Mitsuko Sobue

Production and characterization of monoclonal antibody to dermatan sulfate proteoglycan.

We purified dermatan sulfate proteoglycan (PG) from the capsule of human ovarian fibroma for use as an immunogen. A monoclonal antibody, designated 6B6, was produced which reacts to the intact molecule of dermatan sulfate PG and the chondroitinase AC-treated core molecule on Western-blotted nitrocellulose membrane. Localization of materials showing crossreactivity to this antibody was studied in human tissues by indirect immunohistochemistry. The interstitial elements of almost all tissues examined were positive for the antibody: dermis, submucosal layer of digestive tract, perichondral layer, perivascular connective tissue, perineurium, adventitia of aorta, vessel wall of vein, pleura, and fibrous capsule of kidney and liver. Positive staining was also observed in fibrous elements at post-necrotic foci of cardiac muscle and pancreas, and at atherosclerotic lesions of aorta. The distribution of the antigen, core protein of the dermatan sulfate PG, revealed with 6B6 was compared to that of the dermatan sulfate side chain, which was demonstrated with antibody 9A-2 (Couchman et al.: Nature 307:650, 1984) after treatment with chondroitin sulfate B-lyase. The distribution of both antigens, core protein, and dermatan sulfate side chains showed the same pattern, with minor exceptions. The antibody 6B6 will be a useful tool to study the immunohistochemical localization of dermatan sulfate PG.

The frequency and histology of hepatic tissue in germ cell tumors.

The frequency of hepatic tissue and its histological characteristics were examined in 516 germ cell tumors. Hepatic tissue was observed in 48 cases (9.3%). The incidence of hepatic tissue was low in tumors of the ovary (5%), high in both retroperitoneal (27%) and sacro-coccygeal (24%) tumors, and low in both mature (0.3%) and immature teratomas (11%). It was usually encountered in infancy, and the frequency was high in both yolk sac tumors (48%) and mixed germ cell tumors (52%). The hepatic tissue found mainly in mature or immature grade 1 teratomas was similar to adult normal human liver tissue (Ha-type). Tissue in areas consisting of some immature somatic elements of a mixed germ cell tumor was similar to embryonic or fetal liver tissue (Hf-type). Many hepatic nests found in a polyembryoma were of both Ha- and Hftypes. The hepatic tissue found in close relation to yolk sac elements showed predominantly hepatocellular carcinoma- like features (HCLS). Immunohistochemically, the cytoplasm of adult liver-type cells was positive for alpha-1-antitrypsin (AAT), human albumin (ALB), and the third (C3) and fourth (C4) components of the complement system. The cytoplasm of fetal liver-type cells showed the same positivity; in addition, these cells were positive for alpha-fetoprotein (AFP) in 25% of the cases. The cytoplasm of hepatic cells of HCLS was positive for AFP, AAT, ALB, C3, and C4. A weakly positive reaction for CEA and CA19-9 was observed in bile duct-like structure in some Hf-type cases

Histochemical studies of intercellular components of salivary gland tumors with special reference to glycosaminoglycan, laminin and vascular elements

In 41 salivary gland tumors, the characteristics of the intercellular components and vascular endothelial cells were surveyed by immunohistochmical staining for laminin and factor VIII-related antigen (VIII R:Ag), and by mucopolysaccharidase-digestion for glycosaminoglycan (GAG). In myxomatous areas of pleomorphic adenomas, small vessels (diameter 6.5 ± 0.11 µm) were frequent and found to be negative or weakly positive by VIIIR:Ag staining although endothelial cells were clearly positive for VIIIR:Ag in capsule surrounding the tumor tissues. Alcian blue stainability was diminished by treatment with both Streptomyces hyaluronidase and chondroitinase. By laminin staining, a vascular pattern was clearly detected, but the majority of tumor cells were not stained. In adenomatous areas, the basement membrane-like linear laminin-staining reaction was observed to be weak and inconsistent around some tumor cell nests. However, in adenoid cystic carcinomas, laminin-positivity was much more intense than in other tumors such as pleomorphic adenoma, mucoepidermoid tumor and adenocarcinoma. In cylindromatous areas, the inner luminal surface in the pseudocysts was markedly positive for laminin, and there was weak positivity around tumor cell nests having a trabecular pattern. By immunoelectron microscopy, a juxtacellular network of replicated basal lamina of tumor cells which lined the inner surface of pseudocysts was positive for laminin. Alcian blue-positivity in the pseudocyst was abolished with heparitinase and chondroitinase, but not with hyaluronidase.

Production and immunohistochemical characterization of a monoclonal antibody raised to proteoglycan purified from a human yolk sac tumour

SummaryA large proteoglycan with chondroitin sulphate and dermatan sulphate side chains has been isolated and purified from a yolk sac tumour of the left ovary from a 23-year-old female. A monoclonal antibody, designated 2B1, was produced which reacted specifically with the intact molecule of the large proteoglycan and the chondroitinase ABC-treated core molecule. The localization of substances showing cross-reactivity to this antibody was studied in a variety of human tissues by means of indirect immunohistochemistry. The interstitial elements of nearly all tissues of a 5-month-old foetus were intensely reactive with the antibody, but in adult tissues structures that gave positive reactions were limited; only the perivascular and perimuscular fibrous elements were reactive, except for the aorta, which reacted extensively. In contrast, the interstitial elements of the carcinoma tissues tested were intensely reactive. Thus antibody 2B1 can be regarded as a useful tool for studies on the immunohistochemical localization of large proteoglycan in various human tissues.

Glycosaminoglycan-synthetic activity of pleomorphic adenoma. Adenoid cystic carcinoma and nonneoplastic tubuloacinar cells of the salivary gland.

An analysis was carried out on glycosaminoglycan produced in pleomorphic adenoma, adenoid cystic carcinoma, sialadenitis and normal tissue of the salivary gland. After incubation of the tissue segments in a medium containing 35SO4, a radioautograph of the tissue section was made to observe the localization of 35SO4 incorporation, and 35S‐labelled materials were purified from the tissues, and analyzed. High 35S‐radioactivity was observed in the ductal cells of the inflammatory gland tissue and in the acinar cells of normal palatinal gland, but little radioactivity was observed in the interstitial components in these tissues, and the amount of 35SO4 incorporated in the tumor cells was also significant. Eighty to 90% of the 35S‐radioactivity incorporated could be detected as 35S‐glycosaminoglycans in all tissues except for the normal palatinal gland, which contained a large amount of 35S‐sulfated glycoprotein. No significant differences in the synthetic activity of 35S‐glycosaminoglycans and in their components were observed between nonneoplastic and neoplastic cells. These results suggest that glycosaminoglycan‐producing cells in pleomorphic adenoma as well as in adenoid cystic carcinoma are derived from the tubuloacinar cells of the salivary gland.

Morphologic and biologic characteristics of adenoid cystic carcinoma cells of the salivary gland.

Adenoid cystic carcinoma cells cultivated in monolayer and sponge matrix culture, or implanted on the chorioallantoic membrane (CAM) of embryonated eggs, were observed morphologically, and the glycosaminoglycan components in the tumor tissue were analyzed. This tumor tissue contained a large amount of glycosaminoglycans, composed of chondroitin 4‐ and 6‐sulfate, heparan sulfate, hyaluronic acid, and a small amount of dermatan sulfate. In monolayer culture spindle cells proliferated vigorously as multilayer, and secreted mucinous material. In sponge matrix culture, the proliferating cells became embedded in the material produced by the cells themselves. A trace of fine fibers stained with orceine was observed in the intercellular material in culture. Histologic sections of the implants grown on CAM showed that the tumor cells arranged in various structures produced a large amount of mucinous material that spread into the stromal area without any contribution from the mesenchymal element. The morphologic and biologic characteristics of these tumor cells are quite similar to those of pleomorphic adenoma.

Establishment of a cell line producing basement membrane components from an adenoid cystic carcinoma of the human salivary gland

SummaryA new cell line has been established from an adenoid cystic carcinoma arising in the submandibular gland of a 63-year-old woman. The cultured epithelial-like cells grew vigorously and adhered together to form a sheet. Immunohistochemical stainings for type IV collagen, laminin and fibronectin were clearly positive in the intercellular matrix and on the surface of the culture cells. Chondroitin 6-sulfate proteoglycan and heparan sulfate were also detected. Ultrastructural studies showed that the cells had abundant rough endoplasmic reticulum and a well-developed Golgi apparatus. Rough endoplasmic reticulum near the cell surface was markedly dilated, and contained material of low electron density. This cell line would be useful for biological and biochemical studies on the mechanisms by which the stromal component is formed.

Histochemical studies on pseudocysts in adenoid cystic carcinoma of the human salivary gland

SummaryPseudocysts are unique structures found in adenoid cystic carcinomata of human salivary glands. They were studied in 13 such cases by histochemical and immunohistochemical means. The pseudocysts contained an abundance of mucoid materials which reacted strongly with both Alcian Blue and dialysed iron ferrocyanide. The mucoid material was digested with chondroitinase ABC and heparitinase, but was resistant toStreptomyces hyaluronidase. The inner surfaces of the pseudocysts were strongly reactive for laminin, whereas the interface between the tumour cell nests and the outer stromal area was intensely reactive for fibronectin. Numerous fibronectin-reactive fibrils and blood coagulation factor XIII (F-XIII)-positive cells were distributed extensively in the outer stromal area. The F-XIII-positive cells were also found within some pseudocysts. The results obtained in the present study have shown that the pseudocysts represent a peculiar structure consisting of basement membrane components; laminin, fibronectin, heparan sulphate and chondroitin sulphate.

Immunohistochemical Techniques for Detection of Dermatan Sulfate Proteoglycan in Tissue Sections

Dermatan sulfate proteoglycan chains were detected in tissue sections treated with chondroitin B-lyase (0.01 units/ml) in 20 mM Tris-HCl (pH 8.0) for 1 hr, followed by staining with antibody 9A2 specific for unsaturated uronic acid coupled to N-acetylgalactosamine-4 sulfate. In contrast, after treatment with chondroitin B-lyase, no positive staining was observed with antibodies 3B3 and 1B5 which react to the unsaturated uronic acid coupled to N-acetylgalactosamine 6-sulfate and unsaturated uronic acid coupled to N-acetylgalactosamine, respectively. The distribution of dermatan sulfate thus revealed was confirmed by comparison with that found by monoclonal antibody 6B6 which reacts with small proteoglycans carrying dermatan sulfate side chains. The localization of positive staining in fibrous connective tissues was almost identical with these two procedures.

Immunohistochemical characterization of extracellular matrix components of yolk sac tumors

SummaryProteoglycans (PGs) were isolated from yolk sac tumor and chondroitin sulfate large PG (core molecule with a molecular weight ≃ 200,000) and small PG (core molecule with a molecular weight ≃ 50,000) were detected. Immunohistochemical localization of PGs in three yolk sac tumors was investigated using monoclonal antibodies raised against both small and large PGs, which were purified from human ovarian fibroma capsule and a yolk sac tumor, respectively. The localization of large PG was observed to be distinct from that of small PG. A markedly positive reaction for antibody against large PG was observed in myxomatous areas, perivascular and perivesicular portions; hyaline globules were the most intensely reactive. In the areas showing a polyvesicular vitelline tumor pattern, the compact connective tissue stroma consisted of small PGs. It is conceivable that large PGs are synthesized by immature mesenchymal cells and also by epithelial-like cells as a basement membrane component, whereas small PGs are synthesized by mature fibroblastic cells synthesizing collagen. Immunohistochemical localization of other extracellular matrix components (laminin, fibronectin, type I-IV collagen) was also studied in relation to PG localization.