose amyloid was associated with familial Mediterranean fever, has survived for 24 months and showed no recurrence of amyloid on biopsy of the donor kidney 12 months after transplant. The second patient, with amyloid secondary to osteomyelitis, became normotensive, but died from an abscess 5 months after transplantation. The donor kidney in this individual also showed no amyloid. It is recommended that renal transplantation in amyloid disease still be regarded as an experimental procedure to be done in selected patients only.

AMYLOIDOSIS: AN EXPRESSION OF IMMUNOLOGICAL TOLERANCE ?

Abstract Specific cellular unresponsiveness to casein has been shown to develop during the induction of amyloidosis in guineapigs while cellular immunity to other antigens remains intact. It is suggested that tolerance to a specific immunogen may play a central role in the pathogenesis of amyloidosis.

Isolation and Characterization of a x Amyloid Fibril Protein

The fibril in primary amyloidosis (AL) is composed of a monoclonal light chain or portions thereof. No unique primary structure has been identified that predisposes certain light chains to form amyloid fibrils. Currently, classification of amyloidosis is based on the biochemistry of the amyloid fibril. We determined the NH2‐terminal sequence of an amyloid fibril and found it to be of the xI immunoglobulin subgroup. No structural alterations were detected to account for the conversion of the light‐chain fragment to an amyloid fibril. Antiserum produced to the fibril protein did not react in immunodiffusion with purified LEP or MAG antigens, which are xI proteins. This antiserum may be directed to antigenic sites unique to the immunizing protein and is unable to recognize homologous proteins, rendering it unsuitable for immunochemical identification of amyloid deposits of light‐chain origin. PAG represents the 10th reported variable xI amyloid fibril protein subjeeted to partial sequence analysis. Antisera that recognize antigenic determinants present in all members of an immunoglobulin subgroup need further development.

The carbohydrate composition of human serum amyloid P component

The carbohydrate moiety of human serum amyloid P component was analyzed and found to consist of equal amounts of galactose and mannose (total 4.0%), of glucosamine and galactosamine in a ratio of 7:1 (total 2.7%) and sialic acid (3.9%). It should be noted that this is the first report on the separate quantification of the neutral hexoses and the demonstration of the presence of galactosamine. The contents of glucosamine and galactosamine suggest that this protein possesses both an N- and an O-glycan.

The Effect of Levamisole in Experimental Murine Amyloidosis

In casein-induced murine amyloidosis various lines of investigation have implicated immunodeficiency as playing a role in amyloid formation. In this study, the immunopotentiating agent levamisole failed to prevent amyloidogenesis or to accelerate resolution of preformed amyloid deposits in the mouse model. The serum precursor of amyloid, serum amyloid protein A (SAA), was increased by levamisole in both normal and amyloidotic mice.