Primary Sjögren's syndrome (pSS) is an autoimmune disorder characterized by lymphocytic infiltration of the salivary glands. Most of the infiltrating cells are T cells, but other features of the disease include polyclonal B-cell activation, systemic production of autoantibodies, and increased risk of developing B-cell non-Hodgkin's lymphoma. Recently, a new tumour necrosis factor, the B-cell activating factor (BAFF; also known as BLyS), has been implicated in the polyclonal activation of B cells. Using immunohistochemistry, this study evaluated BAFF expression in labial salivary gland biopsies from 14 patients with pSS, 14 normal controls, and two patients with sarcoidosis. Labial salivary gland samples from seven patients with pSS, seven controls, and one patient with sarcoidosis were double-stained using indirect immunofluorescence. RT-PCR analysis was also performed on lip biopsy samples from two patients and two controls. In all 14 pSS specimens, infiltrating inflammatory cells strongly expressed BAFF protein, as did some ductal epithelial cells, but acinar cells were negative. Some B cells were present in the vicinity of the BAFF-positive cells. In the 14 normal labial salivary glands, some ductal cells were moderately positive, but acinar cells were negative. In the labial salivary glands from the two patients with sarcoidosis, infiltrating lymphocytes were not stained. BAFF mRNA expression was confirmed by RT-PCR in salivary glands from pSS patients. Double immunofluorescence revealed T cells and macrophages to be the main cell types expressing BAFF in salivary glands from pSS patients. In conclusion, BAFF may be expressed by T cells at the site of autoimmune damage and could play a role in the pathogenesis of pSS, particularly by triggering the activation of self-antigen-driven autoimmune B cells.
Summary. The new members of the tumour necrosis factor (TNF) receptor-ligand family, receptor activator of nuclear factor-jB ligand (RANKL) and its receptor RANK, play a crucial role in osteoclast differentiation and activation. An increased expression of RANKL and/or RANK may be involved in the excessive bone resorption observed in multiple myeloma (MM). We used immunohistochemistry to study RANK and RANKL expression in bone marrow (BM) biopsies obtained at diagnosis in 15 MM patients, six patients with monoclonal gammopathy of undetermined significance (MGUS) and 10 normal BM biopsies. Plasma cells were not labelled with anti-RANKL or anti-RANK antibodies. In all biopsies, RANKL was expressed in endosteal bone surface, around vessels and in cells characterized by cytoplasmic expansions. These last cells did not express CD45 and were vimentin positive, corresponding to bone marrow stromal cells. Numerous stromal cells expressed RANKL in MM and MGUS specimens, with a greater expression in MM than in MGUS. Very few cells were stained with anti-RANKL in normal BM specimens. With the anti-RANK antibody, small mononuclear cells in the bone microenvironment were positive and were identified as erythroblast cells. In conclusion, we showed that RANKL was expressed in reticular stromal cells, with a greater intensity in myeloma specimens. These results suggest that RANKL overexpressed by bone marrow stromal cells may contribute to the high rate of bone resorption observed in MM.
This study was designed to determine the effects of PPARalpha lack on cardiac mechanical performance and to identify potential intracellular mechanisms linking PPARalpha pathway deficiency to cardiac contractile dysfunction. Echocardiography, ex vivo papillary muscle assays, and in vitro motility assays were used to assess global, intrinsic ventricular muscle performance and myosin mechanical properties, respectively, in PPARalpha(-/-) and age-matched wild-type mice. Three-nitrotyrosine formation and 4-hydroxy-2-nonenal protein-adducts, both markers of oxidative damage, were analyzed by Western blot analysis and immunolabeling. Radical scavenging capacity was analyzed by measuring protein levels and/or activities of the main antioxidant enzymes, including catalase, glutathione peroxidase, and manganese and copper-zinc superoxide dismutases. Echocardiographic left ventricular fractional shortening in PPARalpha(-/-) was 16% lower than that in wild-type. Ex vivo left ventricular papillary muscle exhibited reduced shortening velocity and isometric tension (three- and twofold, respectively). In vitro myosin-based velocity was approximately 20% slower in PPARalpha(-/-), indicating that myosin itself was involved in the contractile dysfunction. Staining of 3-nitrotyrosine was more pronounced in PPARalpha(-/-), and myosin heavy chain was the main nitrated protein. Formation of 3-nitrotyrosine myosin heavy chain was twofold higher in PPARalpha(-/-) and 4-hydroxy-2-nonenal protein-adducts were threefold higher. The expression and activity of manganese superoxide dismutase were respectively 33% and 50% lower in PPARalpha(-/-), with no changes in copper-zinc superoxide dismutase, catalase, or glutathione peroxidase. These findings demonstrate that PPARalpha pathway deficiency impairs cardiac function and also identify oxidative damage to myosin as a link between PPARalpha deficiency and contractile dysfunction.
The p38 family of MAP kinases (p38-MAPKs) is involved in regulating the proliferation, survival, and migration of various cancer cells. The present study has investigated the expression, subcellular localization, phosphorylation, and activity of p38-MAPKs in normal and tumoural human thyroid tissues and in thyroid cell lines. The expression and nucleo-cytosolic compartmentalization of the alpha-isoform of p38-MAPKs (p38alpha-MAPK) were studied by western blotting in the WRO and B-CPAP cell lines, which are derived from human follicular and papillary thyroid carcinomas, respectively, and in the non-transformed rat thyroid cell lines FRTL-5 and PCCL3. Immunohistochemistry was used to study the expression and subcellular localization of p38alpha-MAPK, and of the phosphorylated forms of p38-MAPKs (P-p38-MAPKs) in human toxic adenomas (TAs), follicular adenomas (FAs), papillary thyroid carcinomas (PTCs), and follicular thyroid carcinomas (FTCs). The activity of p38-MAPKs in PTCs and FTCs was revealed by immunohistochemical detection of their typical phosphorylated substrate, MAPK-activated protein kinase 2/3 (MK2/3). p38alpha-MAPK was expressed in all cell lines and this expression was restricted to the cytosolic compartment. p38 MAPK activity was involved in regulating DNA synthesis in B-CPAP cells. p38alpha-MAPK and P-p38-MAPKs were strongly expressed in PTC and FTC cells, although only in the cytoplasm, whereas they were only very weakly expressed in FA cells, and absent in adjacent normal tissues. They were also expressed at a high level in TAs, but they were found in both nucleus and cytoplasm. Finally, phospho-MK2/3 immunostaining followed very similar patterns to those of p38alpha-MAPK and P-p38-MAPKs in PTCs and FTCs. Taken together, these results show for the first time that the p38-MAPK signalling cascade is functional in two types of differentiated carcinoma of the thyroid. The observation that p38-MAPK hyper-expression occurs in FTC, but not in FA, may provide an additional diagnostic tool for malignancy in some thyroid nodules.
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