132-Microglobulin (f82m) is a major constituent of amyloid fibrils in patients with dialysis-related amyloidosis (DRA). Recently, we found that the pigmented and fluorescent adducts formed nonenzymatically between sugar and protein, known as advanced glycation end products (AGEs), were present in f32m-containing amyloid fibrils, suggesting the possible involvement of AGE-modified f32m in bone and joint destruction in DRA. As an extension of our search for the native structure ofAGEs in 132m of patients with DRA, the present study focused on pentosidine, a fluorescent cross-linked glycoxidation product. Determination by both HPLC assay and competitive ELISA demonstrated a significant amount of pentosidine in amyloid-fibril J2m from longterm hemodialysis patients with DRA, and the acidic isoform Of f82m in the serum and urine of hemodialysis patients. A further immunohistochemical study revealed the positive immunostaining for pentosidine and immunoreactive AGEs and f82m in macrophage-infiltrated amyloid deposits of long-term hemodialysis patients with DRA. These findings implicate a potential link of glycoxidation products in long-lived 82m-containing amyloid fibrils to the pathogenesis of DRA.

Intervertebral discs of 41 chronic renal failure autopsy cases were examined histologically and immunohistochemically to assess the distribution of beta 2-microglobulin-associated (beta 2m) amyloid in the vertebral column. The results demonstrated beta 2m amyloid to appear first in the cervical discs, then in the lumbar and upper thoracic discs, and finally in the middle and lower thoracic discs as the dialysis period is prolonged. The shortest dialysis period for which beta 2m amyloid was detected was one year and seven months. Deposition of beta 2m amyloid was most remarkable in the C4-5, 5-6, and 6-7 levels, which are known to sustain severe mechanical stress in daily life. Thus it is suggested that local mechanical stress accelerates beta 2m amyloidosis. A marked macrophage reaction was observed around the amyloid in cases of severe amyloidosis, the macrophages themselves being immunohistochemically positive for IL-1 beta and TNF-alpha. Amyloid deposition and reactive inflammation mediated by cytokines appear to be closely related to the pathogenesis of destructive spondyloarthropathy.

We studied two patients from a Japanese family with carpal tunnel syndrome (CTS). The biopsy samples obtained during CTS surgical release revealed deposits of amyloid that stained with antihuman transthyretin (TTR) antiserum. Single-strand conformation polymorphism analysis and sequence analysis of polymerase chain reaction (PCR)-amplified exons of the proband's TTR gene revealed a point mutation resulting in a substitution of histidine for tyrosine at position 114. The mutation was confirmed by PCR-primer-induced restriction analysis. Our findings account for clinical heterogeneity of TTR-derived amyloidosis, and suggest the importance of substitution itself for deposits of amyloid in CTS.

Advanced glycation end-products (AGEs) are formed in long-lived matrix proteins by a non-enzymatic reaction with sugar. We recently demonstrated the presence of AGEs in amyloid fibrils of dialysis-related amyloidosis, one of the characteristic features of which is an accelerated bone resorption around amyloid deposits. This suggested a potential link of AGEs in bone resorption and led us to investigate whether AGEs enhance bone resorption. An immunohistochemical study using anti-AGE antibody revealed positive immunostaining for AGEs in bone tissues from elderly subjects. AGE-modified proteins were shown to stimulate monocyte/macrophage to secrete bone-resorbing cytokines such as interleukin-1 beta, interleukin-6 and tumour necrosis factor- alpha. AGE-modified proteins enhanced net calcium efflux in cultured neonatal mouse calvariae to a much greater extent than unmodified proteins. Furthermore, when mouse unfractionated bone cells containing osteoclasts were cultured on dentin slices, AGE-modified proteins increased the number of resorption pits formed by osteoclasts, whereas their normal counterparts or those modified with the early glycation products did not. These findings suggest that AGEs enhance bone resorption by osteoclasts. The modification of bone matrices with AGEs might, therefore, play a pathophysiological role not only in the remodelling of senescent bone matrix tissues, but also in dialysis-related amyloidosis or osteoporosis associated with diabetes and ageing.

Changes in extracellular matrices of articular tissue, intervertebral discs and systemic organs in patients with haemodialysis-related amyloidosis were investigated by immunohistochemical and biochemical examination of proteoglycans. Increased staining for chondroitin sulfate (CS) was detected in the amyloid deposits of all patients, ranging from early to advanced stages. Degenerative tissue changes around early-stage amyloid deposits in the intervertebral discs also showed positive staining for CS. Heparan sulfate (HS) was detected in amyloid deposits, especially in the synovial membrane. Biochemical analysis of connective tissues containing amyloid supported the immunohistochemical studies; CS was the major glycosaminoglycan species in these tissues, accounting for 55-81% of the total glycosaminoglycans. Although previous studies have stressed the importance of HS in amyloidogenesis, the present study showed that CS, which increased significantly in articular tissues associated with mechanical stress, also has a close relationship with amyloidogenesis.

A case of disseminated cryptococcosis with features of primary adrenal insufficiency and meningitis in an immunocompetent host is presented. Despite antifungal chemotherapy, neither meningitis nor bilateral adrenal gland enlargement was improved. Aspiration biopsy of the adrenal gland revealed necrotic tissue with numerous fungi, suggesting that the adrenal glands were the focus of the persistent fungemia. Removal of bilateral adrenal glands led to improve ment by making the patient more sensitive to antifungal chemotherapy.

Immunolocalization of matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) in periarticular tissues of beta 2-microglobulin amyloidosis patients was investigated. MMP-1 (interstitial collagenase) the most strongly expressed of the MMPs, was localized in the synovial lining cells, mesenchymal cells in granulation tissue and nodular amyloid deposits, and chondrocytes within areas of cartilage erosion. Expression of MMP-1 was correlated with the degree of macrophage infiltration and synovial cell hyperplasia, but it was not correlated with the degree of amyloid deposition or haemodialysis period. Expression of MMP-1 appeared more intense than that of TIMP-1 and TIMP-2 in highly inflammatory cases. MMP-2 was mildly expressed in the interstitial fibroblasts and MMP-3 was faintly stained in the extracellular matrix of the synovial membrane. MMP-9 (gelatinase B) was found to be strongly positive in the osteoclasts which increased in the progressing osteolytic lesion from the destructive arthropathy. These results suggest involvement of MMPs in inflammation with an imbalance between expression of MMPs and TIMPs being closely related to pathogenesis of the destructive arthropathy.

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