Calcium pump expression in human bone and soft tissue tumours

Ninety-one cases of human bone and soft tissue tumours were studied for calcium pump expression by strepto-avidin-biotin immunohistochemical staining with a monoclonal antibody against sarcoplasmic reticulum calcium-ATPase (mAb6F5). Two out of 5 cases of embryonal rhabdomyosarcoma, 1 out of 5 cases of biphasic synovial sarcoma, 4 of 4 cases of chordoma and all of 3 chondrosarcoma cases were positive for mAb6F5. Although this novel monoclonal antibody can be used as a marker of myogenic tumours, the present positive result for endoplasmic reticulum calcium-ATPase (calcium pump) in other tumours including chordoma, chondrosarcoma and synovial sarcoma indicates a wider immunoreactivity. The findings further suggest that intracellular calcium may play an important role in cell proliferation and/or differentiation.     

Immunohistochemical expression of MAM-3 and MAM-6 antigens in salivary gland tumours

Summary MAM-3 and MAM-6 antigens of human milk fat globule membrane were detected immunohistochemically in 93 cases of salivary gland tumours as well as in normal glands. The antigens were visualized in 10% formalin-fixed paraffin sections. MAM-3 (MoAbs 115G3, 67D11) antigen was distributed in intercalated and striated duct cells of the normal salivary glands, and in luminal tumour cells and squamous metaplastic cells of pleomorphic adenomas. In pleomorphic adenomas the frequency of positive staining with MoAb 67D11 (54/67; 80.6%) was higher than that with MoAb 115G3 (36/67; 53.7%). MAM-6 (MoAbs 115D8, 115F5) antigen was expressed in luminal and lateral borders of serous acinar cells and ductal of the normal glands, and also in luminal borders of tubulo-ductal and glandular structures of salivary gland tumours. Ductal basal cells were characterized by existence of positive staining for MAM-6 antigen, in adenolymphomas MAM-6 antigen was restricted to the basal tumour cells. Some mucous cells of mucoepidermoid tumours were stained specifically with MoAb 115G3, and epidermoid cells of mucoepidermoid carcinomas manifested MAM-6 antigen staining. Immunohistochemical localization of MAM-6 antigen resembled that of epithelial membrane antigen (EMA) detected with MoAb.     

Production of human interferon-gamma in serum-free medium.

Interferon (IFN)-gamma was produced with a high yield in cultures of human peripheral mononuclear cells by combined stimulation with OK-432 and staphylococcal enterotoxin B. Human mononuclear cells cultured in serum-free medium produced several times as much IFN as those in RPMI-1640 medium containing 10% fetal bovine serum. A synergistic effect of OK-432 and staphylococcal enterotoxin B on the production of IFN-gamma was demonstrated. Ultrogel AcA54 column chromatography of crude IFN showed a single peak with an apparent molecular weight of 43,000. Our production system for human IFN-gamma offers a feasible approach to preparation of large quantities of purified IFN-gamma for structure studies, antibody production, and clinical applications.     

Breast milk macrophages spontaneously produce granulocyte-macrophage colony-stimulating factor and differentiate into dendritic cells in the presence of exogenous interleukin-4 alone

Peripheral blood monocytes extravasate and differentiate into tissue macrophages to mediate effective local defence, but how tissue-specific stimuli and environments may influence their functions remains unknown. Here, we found that peripheral blood monocytes gained the ability to produce granulocyte-macrophage colony-stimulating factor (GM-CSF) upon exposure to breast milk and differentiated into CD1+ dendritic cells (DCs) in the presence of exogenous interleukin-4 (IL-4) alone. This in vitro observation appeared physiologically relevant since macrophages that were freshly isolated from breast milk were also found to produce GM-CSF spontaneously. Furthermore, in contrast to peripheral blood monocytes that differentiated into DCs only in the presence of both exogenous GM-CSF and IL-4, differentiation of breast milk macrophages into DCs was induced by incubation with exogenous IL-4 alone. These IL-4-stimulated breast milk macrophages were efficient in stimulating T cells, suggesting their potential role in mediating T-cell-dependent immune responses in situ. On the other hand, unexpected expression of DC-SIGN, a DC-specific receptor for human immunodeficiency virus (HIV), even in unstimulated breast milk macrophages, may favour HIV infection, resulting in an increased risk of mother-to-infant vertical transmission of the virus via breast milk. Thus, tissue-specific development of macrophages is often linked to effective local immunity, but may potentially provide an opportunity for a pathogen to spread and transmit.     

Catalase, a Specific Antigen in the Feces of Human Subjects Infected with Helicobacter pylori

Recently, we reported the production of three new monoclonal antibodies with high specificity for a Helicobacter pylori antigen suitable for diagnosis of H. pylori infection. The aim of the present study was to identify the antigen recognized by these monoclonal antibodies concerning both H. pylori and the feces of human subjects infected with H. pylori. The cellular antigen was purified from an H. pylori cell extract by immunoaffinity column chromatography with the monoclonal antibody as a ligand. The amino-terminal amino acid sequences (eight residues) of the purified antigen and H. pylori catalase were the same. The molecular weights of native and subunit, specific catalase activity, and UV and visible spectra of the purified antigen were in good agreement with those of H. pylori catalase. The human fecal antigens were purified from two fecal samples of two H. pylori-positive subjects by ammonium sulfate precipitation, CM-Sephadex C₅₀ chromatography, and the same immunoaffinity chromatography used for the H. pylori cellular antigen. The fecal antigens had catalase activity. The amino-terminal amino acid sequences (five residues) of the human fecal antigen and H. pylori catalase were the same. The monoclonal antibodies reacted with the native cellular antigen, but did not react with the denatured antigen, human catalase, and bovine catalase. The results show that the target antigen of the monoclonal antibodies is native H. pylori catalase and that the monoclonal antibodies are able to specifically detect the antigen, which exists in an intact form, retaining the catalase activity in human feces.     

Contractile effects of bacterial cell walls, their enzymatic digests, and muramyl dipeptides on ileal strips from guinea pigs.

Cell walls isolated from four bacterial species (Streptococcus pyogenes, Lactobacillus plantarum, Streptomyces gardneri, and Nocardia corynebacteriodes), which exhibited the adjuvant effect of stimulating cellular and humoral immune responses against ovalbumin in guinea pigs, caused the slow-starting and long-lasting contraction of guinea pig ileal strips suspended in Tyrode solution. In contrast to these cell walls active in immunoadjuvancy, those isolated from five bacterial species (Micrococcus lysodeikticus, Staphylococcus epidermidis, Arthrobacter atrocyaneus, Corynebacterium insidiosum, and Ampullariella regularis), which lacked immunoadjuvancy at least in intact walls, caused no or very weak contraction of the ileal strips. Further study demonstrated that both a monomer and a polymer of disaccharide-stem peptides, which were obtained by enzymatic degradation of S. epidermis cell wall peptidoglycans, displayed similar contractile effects. It was finally revealed that guinea pig ileum strips showed a definite contractile response to N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP) and 6-O-stearoyl- and 6-O-(2-tetradecylhexadecanoyl)-MDPs, but not to their analogs, whose C-terminal amino acid was L-isoglutamine or D-isoasparagine in place of D-isoglutamine and which lacked adjuvancy. 6-O-(3-Hydroxy-2-docosylhexacosanoyl)-MDP, on the other hand, caused a slow and lasting relaxation of the ileum strips, but its L-isoglutamine and D-isoasparagine analogs did not.     

Immunohistological study on malignant fibrous histiocytoma

Malignant fibrous histiocytoma (MFH) shows a mixed proliferation of both fibroblastic and histiocytic cells. Because of their complex morphologic appearances, the nature of truly neoplastic cells in MFH has been controversial. In the present study, immunoperoxidase method (PAP method) was used to examine the intracytoplasmic lysozyme (LY), alpha-1-antitrypsin (A1AT), fibronectin (FN), and polyvalent immunoglobulin (PI) in the fibroblastic and histiocytic cells. Twenty-three cases with MFH were histologically divided into three groups; predominantly fibroblastic type (Group I; 5 cases), mixed fibroblastic and histiocytic type (Group II; 15 cases), and almost pure histiocytic type (Group III; 3 cases). Fibroblastic cells showed a strong positive reaction for LY and A1AT, suggesting the histiocytic nature, while the proliferating cells in Group II were more intensely stained by each of the antibodies than in Groups I and III. Enzyme histochemical examinations on fresh materials were available in 3 cases. These findings suggest that proliferating cells in MFH possess a histiocyte nature.     

Actin assembly is a crucial factor for superoxide anion generation from adherent human eosinophils

BACKGROUND: Cellular adhesion is crucial for eosinophil effector functions. OBJECTIVE: We sought to elucidate the role of the actin cytoskeleton in cellular adhesion and superoxide anion generation by human eosinophils. METHODS: Eosinophils were stimulated with platelet-activating factor (PAF) or complement component 5a on human serum albumin-coated plates with or without an actin-polymerization inhibitor, cytochalasin B (CB), or cytochalasin D (CD). Superoxide anion generation was measured on the basis of reduction of absorbance associated with cytochrome c.2 Eosinophil adhesion was assessed on the basis of eosinophil protein X content in adherent cells. Transient stimulus-induced increase of intracellular calcium and translocation of protein kinase C (PKC) betaII, PKC delta, PKC zeta, and p47 phagocyte oxidase (a component of nicotinamide adenine dinucleotide phosphate oxidase) were also investigated. RESULTS: CB, CD, or antibodies against CD18 (the beta2 chain of integrin, alphaMbeta2) inhibited stimulus-induced eosinophil superoxide anion generation. Stimulus-induced eosinophil adhesion was unaltered by CB, whereas it was significantly suppressed by CD or anti-CD18 antibodies. Transient PAF-induced intracellular calcium increase was also unaffected by CB or CD, but stimulus-induced eosinophil shape changes and translocation of PKCs and p47 phagocyte oxidase to the cell membrane region were completely inhibited by CB. PAF-induced eosinophil degranulation was inhibited by CB, CD, or anti-CD18 antibodies, whereas complement component 5-induced degranulation was not suppressed by CB. CONCLUSION: By itself, beta2 integrin-dependent cellular adhesion is not sufficient for promoting eosinophil effector function. Adequate actin assembly is required for eosinophil adhesion and also for full superoxide anion generation in eosinophils.     

Expression of CD10 by stromal cells during colorectal tumor development

CD10 is a cell surface metalloprotease expressed by a variety of normal cell types, including lymphoid precursor cells, germinal center B lymphocytes, and some epithelial cells. We noticed that stromal cells of some cancers are positive for CD10. In this study, we investigated the role of CD10 produced by the stromal cells of colorectal neoplasms in the progression of colorectal neoplasms. Immunohistochemical examination of CD10 and p53 was performed in 169 colorectal epithelial neoplasms representing various stages of carcinogenesis. The results were correlated with the morphologic characteristics of the neoplasms. There was no expression of CD10 in the stromal cells of normal colorectal tissue. CD10-positive stromal cells were present adjacent to the tumor cells in 16 of 73 adenomas with mild or moderate dysplasia. More frequent expression of CD10 by the stromal cells was detected in adenomas with severe dysplasia (12 of 17), intramucosal carcinomas (10 of 16), and invasive carcinomas (50 of 63) than in adenomas with mild or moderate dysplasia (P < 0.0001). Expression of CD10 by > 10% of the stromal cells was detected only within the area of the invasive growth front of invasive carcinomas, not in adenomas and in only 1 of the intramucosal carcinomas. The difference between invasive and non invasive tumors was significant (P < 0.0001). The stromal expression of CD10 was significantly associated with the accumulation of p53 and a larger tumor size. These results indicate that CD10 expression is an integral part of colorectal carcinogenesis. CD10 expression seems to contribute to the invasion and thus probably facilitates metastasis.     

Nuclear accumulation of beta-catenin in intestinal-type gastric carcinoma: correlation with early tumor invasion

Mutation of the adenomatous polyposis coli gene, which is known to be an early event in the carcinogenesis of intestinal-type gastric carcinoma, leads to accumulation of beta-catenin. In addition, beta-catenin has been found to activate down stream signaling molecules in the wingless/Wnt pathway. In this study, the clinical significance of nuclear accumulation of beta-catenin was evaluated in gastric carcinoma. Immunohistochemical staining showed nuclear localization in 16 (12%) of 139 (94 intestinal-type and 45 diffuse-type) gastric carcinomas, and all 16 lesions with nuclear staining were intestinal-type adenocarcinomas. Of the 16 cases, 15 were in the early clinical stage. In the remaining case, the lesion had invaded the subserosal layer and showed strong nuclear staining at the invasive front. In 14 of the 16 cases with nuclear localization, there were no abnormal mobility shifts detected using polymerase chain reaction-single strand conformational polymorphism analysis. This was confirmed using direct sequencing analysis, which revealed the wild-type sequence in the 12 cases tested. Nuclear accumulation of beta-catenin did not correlate with lymph node metastasis or 5-year survival. These findings suggest that high intranuclear levels of beta-catenin protein play an important role in early tumor growth and may function in initiation of invasive processes in intestinal-type gastric carcinoma.