Mie Kurosawa

Vizantin Inhibits Endotoxin-Mediated Immune Responses via the TLR 4/MD-2 Complex

Vizantin has immunostimulating properties and anticancer activity. In this study, we investigated the molecular mechanism of immune activation by vizantin. THP-1 cells treated with small interfering RNA for TLR-4 abolished vizantin-induced macrophage activation processes such as chemokine release. In addition, compared with wild-type mice, the release of MIP-1β induced by vizantin in vivo was significantly decreased in TLR-4 knockout mice, but not in TLR-2 knockout mice. Vizantin induced the release of IL-8 when HEK293T cells were transiently cotransfected with TLR-4 and MD-2, but not when they were transfected with TLR-4 or MD-2 alone or with TLR-2 or TLR-2/MD-2. A dipyrromethene boron difluoride–conjugated vizantin colocalized with TLR-4/MD-2, but not with TLR-4 or MD-2 alone. A pull-down assay with vizantin-coated magnetic beads showed that vizantin bound to TLR-4/MD-2 in extracts from HEK293T cells expressing both TLR-4 and MD-2. Furthermore, vizantin blocked the LPS-induced release of TNF-α and IL-1β and inhibited death in mice. We also performed in silico docking simulation analysis of vizantin and MD-2 based on the structure of MD-2 complexed with the LPS antagonist E5564; the results suggested that vizantin could bind to the active pocket of MD-2. Our observations show that vizantin specifically binds to the TLR-4/MD-2 complex and that the vizantin receptor is identical to the LPS receptor. We conclude that vizantin could be an effective adjuvant and a therapeutic agent in the treatment of infectious diseases and the endotoxin shock caused by LPS.

Clostridium perfringens Alpha-Toxin Induces Gm1a Clustering and Trka Phosphorylation in the Host Cell Membrane.

Clostridium perfringens alpha-toxin elicits various immune responses such as the release of cytokines, chemokines, and superoxide via the GM1a/TrkA complex. Alpha-toxin possesses phospholipase C (PLC) hydrolytic activity that contributes to signal transduction in the pathogenesis of gas gangrene. Little is known about the relationship between lipid metabolism and TrkA activation by alpha-toxin. Using live-cell fluorescence microscopy, we monitored transbilayer movement of diacylglycerol (DAG) with the yellow fluorescent protein-tagged C1AB domain of protein kinase C-γ (EYFP-C1AB). DAG accumulated at the marginal region of the plasma membrane in alpha toxin-treated A549 cells, which also exhibited GM1a clustering and TrkA phosphorylation. Annexin V binding assays showed that alpha-toxin induced the exposure of phosphatidylserine on the outer leaflet of the plasma membrane. However, H148G, a variant toxin which binds cell membrane and has no enzymatic activity, did not induce DAG translocation, GM1a clustering, or TrkA phosphorylation. Alpha-toxin also specifically activated endogenous phospholipase Cγ-1 (PLCγ-1), a TrkA adaptor protein, via phosphorylation. U73122, an endogenous PLC inhibitor, and siRNA for PLCγ-1 inhibited the formation of DAG and release of IL-8. GM1a accumulation and TrkA phosphorylation in A549 cells treated with alpha-toxin were also inhibited by U73122. These results suggest that the flip-flop motion of hydrophobic lipids such as DAG leads to the accumulation of GM1a and TrkA. We conclude that the formation of DAG by alpha-toxin itself (first step) and activation of endogenous PLCγ-1 (second step) leads to alterations in membrane dynamics, followed by strong phosphorylation of TrkA.

Streptococcus pyogenes CAMP factor attenuates phagocytic activity of RAW 264.7 cells

Streptococcus pyogenes produces molecules that inhibit the function of human immune system, thus allowing the pathogen to grow and spread in tissues. It is known that S. pyogenes CAMP factor increases erythrocytosis induced by Staphylococcus aureus β-hemolysin. However, the effects of CAMP factor for immune cells are unclear. In this study, we investigated the effects of CAMP factor to macrophages. Western blotting analysis demonstrated that all examined strains expressed CAMP factor protein. In the presence of calcium or magnesium ion, CAMP factor was significantly released in the supernatant. In addition, both culture supernatant from S. pyogenes strain SSI-9 and recombinant CAMP factor dose-dependently induced vacuolation in RAW 264.7 cells, but the culture supernatant from Δcfa isogenic mutant strain did not. CAMP factor formed oligomers in RAW 264.7 cells in a time-dependent manner. CAMP factor suppressed cell proliferation via G2 phase cell cycle arrest without inducing cell death. Furthermore, CAMP factor reduced the uptake of S. pyogenes and phagocytic activity indicator by RAW 264.7 cells. These results suggest that CAMP factor works as a macrophage dysfunction factor. Therefore, we conclude that CAMP factor allows S. pyogenes to escape the host immune system, and contribute to the spread of streptococcal infection.

STO Feeder Cells Are Useful for Propagation of Primarily Cultured Human Deciduous Dental Pulp Cells by Eliminating Contaminating Bacteria and Promoting Cellular Outgrowth.

STO feeder cells, a line established from mouse SIM embryonic fibroblasts, have been frequently used for establishing embryonic stem cells and maintaining them in an undifferentiated state. There are some reports demonstrating that fibroblastic cells have the ability to phagocytose Gram-positive bacterium (e.g., streptococci and staphylococci). In this study, we examined the possibility that STO cells could phagocytose Streptococcus mutans (a bacteria causing tooth decay), which always contaminates cultures of primarily isolated human deciduous dental pulp cells (HDDPCs). Simple cultivation of the primary HDDPCs in the absence of STO cells allowed S. mutans to massively propagate in the medium, thus leading to an opaque medium. In contrast, there was no bacterial contamination in the cultures containing mitomycin C (MMC)-inactivated STO cells. Furthermore, STO cells indicated bacterial phagocytic activity under fluorescent microscopy with the dye pHrodo. Besides removal of contaminating bacteria, STO feeder cells allowed the HDDPCs to spread out. These data suggest that MMC-treated STO cells can be useful for propagation of HDDPCs by eliminating contaminating bacteria and by promoting cellular outgrowth.

Streptococcus pyogenes Phospholipase A2 Induces the Expression of Adhesion Molecules on Human Umbilical Vein Endothelial Cells and Aorta of Mice

The Streptococcus pyogenes phospholipase A2 (SlaA) gene is highly conserved in the M3 serotype of group A S. pyogenes, which often involves hypervirulent clones. However, the role of SlaA in S. pyogenes pathogenesis is unclear. Herein, we report that SlaA induces the expression of intercellular adhesion molecule 1 (ICAM1) and vascular cell adhesion molecule 1 (VCAM1) via the arachidonic acid signaling cascade. Notably, recombinant SlaA induced ICAM1 and VCAM1 expression in human umbilical vein endothelial cells (HUVECs), resulting in enhanced adhesion of human monocytic leukemia (THP-1) cells. However, C134A, a variant enzyme with no enzymatic activity, did not induce such events. In addition, culture supernatants from S. pyogenes SSI-1 enhanced the adhesion of THP-1 cells to HUVECs, but culture supernatants from the ΔslaA isogenic mutant strain had limited effects. Aspirin, a cyclooxygenase 2 inhibitor, prevented the adhesion of THP-1 cells to HUVECs and did not induce ICAM1 and VCAM1 expression in HUVECs treated with SlaA. However, zileuton, a 5-lipoxygenase inhibitor, did not exhibit such effects. Furthermore, pre-administration of aspirin in mice intravenously injected with SlaA attenuated the transcriptional abundance of ICAM1 and VCAM1 in the aorta. These results suggested that SlaA from S. pyogenes stimulates the expression of adhesion molecules in vascular endothelial cells. Thus, SlaA contributes to the inflammation of vascular endothelial cells upon S. pyogenes infection.

Sulfated vizantin induces formation of macrophage extracellular traps: Sulfated vizantin induces ET formation

Vizantin is an insoluble adjuvant that activates macrophages and lymphocytes. Recently, 2,2′,3,3′,4,4′‐hexasulfated‐vizantin (sulfated vizantin), which enables solubilization of vizantin, was developed by the present team. Sulfated vizantin was found to enhance bactericidal activity against multi‐drug resistant Pseudomonas aeruginosa in RAW264.7 cells. In addition, spread of P. aeruginosa was inhibited in RAW264.7 cells treated with sulfated vizantin. When only sulfated vizantin and P. aeruginosa were incubated, sulfated vizantin did not affect growth of P. aeruginosa. Formation of DNA‐based extracellular traps (ETs), a novel defense mechanism in several types of innate immune cells, helps to eliminate pathogens. In the present study, ET‐forming macrophages constituted the majority of immune cells. Sulfated vizantin induced ET formation in RAW264.7 cells, whereas a Ca‐chelating reagent, EDTA, and T‐type calcium channel blocker, tetrandrine, inhibited ET formation and attenuated inhibition of spread of P. aeruginosa in sulfated vizantin‐treated cells. Thus, sulfated vizantin induces ET formation in phagocytic cells in a Ca‐dependent manner, thus preventing spread of P. aeruginosa. Hence, sulfated vizantin may be useful in the management of infectious diseases.

Streptococcus pyogenes CAMP factor promotes calcium ion uptake in RAW264.7 cells: CAMP factor induces Ca2+ uptake in cells

Streptococcus pyogenes is a bacterium that causes systemic diseases such as pharyngitis and toxic shock syndrome. S. pyogenes produces molecules that inhibit the function of the human immune system, thus allowing growth and spread of the pathogen in tissues. It is known that S. pyogenes CAMP factor induces vacuolation in macrophages; however, the mechanism remains unclear. In the current study, the mechanism by which CAMP factor induces vacuolation in macrophages was investigated. CAMP factor was found to induce calcium ion uptake in murine macrophage RAW264.7 cells. In addition, EDTA inhibited calcium ion uptake and vacuolation in the cells. The L‐type voltage‐dependent calcium ion channel blockers nifedipine and verapamil reduced vacuolation. Furthermore, the phosphoinositide 3‐kinase inhibitors LY294002 and wortmannin also inhibited the vacuolation induced by CAMP factor. Fluorescent microscopy revealed that clathrin localized to the vacuoles. These results suggest that the vacuolation is related to calcium ion uptake by RAW264.7 cells via L‐type voltage‐dependent calcium ion channels. Therefore, it was concluded that the vacuoles induced by S. pyogenes CAMP factor in macrophages are clathrin‐dependent endosomes induced by activation of the phosphoinositide 3‐kinase signaling pathway through calcium ion uptake.

Change in masticatory movement according to food size in young Japanese females

The present study aimed to investigate the effect of food size on chewing behavior (number of chewing cycles, cycle duration and displacement of chin movement) from food intake to the terminal swallow. Twenty healthy females were recruited. Food intake behavior was recorded using two digital video cameras and threedimensional kinematic data were collected using three-dimensional image processing software. Multilevel model analysis was applied to describe the fit of the chin marker displacement curves during the chewing cycles. Our results showed that there was a significant tendency for the number of chewing cycles to increase with increasing rice ball weight. However, in contrast, the number of chews per rice weight decreased gradually with increasing weight. The displacement of the chin marker increased from the first to last chewing cycle as the rice ball weight increased. For time-dependent changes in displacement, the differences of displacement in the first cycle were greater than in the last cycle. There were significant differences in the vertical, lateral and anteroposterior displacement curves depending on rice ball weight. Vertical and lateral displacement generally decreased with time, and the curves showed steep slopes from the start to middle points of an intake. This study suggested that change in masticatory movement according to food size may be associated with appropriate eating behavior. Correspondence to: Yuki Nakamura, Division of Pediatric Dentistry, Niigata University Graduate School of Medical and Dental Sciences, Japan, Tel/Fax: +81-25227-2912; E-mail: nakayuki@dent.niigata-u.ac.jp

Comparison of dynamic occlusal contacts during chewing between working and balancing sides

Objectives: Mastication is a crucial function for the elderly, and promotes oral health status, cognitive function and the physical constitution. Most reports about occlusion patterns and occlusal glide of adults have reported the jaw movement at the lower incisal point due to easiness of evaluating masticatory performance. The purpose of this study was to test the hypothesis that dynamic occlusal contact area (OCA) during chewing differ for each tooth on the working vs. the balancing chewing side. Design: In thirteen healthy Japanese females, OCA was estimated with a measurement system combining 3-D tracking of mandibular movements with 3-D digitization of tooth shape. Results: The starting of occlusal contact between teeth at working side and balancing side did not differ significantly. In contrast, ending of occlusal contact of teeth at balancing side were markedly longer than that of teeth at working side at lateral incisor, canine, and first premolar. The dynamic sum of OCAs for all teeth was symmetrical around maximum closed position (MCP) when chewing on the working side. In contrast, the dynamic sum of OCA peaked after MCP when chewing on the balancing side. In working and balancing side, sums of maximum OCA at all posterior teeth accounted for 93%, 86% of sum OCA for all teeth at working and balancing sides, respectively. Conclusion: Our result suggested that the hypothesis that dynamic OCA during chewing differ for each tooth on the working vs. the balancing chewing side was not accepted at molars. Correspondence to: Issei Saitoh, DDS, Ph.D, Division of Pediatric Dentistry, Niigata University Graduate School of Medical and Dental Sciences, 2-5274, Gakkocho-Dori, Chuo-ku, Niigata, 951-8514, Japan, Tel/Fax: +81 – 25 – 227 – 2911 / +81 –25 – 227 – 2911; E-mail: isaito@dent.niigata-u.ac.jp

Oral feeding behavior during a whole meal

Eating is an instinctive behavior and a fundamental oral function for energy intake. However, the changes in the variables involved in intake volume and timedependent changes during a meal have yet to be sufficiently clarified. This study aimed to elucidate the changes in normal food intake behavior during a meal. We recruited 20 healthy females with normal body mass index. Food intake behavior and intake volume were recorded using a three-dimensional motion capture system and electric weighing instrument. We applied multilevel model analysis to describe fitting curves of the variables related to food intake. Correlations of these variables were also estimated. We described time-dependent statistically significant polynomial curves of variables during a meal. The mouthful weight and intake interval were not constant throughout the meal. The mouthful weight was larger at the beginning of the meal than at the end; the intake interval was the shortest at the start of a meal; it gradually increased until the 80% point. Most of the variables were statistically and strongly correlated with one another. The total intake time had a significantly high positive correlation with the number of mouthfuls, mouthful weight, intake interval, total chewing number, and mouthful chewing number; the total intake time had a significantly negative correlation with mouthful weight. We established that in the healthy subjects, food intake behavior changed according to the consumed volume and with time. A small mouthful weight leads to a large number of mouthfuls, thereby extending the mealtime. These findings may be helpful in assessing eating behaviors toward proposing a behavioral strategy to control meal durations and eating rates. Correspondence to: Yuki Nakamura, Division of Pediatric Dentistry, Niigata University Graduate School of Medical and Dental Sciences, Japan, Tel/Fax: +81-25227-2912; E-mail: nakayuki@dent.niigata-u.ac.jp