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Syndecan-1 (CD138) is a surface proteoglycan consisting of long unbranched glycosaminoglycan (GAG) chains covalently attached to a protein backbone. High levels of a putatively syndecan-1 isoform have recently been found on neoplastic cells of primary effusion lymphoma (PEL). As opposed to murine systems, studies on syndecan-1 isoforms in humans have been hampered by the lack of a precise characterization of anti-CD138 monoclonal antibodies (mAbs). We have therefore investigated the reactivity of anti-CD138 mAbs (B-B4, B-B2, 1D4, MI15 and 104-9) with either intact native proteoglycans or a recombinant unglycosylated form of syndecan-1 core protein, and utilized these reagents to dissect the molecular heterogeneity of syndecan-1 in human lymphoma cells. Our results indicated that: (a) mAb B-B2 recognized only nondenatured syndecan-1, being poorly reactive by immunoblotting with both intact and recombinant syndecan-1 protein; (b) mAb 104-9 was unable to recognize native syndecan-1, but showed a significant reactivity with intact and unglycosylated syndecan-1 protein upon immunoblotting; (c) mAbs B-B4, 1D4 and MI15 recognized both the intact molecule and the core protein of syndecan-1, and showed a comparable reactivity in flow cytometry and immunoblotting. Cross-blocking experiments indicated these latter mAbs recognizing the same or closely related epitopes of syndecan-1. Using these mAbs, we have demonstrated that: (a) tumour cells from PEL expressed a syndecan-1 isoform with a higher molecular weight than that present on malignant plasma cells; (b) syndecan-1 expressed by PEL cells had a core protein identical in size to that expressed by plasma cells, suggesting that differences in syndecan-1 size were due to different GAG chains attached to an identical protein backbone; (c) the PEL-specific isoform of syndecan-1, which probably represented the major proteoglycan expressed by these cells, was effective in mediating cell adhesion to type I collagen substrates. This data represents the first evidence describing the existence of a molecular polymorphism, of syndecan-1 in human lymphomas.

Vascular invasion of calcified cartilage, during endochondral ossification, is initiated and sustained by invasive cells (endothelial cells and macrophages) which degrade the tissue by releasing lytic enzymes. Concurrently, reactive oxygen species (ROS) are also released by these cells and we hypothesize that ROS also contribute to the degradation of the tissue. As a preliminary approach to this problem, the antioxidant activities and the effect of ROS on hypertrophic cartilage and chondrocytes (HCs) were investigated. Compared to resting or articular chondrocytes, HCs exhibited higher catalase but lower SOD specific activities and lower PHGPx concentration, thus revealing a defence activity specific against H2O2. Moreover, dose-dependent depletion of ATP occurred after few minutes of exposure to ROS, and a long-term treatment (16 h incubation with ROS) promoted the release of LDH activity and a significant variation of the poly- to mono-unsaturated fatty acid ratio. Finally, the incubation of HCs with low ROS doses induced the release of sedimentable alkaline phosphatase activity (matrix vesicles). How the obtained results fit the in vivo occurring events is discussed.

Hodgkin's disease (HD) is a peculiar type of human malignant lymphoma characterized by a very low frequency of tumor cells, the so called Hodgkin and Reed-Sternberg (H-RS) cells, embedded in a hyperplastic background of non-neoplastic (reactive) cells recruited and activated by H-RS cells-derived cytokines. H-RS cells can be functionally regarded as antigen-presenting cells (APC) able to elicit an intense, but anergic and ineffective, T-cell mediated immune response along with a hyperplastic inflammatory reaction which involves several cell types including T- and B-cells, neutrophils, eosinophils, plasma cells, fibroblasts and stromal cells. In tissues involved by HD, malignant H-RS cells and their reactive neighboring cells are able to cross-talk via a complex network of cytokine- and cell contact-dependent interactions. As a result of such interactions, mediated by specific surface receptors and adhesion molecules on both tumor and non-neoplastic cells, H-RS cells may receive several proliferative and anti-apoptotic signals favoring the cellular expansion and tumor cell survival in HD. The ineffective T-cell immune response elicited by the abnormal APC function of H-RS cells may further contribute to the biologic and clinical progression of HD. Innovative therapeutic strategies aimed at blocking the pathways of dysregulated cellular cross-talk among H-RS cells and bystander reactive cell populations might be beneficial in the treatment of HD patients.

The selenoenzyme phospholipid hydroperoxide glutathione peroxidase (PHGPx, EC 1.11.1.12) is present, in both free and membrane-bound form, in several mammalian tissues. It utilizes thiols such as glutathione to specifically scavenge phospholipid hydroperoxides. The testis exhibits the highest PHGPx-specific activity so far measured, and interest in the presence and function of the enzyme in this tissue has recently grown. Here we report the localization of PHGPx in rat epididymal spermatozoa and its distribution in subfractions obtained by sucrose density gradient centrifugation. Immunochemical evidence and enzymatic activity revealed for the first time that PHGPx is present in sperm heads and tail midpiece mitochondria. The binding of the enzyme to spermatozoa, head, and mitochondria was barely affected by ionic strength or thiols or detergents, as compared to the detachment of PHGPx obtained from testis nuclei. Moreover, we demonstrated that pure PHGPx exhibits a higher thiol-oxidase activity toward isolated epididymal caput protamines than toward protamines from epididymal cauda. These results suggest a role for the enzyme in the maturation of spermatozoa through the metabolism of hydroperoxides and sperm thiol oxidation, in addition to its serving as an antioxidant protector.

In rat testis nuclei the activity of the selenoenzyme phospholipid hydroperoxide glutathione peroxidase (PHGPx, EC 1.11.1.12) is much higher than in other tissues and subcellular compartments, with the sole exception of mitochondria. In nuclei, the bound enzyme is solubilized by DNase I treatment, thus suggesting a binding to chromatin. Treatment with ionic strength releases about 70% of bound PHGPx, suggesting that electrostatic bonds are involved. Immunogold electron microscopy indicates the association of PHGPx with chromatin structures in isolated nuclei. A possible interpretation of these data is a PHGPx protective role against DNA peroxidative damage. Furthermore, in agreement with kinetic and structural information, PHGPx-chromatin binding could suggest an hypothetical thiol oxidase activity toward specific thiol bearing proteins which could substitute for GSH as alternative donor substrates. Such activity could give to the enzyme a new important function which is not only protective but also has a specific regulatory function in chromatin condensation.

The distribution of phospholipid hydroperoxide glutathione peroxidase (PHGPx) in isolated rat testis mitochondria was investigated, using a reverse sucrose density gradient centrifugation procedure for the separation of the inner and outer membranes and the contact sites between the two membranes. The results indicate that PHGPx is largely localized in the contact sites fraction. This finding might therefore suggest that the enzyme has more than just an antioxidant function.

Some aspects of the physiology of chondrocytes from horse articular cartilage were studied, since this animal model can be helpful in understanding arthritic processes. The replicative ability of articular chondrocytes, measured by the incorporation of [3H]thymidine, and their capacity of proteoglycan production, evaluated from the incorporation of [35S] sulfate, are very low. In addition, these cells do not differentiate in vitro as shown by the constant specific activity of alkaline phosphatase measured at different times in culture. Two types of potassium channels were identified by patch clamp experiments in the cell-attached configuration, one characterized by a conductance of 40 pS and the other of 100 pS. No active K+ channels were found at Vpip = 0. It was shown by Fura-2 experiments that the low replicative ability is paralleled by a modest variation of the intracellular calcium concentration after a mitogenic stimulus. 31P NMR experiments, both on slices of whole articular cartilage and on isolated cells, demonstrate that chondrocytes derive their energy mainly from the glycolytic pathway.

SDS-PAGE and Western immunoblot profiles have been determined for different strains of Borrelia burgdorferi. Major proteins of 60 kDa, 41 kDa corresponding to flagellin, 34-36 kDa and 30-31 kDa corresponding to OspB and OspA respectively, and 18-20 kDa corresponding to 'pC' fractions were detected. A "rough" lipopolysaccharide which we called lipooligosaccharide (LOS) of 8-11 kDa appeared to be present, being detected by specific silver staining, as in crude Borrelia lysates as in proteinase K digested Borrelia strains, quite similar in shape among the different strains examined. The LOS reacted in Western blotting with immune anti-B. burgdorferi rabbit serum and also with sera collected from humans affected by Lyme borreliosis. The LOS did not react with sera positive for syphilis or leptospirosis, and their immunological specificity is discussed.