Aminobisphosphonates, synthetic analogues of endogenous pyrophosphate, have been widely used for the treatment of osteoporosis and bone metastases characterized by excessive osteoclastic bone resorption. Such aminobisphosphonates can be recognized by human γδ T cells having inherent Vγ2Vδ2 T cell receptors (TCRs) circulating in the blood. Here we show that risedronate, one of the most potent aminobisphosphonates, together with 10 U/ml IL-2, strongly expanded both CD8αα-positive and double negative Vγ2Vδ2 T cells among freshly isolated peripheral blood mononuclear cells (PBMCs) from healthy individuals in vitro. The optimal concentration of risedronate was 5 μM and the expanded Vγ2Vδ2 T cells secreted a good amount of IFN-γ as well as small amount of TNF-α but not IL-4. Complete abrogation of IFN-γ production was observed in Vδ2-depleted PBMCs when stimulated with 5 μM of risedronate for 7 days. However, oral administration with risedronate had no effect on the absolute number of Vγ2Vδ2 T cells in PBMCs and risedronate medication for 8 weeks did not prime those γδ T cells for proliferation by in vitro restimulation. Nonetheless, we did observe transient enhancement of IFN-γ production in the supernatant 48 h after the risedronate stimulation among 3 out of 6 patients on either two or four weeks after the medication. These data suggest that orally administered risedronate can commit Vγ2Vδ2-positive γδ T cells to IFN-γ-secreting effectors in vivo.
Polychlorinated dibenzo-p-dioxins belong to a group of widespread environmental endocrine disruptors (EEDs). They are highly stable and have diverse biological activities, especially strong toxicity and carcinogenecity. As a part of our studies on EEDs microquantitation in biological and environmental specimens, the present study developed a simple, sensitive and rapid enzyme-linked immunosorbent assay (ELISA) method for the dioxins with a novel type of labeled antigen, in which two Arg residues were introduced to improve the solubility of highly hydrophobic dioxins in aqueous medium. The assay system was proved to recognize the tetra- and trichlorinated dibenzo-p-dioxins to the same extent, so that the latter of little toxicity could be used as standard antigen. The system could detect the standard antigen in a range of 100 pg∼1,000 ng/mL, which supports usefulness of the system for practical microquantitation of the dioxins in biological and environmental specimens. The dioxins concentrations in soil samples measured by the ELISA showed high correlation (r = 0.978) with those by the conventional gas chromatography / mass spectrometry method. It was concluded that the ELISA system specific for the dioxins is rapid and simple method and will be useful especially for screening of large numbers of samples in terms of dioxins contamination.
Hepatocyte growth factor (HGF) plays a role in development and regeneration of a variety of tissues, whereas, regulatory mechanisms for HGF expression have been not fully understood. We here found that hydrocortisone potentiate HGF expression in vascular endothelial cells. In rat aortic endothelial cells in culture, hydrocortisone alone had no stimulatory effect on HGF production, whereas it largely potentiates the enhancement of HGF production by dibutyryl cyclic AMP (dbcAMP). Likewise, hydrocortisone markedly potentiated the stimulatory effect of phorbol 12-myristate 13-acetate on HGF production in human umbilical vein and pulmonary artery endothelial cells. Hydrocortisone alone did not induce HGF mRNA expression, whereas it augmented HGF mRNA expression induced by dbcAMP in rat aortic endothelial cells, suggesting that the potentiation by hydrocortisone was attributable to transcriptional regulation of HGF gene. In contrast, transforming growth factor-β1, endothelin-1, and nitric oxide suppressed HGF expression in endothelial cells. Previous studies demonstrated that hydrocortisone suppresses HGF expression in non-endothelial cells. Taken together, hydrocortisone bi-directionally regulates HGF expression in cell type-dependent manner and our results provide a unique regulatory mechanism for HGF expression in vascular endothelial cells.
Chelidonine, a tertiary hexahydro-benzophenanthridine alkaloid is an inhibitor of tubulin polymerisation and has been revealed to arrest cells in G2/M. Since enhanced tyrosine kinase (TK) activity is linked to the transition from normal to the immortal malignant phenotype, the effect of 10 mM chelidonine was evaluated on TK activity in normal, transformed and malignant cell lines after 2 hours of exposure. Chelidonine caused a stimulation of TK activity in two normal cell lines (human foreskin fibroblast (Hs27) and normal monkey kidney (NMK)). In contrast, an inhibition of TK activity was observed in transformed human embryonic kidney (Graham 293) and transformed African green monkey kidney (Vero), as well as in human cervical carcinoma (HeLa) and squamous oesophageal carcinoma (WHCO5) cells. Hs27 cells exposed to chelidonine, revealed an increase in TK activity of 1.27-fold (P < 0.05). NMK cells showed a 1.15-fold increase in TK activity. A decrease in TK activity was observed in Graham 293 (0.91-fold) and Vero (0.45-fold) (P < 0.005) cells. In both HeLa and WHCO5 cells, the TK activity was reduced to 0.68-fold (P < 0.05) and 0.56-fold (P < 0.005) respectively. These data, including results from our previous studies, suggest a potential cross talk between the SAPK/JNK and TK signal transduction pathways and a possible differential effect of chelidonine on the phosphorylation status of role players involved in determining the length of G2 arrest in normal versus transformed and malignant cells.
Previous studies have confirmed the presence of corticosteroid receptors including mineralocorticoid (MCR) and glucocorticoid receptors (GCR) in the glia cells of both central and peripheral nervous systems. However, no report has been offered as yet on the detailed localization of these receptors in the specialized Schwann cells associated with mechanoreceptors. Thus, the present study examined the immunolocalization of MCR and GCR in the specialized Schwann cells (terminal and lamellar Schwann cells) associated with the mechanoreceptors in rat periodontal ligament and palatal mucosa by the use of immunohistochemical techniques and confocal laser scanning microscopy. It further attempted to detect the localization of 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD II), a key enzyme for signal transduction via MCR. Immunostaining with antisera against GCR and MCR demonstrated intense immunoreactions in the terminal or lamellar Schwann cells but not nerve fibers associated with the periodontal and palatal mechanoreceptors. A double staining with propidium iodide and either GCR- or MCR-antiserum revealed the intranuclear and cytoplasmic localization of these receptors in the terminal Schwann cells. 11β-HSD II-immunoreactivity was found in the cytoplasm and on the nuclear envelope of the terminal Schwann cells, indicating the co-localization of MCR and 11β-HSD II in them. These findings suggest that the terminal or lamellar Schwann cells are target tissues for corticosteroid hormones. Taken together with previous reports on the functional significance of the corticosteroids, the results indicate that glucocorticoids and mineralocorticoids might be respectively involved in the proliferation and/or differentiation and Na+/K+-homeostasis in the terminal or lamellar Schwann cells associated with the mechanoreceptors.
A histological investigation of the anal submucosal muscle was performed on 73 specimens obtained from 30 elderly Japanese cadavers. On immunohistochemistry, the submucosal muscle (maximum thickness of the muscle layer, 0.3—5.0 mm) was positive for smooth muscle actin and desmin. This smooth muscle was accompanied by abundant elastic fibers, and clearly differed from the rectal muscularis mucosae. It was present around the entire circle of the anal canal wall, except for the mid-dorsal portion, and covered the internal aspect of the internal anal sphincter. The sphincter consistently issued several muscle fibers to the submucosal muscle. The distal end of the rectal muscularis mucosae almost corresponded with the level of the squamous-columnar epithelial junction. The muscularis mucosae either intermingled with (11/30) or did not co-exist with (19/30) the submucosal muscle. The submucosal muscles were usually more sparsely distributed than the internal sphincter, but they sometimes (5/30) included a dense, plate-like part adjacent to the lateral portion of the sphincter. Physiologically, the anal submucosal muscle seemed to provide a cushion for smooth evacuation; however, under pathological conditions, hemorrhoidal venous plexuses developed in this area. For functional preservation during hemorrhoidectomy, restricted treatment of the submucosal layer, based on individualized presurgical evaluations of its morphology, is required.
Both the production of nitric oxide (NO) through the inducible NO synthase (iNOS) pathway and the production of carbon monoxide (CO) through the inducible heme oxygenase (HO-1) pathway have been implicated as major contributors in the process of many pathological inflammatory conditions, however, the exact role of CO and the interaction between NO and CO production systems during inflammatory cytokine-induced myocardial depression are not yet clarified. Using the isolated rat working heart model, the effects of iNOS inhibitor (L-canavanine) and HO inhibitor (zinc protoporphyrin) on inflammatory cytokines (tumor necrosis factor-α and interleukin-1β)-induced cardiac depression as well as the changes in gene expression of NOS and HO were evaluated. Administration of cytokines significantly increased iNOS and HO-1 mRNA expression in the left ventricle and depressed cardiac contraction. L-canavanine attenuated cytokine-induced cardiac depression and increase in HO-1 mRNA expression. Zinc protoporphyrin did not worsen cytokine-induced cardiac depression and increased iNOS mRNA expression. These data suggest that NOS/NO pathway system and HO/CO pathway system are interrelated at the gene expression levels even when those systems are highly promoted. However, the interaction of both systems on cardiac contractility did not make clear and other factors are speculated to be important under excessive production of those gaseous molecules.