Masatoshi Kataoka

Cyclosporin A decreases the degradation of type I collagen in rat gingival overgrowth

Cyclosporin A (CsA) is used as an immunosuppressive agent and its prominent side effect is the induction of fibrous gingival overgrowth. The purpose of this study was to investigate the effect of CsA on the type I collagen metabolism in the gingiva of rats fed a powdered diet either containing or lacking CsA. Immunohistochemical analysis revealed that type I collagen was more prevalent in the connective tissue of CsA‐treated gingiva than in those of control rats on days 15, 30, and 55 after the start of feeding. Total RNAs were isolated from mandibular molar gingiva on days 0, 3, 8, 15, 30, and 55. Quantitative analysis of mRNA by reverse transcriptase‐polymerase chain reaction revealed that the CsA‐treated groups showed a gradual decrease in expression of type I collagen and collagenase mRNAs, 0.4% and 18.0% on day 55 compared with those on day 0, respectively. In the control groups, type I collagen and collagenase mRNAs also decreased to 19.7% and 63.0%, respectively, however, both mRNA expressions were significantly lower in the CsA‐treated group than in the controls. An electron microscopic analysis of fibroblasts was performed to count the number of cells with collagen fibrils in the cytoplasm, a marker of phagocytosis of collagen by fibroblasts. The collagen fibrils were detected in 4.7% ± 2.7% and 24.3% ± 13.7% of fibroblasts in the overgrown gingiva treated with CsA rat for 8 days and 30 days, but in 57.0% ± 5.3% and 81.3% ± 9.2% of fibroblasts in the each control group gingiva, respectively. Furthermore, in vitro analysis was performed to measure the phagocytosis of cultured fibroblasts by flow cytometry using collagen‐coated latex beads. Fibroblasts isolated from CsA‐treated gingiva on day 8 and day 30 contained 5.7% ± 0.6% and 9.9% ± 1.5% phagocytic cells, whereas control fibroblasts contained 50.3% ± 5.5% and 33.3% ± 4.9% phagocytic cells, respectively. The inhibition rate of phagocytic activity was similar between in vivo and in vitro assays. These findings suggest that the decrease of the collagen degradation due to the lower phagocytosis and the lower collagenase mRNA expression are closely associated with the increase of type I collagen accumulation in CsA‐treated rat gingiva. J. Cell. Physiol. 182:351–358, 2000.

Interleukin-1α regulates antimicrobial peptide expression in human keratinocytes

Human epidermis and epithelium serve as physiologic barriers to protect against noxious and infectious agents. Contributing to the defense against infection, epithelial cells express antimicrobial peptides (AMPs). The expression of AMPs in keratinocytes is generally regulated directly by bacteria and indirectly by proinflammatory cytokines. Bacteria may also regulate AMP expression by inducing keratinocyte expression of the autonomous proinflammatory cytokine, interleukin‐1α (IL‐1α). To test the hypothesis that AMP expression may be regulated by cell autonomous cytokines, we investigated the effect of IL‐1α on the expression of AMPs in human keratinocytes (HaCaT cells) by microarray, northern blot, reverse transcriptase (RT)–PCR and western blot analyses. IL‐1α increased expression of mRNA in a dose‐ and time‐dependent manner specific for lipocalin 2, S100A8, S100A9 and secretory leukocyte protease inhibitor (SLPI) more than twofold relative to nonstimulated cells (control), and slightly upregulated S100A7 and β‐defensin‐2. Furthermore, the expression of lipocalin 2, S100A7, S100A8, S100A9 and SLPI proteins were upregulated by IL‐1α. On the other hand, HaCaT cells expressed mRNA specific for other AMPs, including cystatin 3, adrenomedullin, RNase‐7 and mucin 5, which were unaffected by IL‐1α treatment. These results suggest that the autonomous keratinocyte cytokine, IL‐1α, selectively upregulates the expression of AMPs which may modulate innate epithelial cell immunity in skin and mucosa.

Ca2+-induced permeability transition can be observed even in yeast mitochondria under optimized experimental conditions

Yeast mitochondria have generally been believed not to undergo the permeability transition (PT) by the accumulation of Ca(2+) within the mitochondrial matrix, unlike mammalian mitochondria. However, the reason why the yeast PT is not induced by Ca(2+) has remained obscure. In this study, we examined in detail the effects of Ca(2+) on yeast mitochondria under various conditions. As a result, we discovered that the PT could be induced even in yeast mitochondria by externally added Ca(2+) under optimized experimental conditions. The 2 essential parameters for proper observation of the PT-inducing effects of Ca(2+) were the concentrations of the respiratory substrate and that of inorganic phosphate (Pi) in the incubation medium. The yeast mitochondrial PT induced by Ca(2+) was found to be insensitive to cyclosporin A and suppressed in the presence of a high concentration of Pi. Furthermore, when the PT was induced in yeast mitochondria by Ca(2+), the release of cytochrome c from mitochondria was also observed.

Calprotectin expression in human monocytes: induction by porphyromonas gingivalis lipopolysaccharide, tumor necrosis factor-alpha, and interleukin-1beta.

BACKGROUND Calprotectin is a major cytosolic protein of monocytes and granulocytes. It is increased in inflammatory tissues and is detected at high levels in the gingival crevicular fluid (GCF) of periodontitis patients. We previously reported that lipopolysaccharide of Porphyromonas gingivalis (P-LPS) and cytokines induced the release of calprotectin from monocytes isolated from human peripheral blood. The mechanisms of calprotectin expression and presence of its regulation factors in periodontal disease are unknown. On the other hand, P-LPS and cytokines are significant etiologic factors in the initiation and progression of periodontal diseases. In this study, we investigated the expression and production of calprotectin from human monocytes by examining the effects of lipopolysaccharide of P-LPS, tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β). METHODS Monocytes were isolated from the peripheral blood of healthy donors and cultured in the presence or absence of P-LPS, TNF-α, or IL-1β. The expressions of calprotectin mRNAs (MRP8 and MRP14) were detected by Northern blotting. The contents of calprotectin in the cells and medium fractions were determined by enzyme-linked immunosorbent assay (ELISA). The DNA binding activity of C/EBPα, a transcription factor of MRP14, was assessed by electrophoretic mobility shift DNA-binding assay (EMSA). RESULTS P-LPS, TNF-α, and IL-1β induced MRP8/14 mRNAs and calprotectin production in monocytes. These factors also induced DNA CEBPα binding activity in monocytes. P-LPS increased MRP14 mRNA expression in monocytes to the maximum level, about two times the control level after 24 hours treatment, but did not enhance the basal level of MRP8. When the effects of TNF-α and IL-1β on those mRNAs were investigated, both MRP8 and MRP14 significantly increased to about 2- and 2.5-fold the control level, respectively. Increases of MRP8/14 mRNA expression were followed by their protein production at about 2-fold the basal amount. DNA binding activity of C/EBPα was increased in P-LPS, TNF-α, and IL-1β-treated monocytes. CONCLUSIONS These results demonstrate that P-LPS, TNF-α, and IL-1β induce calprotectin production from human monocytes and that this production is associated with the activation of DNA C/EBPα binding complex. J Periodontol 2005;76:437-442.

Effect of Porphyromonas gingivalis Lipopolysaccharide, Tumor Necrosis Factor-α, and Interleukin-1β on calprotectin release in human monocytes

BACKGROUND Calprotection is a major cytosolic protein of monocytes, granulocytes, and epithelial cells. It is known that calprotectin is released in inflammatory tissues and detected in gingival crevicular fluid (GCF) of periodontitis patients at high levels. The origin of calprotectin in GCF and its regulation in periodontal disease are unknown. In this study, we investigated the distribution of calprotectin in gingiva tissues with inflammation and the induction of calprotectin release from human monocytes by lipopolysaccharide of Porphyromonas gingivalis (P-LPS), tumor necrosis factor-α (TNF-α), or interleukin -1β(IL-1β) Methods: Gingival tissues were obtained from a healthy donor and a periodontitis patient and calprotectin in gingival tissues was examined by immunohistochemical staining. Monocytes were isolated from the peripheral blood of healthy donors and cultured with P-LPS, TNF-α for 30 minutes to 4 hours. The content of calprotectin in the cell and medium fractions was determined by enzyme-linked immunosorbent assay (ELISA) Results: Calprotectin was markedly detected at the epithelial and adjacent connective tissue with many inflammatory cells in the gingival tissue from the periodontitis patient. P-LPS increased calprotectin release from monocytes to the maximum level after 30 minutes of treatment and its level was elevated to about 2- to 3-fold of the control level in a dose-dependent manner (1 to 1,000 ng/ml). When the effect of TNF-α and IL-Iβ on calprotectin release was investigated, calprotectin release significantly increased to about 2.2- and 1.5-fold that of the control level, respectively. CONCLUSION These results demonstrate that calprotectin release from monocytes is induced by P-LPS, TNF-α, and IL-lβ, which in turn, cause and aggravate periodontal disease. J Periodontol 2003;74:1719-1724.

Rapid and Highly Sensitive Detection of Malaria-Infected Erythrocytes Using a Cell Microarray Chip

Background Malaria is one of the major human infectious diseases in many endemic countries. For prevention of the spread of malaria, it is necessary to develop an early, sensitive, accurate and conventional diagnosis system. Methods and Findings A cell microarray chip was used to detect for malaria-infected erythrocytes. The chip, with 20,944 microchambers (105 µm width and 50 µm depth), was made from polystyrene, and the formation of monolayers of erythrocytes in the microchambers was observed. Cultured Plasmodium falciparum strain 3D7 was used to examine the potential of the cell microarray chip for malaria diagnosis. An erythrocyte suspension in a nuclear staining dye, SYTO 59, was dispersed on the chip surface, followed by 10 min standing to allow the erythrocytes to settle down into the microchambers. About 130 erythrocytes were accommodated in each microchamber, there being over 2,700,000 erythrocytes in total on a chip. A microarray scanner was employed to detect any fluorescence-positive erythrocytes within 5 min, and 0.0001% parasitemia could be detected. To examine the contamination by leukocytes of purified erythrocytes from human blood, 20 µl of whole blood was mixed with 10 ml of RPMI 1640, and the mixture was passed through a leukocyte isolation filter. The eluted portion was centrifuged at 1,000×g for 2 min, and the pellet was dispersed in 1.0 ml of medium. SYTO 59 was added to the erythrocyte suspension, followed by analysis on a cell microarray chip. Similar accommodation of cells in the microchambers was observed. The number of contaminating leukocytes was less than 1 on a cell microarray chip. Conclusion The potential of the cell microarray chip for the detection of malaria-infected erythrocytes was shown, it offering 10–100 times higher sensitivity than that of conventional light microscopy and easy operation in 15 min with purified erythrocytes.

Classification of FABP isoforms and tissues based on quantitative evaluation of transcript levels of these isoforms in various rat tissues.

In mammals, 10 isoforms of fatty acid-binding protein (FABP) are expressed in various tissues. To understand the role of multiple FABP isoforms, we have quantitatively examined the transcript levels of individual FABP isoforms in each of various tissues by Northern blotting using synthesized RNAs corresponding to the mRNA of each isoform as external standards. As a result, absolute transcript levels of individual FABP isoforms expressed in each tissue were successfully determined. The 10 FABP isoforms were classified into three categories: (1) isoforms FABP7 and 12 were not markedly expressed in any tissue examined; (2) isoforms showing certain transcript levels in multiple tissues; and (3) isoforms FABP6, 8, and 9, expressed at certain levels in one particular tissue. Based on the expression profiles of the isoforms, individual tissues were also classified into three groups: (1) tissues in which high-level expression of FABP isoforms was not observed, (2) tissues in which multiple FABP isoforms were expressed at certain levels, and (3) tissues in which a single FABP isoform was dominantly expressed at a certain level. These results give a better understanding of the meaning of the presence of multiple FABP isoforms in mammals.

Regulation of antimicrobial peptide expression in human gingival keratinocytes by interleukin-1α.

In the oral cavity, mucosal keratinocytes resist bacterial infection, in part, by producing broad-spectrum antimicrobial peptides (AMPs) including defensin, adrenomedullin and calprotectin. Epidermal keratinocyte expression of many AMPs increases in response to interleukin-1α (IL-1α). IL-1α is produced by epidermal keratinocytes and regulates cell differentiation. To better understand innate immunity in the oral cavity, we sought to determine how IL-1α might regulate expression of AMPs by human gingival keratinocytes (HGKs) using DNA microarray and Western blot analyses. HGKs from three subjects expressed eleven AMPs, including S100A7, S100A8, S100A9, S100A12, secretory leucocyte protease inhibitor, lipocalin 2 (LCN2), cystatin C and β-defensin 2. Of the expressed AMPs, S100A7, S100A12 and LCN2 were up-regulated by IL-1α (inducible AMPs); the other AMPs were considered to be constitutive. Human gingival keratinocytes, therefore, express constitutive and IL-1α-inducible AMPs to provide a rapid and robust innate response to microbial infection.

Differential Permeabilization Effects of Ca2+ and Valinomycin on the Inner and Outer Mitochondrial Membranes as Revealed by Proteomics Analysis of Proteins Released from Mitochondria

It is well established that cytochrome c is released from mitochondria when the permeability transition (PT) of this organelle is induced by Ca2+. Our previous study showed that valinomycin also caused the release of cytochrome c from mitochondria but without inducing this PT (Shinohara, Y., Almofti, M. R., Yamamoto, T., Ishida, T., Kita, F., Kanzaki, H., Ohnishi, M., Yamashita, K., Shimizu, S., and Terada, H. (2002) Permeability transition-independent release of mitochondrial cytochrome c induced by valinomycin. Eur. J. Biochem. 269, 5224–5230). These results indicate that cytochrome c may be released from mitochondria with or without the induction of PT. In the present study, we examined the protein species released from valinomycin- and Ca2+-treated mitochondria by LC-MS/MS analysis. As a result, the proteins located in the intermembrane space were found to be specifically released from valinomycin-treated mitochondria, whereas those in the intermembrane space and in the matrix were released from Ca2+-treated mitochondria. These results were confirmed by Western analysis. Furthermore to examine how the protein release occurred, we examined the correlation between the species of released proteins and those of the abundant proteins in mitochondria. Consequently most of the proteins released from mitochondria treated with either agent were highly expressed proteins in mitochondria, indicating that the release occurred not selectively but in a manner dependent on the concentration of the proteins. Based on these results, the permeabilization effects of Ca2+ and valinomycin on the inner and outer mitochondrial membranes are discussed.

High-turnover osteoporosis is induced by cyclosporin A in rats.

Cyclosporin A (CsA) is used widely as an immunosuppressive agent, but it induces osteoporosis as a prominent side effect. To elucidate the mechanisms involved in CsA-induced osteoporosis, the effects of CsA on bone metabolism were investigated in a rat experimental model. Fifteen-day-old rats were fed a powdered diet containing or lacking CsA for 8–30 days. Analysis was performed by micro-computed tomography (μCT) and light microscopy to examine histomorphometric changes in rat tibiae on days 8, 16, and 30. Plasma parathyroid hormone (PTH) and osteocalcin (OCN) levels were determined by enzyme-linked immunosorbent assay (ELISA) on days 8, 16, and 30. The expression of OCN, osteopontin (OPN), and cathepsin K mRNAs in tibial bone marrow was examined by Northern blot analysis on days 8 and 16. Although no significant differences were observed in tibial length during the experimental periods, or in histomorphometric parameters on day 8, an apparent decrease in bone volume was observed in the CsA-treated group after day 16. Histologic analysis showed that the number of osteoblasts and osteoclasts on the surface of trabecular bone in the CsA-treated group had increased significantly on day 16. Plasma PTH and OCN levels in CsA-treated rats were significantly higher than those in control animals on day 8. Northern blot analysis revealed that the CsA-treated group showed an increase in the expression of OCN, OPN, and cathepsin K mRNAs on day 8 compared with the controls. These findings suggest that bone resorption in CsA-treated rats is induced by high-turnover osteoporosis and that bone remodeling activity may be activated by PTH.