Development of a monoclonal antibody to the aP2 protein to identify adipocyte precursors in tumours of adipose differentiation.

aP2 gene product (aP2 protein) expression has been shown to be a useful diagnostic marker for identification of lipoblasts and fetal fat cells in soft tissue tumours. A monoclonal antibody was developed by a mouse spleen cell-myeloma hybridoma technique to an 18 amino acid segment of the aP2 protein and was used to investigate the immunohistochemical expression of this protein in benign and malignant tumours of adipocytic differentiation and a wide variety of other soft tissue tumours. We found that aP2 protein was expressed by lipoblasts in liposarcomas and lipoblastomas and by brown fat cells in hibernomas and normal periadrenal fat. Other benign adipose tissue tumours and benign and malignant soft tissue tumours were distinguished from liposarcoma by absence of staining for aP2 protein. Immunohistochemical identification of the aP2 protein is likely to prove a useful means of distinguishing liposarcoma from other malignant mesenchymal and epithelial neoplasms, some of which contain cells that morphologically resemble lipoblasts.

Deposition of calcium pyrophosphate in tissue after revision arthroplasty of the hip.

We reviewed histologically the incidence and pathogenesis of the deposition of calcium pyrophosphate dihydrate (CPPD) crystals in the pseudocapsule, femoral and acetabular membranes and periprosthetic tissue at revision of 789 cases of failed total hip replacement. In 13, periprosthetic tissues were found to have deposits of CPPD crystals in areas of cartilaginous metaplasia; four also showed evidence of localised deposition of amyloid. None of the patients had a history of chondrocalcinosis in the hip or other joints. Cartilaginous metaplasia and other changes in periprosthetic tissues may predispose to the deposition of CPPD and associated localised amyloid.

Immunohistochemical identification of tumours of adipocytic differentiation using an antibody to aP2 protein.

AIM: To determine whether aP2 expression is a useful diagnostic marker in soft tissue tumour pathology. METHODS: A polyclonal antibody to aP2 was used to investigate the immunohistochemical expression of this protein in benign and malignant tumours of adipocytic differentiation and a wide variety of other neoplasms. RESULTS: aP2 was only expressed by lipoblasts (in all types of liposarcoma and in lipoblastomatosis) and by brown fat cells (in both hibernomas and normal periadrenal fetal fat). Other benign adipose tissue tumours and malignant connective tissue or epithelial tumours were distinguished from liposarcoma by the absence of staining for aP2. CONCLUSION: Identification of lipoblasts using markers of aP2 expression is of value in the differential diagnosis of benign and malignant adipose tissue tumours and in distinguishing liposarcomas from other malignant mesenchymal and epithelial neoplasms, some of which contain cells that morphologically resemble lipoblasts.

Localized amyloid deposition in cartilage is glycosaminoglycans-associated.

Localized amyloid deposition is known to occur commonly in the articular cartilage of elderly patients. Its pathogenesis is uncertain and it is not known if other cartilage-containing tissues also contain amyloid deposits. Systemic amyloid deposits are known to contain highly sulphated glycosaminoglycans, a major constituent of cartilage. As the composition of articular cartilage glycosaminoglycans is known to change with age, we sought to identify whether localized amyloid deposition in cartilage was glycosaminoglycan-related. We examined specimens of articular cartilage over a wide age range and also examined a variety of cartilaginous tumours and tumour-like lesions for the presence or absence of amyloid deposits. Using mucin histochemistry (alcian blue: MgCl2 critical electrolyte concentration) and immunohistochemistry, we found that highly sulphated glycosaminoglycans (0.9 M and 1 M MgCl2), in particular keratan sulphate, localized to amyloid deposits in both articular cartilage and loose bodies derived from the articular surface. Other cartilaginous lesions (including loose bodies of primary synovial chondromatosis) were negative for amyloid and did not contain highly sulphated glycosaminoglycans. These findings suggest that changes in specific highly sulphated glycosaminoglycans may play a role in localized amyloid deposition in articular cartilage.

Glycosaminoglycans in intervertebral disc amyloid deposits.

Intervertebral discs are a common site of localized articular and some forms of systemic articular amyloid deposition. Whether there is an intrinsic matrix factor that favours amyloid deposition in intervertebral disc connective tissues is uncertain, but it is known that small localized deposits of amyloid in intervertebral discs are largely age related. As the glycosaminoglycans (GAGs) composition of the intervertebral disc is known to change with age, and as some forms of systemic amyloid deposition have been shown to be associated with particular highly sulphated GAGs, we examined the GAGs profile of amyloid deposits in intervertebral discs using mucin histochemistry (Alcian blue: MgCl2 critical electrolyte concentration) and immunohistochemistry. We found strong staining for very highly sulphated GAGs (0.9 M and 1 M MgCl2) and confirmed the presence of keratan sulphate in both localized and systemic, dialysis-associated beta 2-microglobulin amyloid deposits within disc fibrocartilage. These findings suggest that qualitative and quantitative changes in matrix GAGs, particularly strongly sulphated GAGs such as keratan sulphate, may play a role in the pathogenesis of localized and systemic amyloid deposition in intervertebral discs.

Highly sulphated glycosaminoglycans in articular cartilage and other tissues containing beta 2 microglobulin dialysis amyloid deposits.

BACKGROUND: Highly sulphated glycosaminoglycans (GAGs) are a common constituent of amyloid deposits and an integral component of articular connective tissues where beta 2-microglobulin (beta 2M) amyloid is most often found. METHODS: Using alcian blue, magnesium chloride, critical electrolyte concentration, mucin histochemistry, and immunohistochemistry, the GAGs composition of beta 2M amyloid deposits in joint capsule and cartilage, carpal, and heart tissues of 22 uraemic patients was determined. RESULTS: Highly sulphated GAGs were found in beta 2M amyloid deposits not only within cartilage, where such GAGs are normally found in high concentration, but also in other articular and extra-articular connective tissues. Keratan sulphate was often specifically localized to beta 2M amyloid deposits in articular cartilage and to a lesser extent in periarticular tissues, with one case showing colocalization with systemic vascular amyloid deposition. Other sulphated GAGs, chondroitin 4 and 6 sulphate, dermatan sulphate, and heparan sulphate were also identified in tissues containing beta 2M amyloid deposits, but with the exception of heparan sulphate (identified by mucin histochemistry) were not specifically localized to the deposits themselves. CONCLUSIONS: These findings suggest that qualitative or quantitative changes in the composition of highly sulphated GAGs may play a role in localization of beta 2M amyloid deposits in articular and extra-articular tissues.

Immunophenotype of multinucleated and mononuclear cells in giant cell lesions of bone and soft tissue.

AIMS: To compare the antigenic phenotype of giant cells in giant cell lesions of bone and soft tissue with that of osteoclasts and macrophage polykaryons. METHODS: Formalin fixed, paraffin wax embedded sections of 106 giant cell lesions, 19 granulomatous, and 14 osteoclast containing lesions were immuno-histochemically stained for leucocyte common antigen (LCA), CD68, and HLA-DR. RESULTS: Osteoclasts and giant cells of giant cell tumour of bone and giant cell reparative granuloma could be distinguished by their generalised absence of HLA-DR reaction from macrophage polykaryons and giant cells in other giant cell lesions of bone and soft tissue. Staining for LCA, CD68, and HLA-DR was useful in distinguishing reactive histiocytic giant cells and osteoclasts from tumour giant cells. CONCLUSIONS: A panel of macrophage associated antigens should be diagnostically useful in differentiating the histological nature of giant cells in various giant cell lesions of bone and soft tissue.

Use of monoclonal antibodies to recognise osteoclasts in routinely processed bone biopsy specimens.

In decalcified (5% nitric acid) and undecalcified (glycol-methacrylate or resin embedded) routinely processed bone specimens osteoclasts against resorbing surfaces were identified with monoclonal antibodies directed against leucocyte common antigen (LCA) (PD7/26, 2B11), CD68 (KP1), and gpIIIa (Y2/51) but not against HLA-DR (CR3/43 and Ta11B5). Mononuclear cells on resorbing surfaces and occasional mononuclear cells against or near resting surfaces showed a similar pattern of reactivity. This study shows that immunohistochemistry is a sensitive and useful technique for identifying osteoclasts in routinely processed bone specimens. It also suggests a role for mononuclear cells (possibly pre-osteoclasts) in bone resorption.

Bromodeoxyuridine (BrdUrd) immunohistochemistry in undecalcified plastic-embedded tissue. Elimination of the DNA denaturation step.

We examined the application of BrdUrd immunohistochemistry to detect S-phase cells in undecalcified bone and cartilage from the growing rat embedded in Spurr's resin. The effect of fixation on the procedure was studied, and the validity of the technique examined by a comparative study with tritiated thymidine ([3H]-TdR) autoradiography. The use of sodium-ethoxide to remove plastic from tissue sections prior to immunohistochemistry resulted in the production of sufficient ssDNA to make a separate DNA denaturation step unnecessary, thus sparing sections from potentially destructive treatment and shortening the immunohistochemical procedure. Fixation in formalin or Bouin's fluid gave the most satisfactory results. The distribution of BrdUrd labeled cells was restricted to the sites of cell proliferation in growing long bones. Combined studies with BrdUrd immunohistochemistry and [3H]-TdR autoradiography showed that the majority of BrdUrd labeled cells had also incorporated [3H]-TdR, thus attesting to the validity of the technique. This novel approach is suitable for the study of undecalcified hard tissues as well as soft tissues.

The immunohistology of synovial lining cells in normal and inflamed synovium.

The immunohistology of synovial lining cells (SLCs) in normal and inflamed hyperplastic synovium was investigated using monoclonal antibodies directed against leucocyte common antigen (LCA) HLA-DR and other macrophage components. We found that some SLCs in normal synovium express LCA, HLA-DR, and monocyte/macrophage-associated antigens. The number of SLCs expressing these antigens is increased in hyperplastic osteoarthritic (OA) and rheumatoid (RA) synovium. Some SLCs which did not react for LCA or other macrophage markers but were positive for HLA-DR were also noted in normal synovium and some segments of hyperplastic OA synovium. SLCs which are positive for LCA, HLA-DR, and macrophage markers contribute to the intimal hyperplasia in RA where they account for the majority of SLCs in the synovial intima. In OA synovium, the distribution of SLCs showing this pattern of reactivity was less uniform with numerous SLCs which were positive for HLA-DR but negative for LCA and other macrophage markers also present in the synovial intima. These findings indicate that there are some SLCs of bone marrow origin in normal and hyperplastic synovium. They also suggest that recruitment of SLCs of marrow origin is important in the production of intimal hyperplasia in both RA and OA and that there is also a significant local proliferation of non-marrow derived SLCs in OA.

The production of prostaglandins in response to experimentally induced osteomyelitis in rabbits.

Osteomyelitis was induced in the tibiae of rabbits by injection of staphylococcus aureus and sodium tetradecylsulphate (STD); additional rabbits were injected with STD alone. Confirmation of osteomyelitis was based on positive culture of the same phage type bacteria from the tibiae and on the characteristic radiographical and histological appearance of osteomyelitis. Only tibiae which proved to be infected by the above criteria showed significantly increased in vitro release and content of Prostaglandin E and Prostaglandin F2 alpha compared with tibiae injected with STD (P less than 0.05). After two weeks infection, infected tibiae released nine times more Prostaglandin E and five times more Prostaglandin F2 alpha than tibiae injected with STD alone. After four weeks infection, infected tibiae released less Prostaglandin E (P less than 0.05) than after two weeks infection but the release of Prostaglandin F2 alpha was similar. The production of large amounts of prostaglandins by bones in response to infection may be the cause of the rapid bone resorption and sequester formation observed in osteomyelitis.