Brain ischemic stroke is one of the leading causes of death in developed countries, including Japan. Controlling the neuroinflammation in the penumbra region with mild ischemia is crucial for treating ischemic stroke. C-C chemokine receptor 5 (CCR5), noted for its functions in the progression of neuroinflammation, is considered a promising drug target. We recently found that three CCR5-regulated matrix metalloproteinases (MMPs) are detected in various cell types, including neurons, microglia, and blood vessel endothelial cells, in the ischemic brain of the photothrombosis model mouse. However, it is still unclear whether CCR5 is expressed in these cell types. This study examined the presence of CCR5 in the photothrombotic brain. Preceding the analysis, we evaluated and improved the photothrombosis induction protocol to obtain equable results with lower toxicity. Rose bengal, used to induce thrombosis to cause an infarction, is radicalized by laser and exhibits pancreatic toxicity. Therefore, we changed the administration route from the abdomen to the jugular vein and reduced the required dose of rose bengal. With this improved protocol, we found that the level of CCR5 protein was increased in neurons, microglia, and blood vessels in the ischemic core, in the infarct brain. The increase in CCR5 levels was sensitive to NSAIDs, especially to cyclooxygenase-2-selective etodolac. CD4, a collaborative membrane receptor for CCR5, was also detected in the migrating microglia. These results suggest that CCR5 is dynamically regulated and play diverse roles during ischemic stroke.

Green tea contains catechins, possessing anti-obesity and anti-oxidative effects, and has been consumed for hundreds of years. Our previous pilot study reported that Kosen-cha improves obesity and the parameters of metabolic syndromes in obese patients, however, the effect of Kosen-cha on obesity is still unclear in pre-obese subjects. The aim of this study was to investigate the effect of Kosen-cha on obesity and related clinical parameters including blood lipid and liver functions in a randomized placebo-controlled, double-blinded study. In total, 54 subjects with body mass index (BMI) of 25–30 were enrolled and randomized to receive either Kosen-cha or a placebo. The subjects drank Kosen-cha or the placebo thrice-daily for 12 weeks. Thereafter, we examined the effect of Kosen-cha on obesity (body weight, BMI, body fat, waist circumference, and visceral fat), lipid metabolism (triglyceride and high- and low-density lipoprotein cholesterol), and serum liver enzymes (aspartate aminotransferase, alanine aminotransferase (ALT), and γ-glutamyl transpeptidase). None of the subjects reported adverse effects from drinking Kosen-cha. Body weight, BMI, body fat, waist circumference, and visceral fat area remained unchanged in both groups. However, the change ratio of ALT significantly reduced between placebo and Kosen-cha groups after 12 weeks (Kosen-cha: −11.1 ± 32.7% vs. placebo: 8.46 ± 23.4%, p = 0.019). These results show that the consumption of Kosen-cha did not significantly improve obesity and may reduce liver enzyme levels in pre-obese Japanese subjects.

Inappropriate polypharmacy increases the risks of adverse drug reactions and hospitalization. Thus, it is important to evaluate the appropriateness of prescriptions in polypharmacy. We designed a checklist based on previous studies and guidelines for pharmacists in our hospital to evaluate the appropriateness of a prescription of multiple medications. The efficacy of checklist-based standardization was evaluated by investigating inpatient medical records and prescriptions. We designed a checklist for pharmacists in our hospital to evaluate the appropriateness of a prescription and reduce the prescription of medications with multiple administrations for all age groups. When patients using more than six medications were admitted, pharmacists assessed the prescriptions of these patients using the checklist. We examined 729 inpatients over the course of 4 months before and after the standardization. The research protocol was approved by the Human Ethical Committee of Showa University, School of Pharmacy. For prescriptions with six or more medications, the total number of suggestions for all patients significantly increased upon implementation of the checklist (50 vs. 21, P < 0.05). Additionally, the number of changes in prescriptions by doctors increased while using the checklist (44 vs. 17, P < 0.05), whereas the rate of changes per suggestions did not change. The most common reason for the increase in prescription suggestions after the standardization was a medication was prescribed to patients despite the absence of symptoms. Our checklist was effective in reducing the prescription of inappropriate medications in patients of all ages.

Increased permeability of vascular endothelial cells in the brain is an underlying cause of stroke, which is associated with high mortality rates worldwide. Vascular permeability is regulated by tight junctions (TJs) formed by claudin family and occludin proteins. In particular, increased vascular permeability is associated with decreased claudin domain-containing 1 (CLDND1) expression, which belongs to the TJs family. We previously reported that myeloid zinc finger 1 (MZF1) acts as an activator of CLDND1 expression by binding to its first intron. Several transcription factors regulate transcription by acting on the promoter regions of target genes. However, transcription factors acting on the promoter of CLDND1 are not completely elucidated. Thus, we focused on the promoter region of human CLDND1 to identify factors that could regulate its transcription. Reporter analysis of CLDND1 promoter region revealed an enhancer in the -742/-734 region with MZF1 and specificity protein 1 (SP1) binding sites. Chromatin immunoprecipitation assays confirmed that both MZF1 and SP1 could bind to CLDND1 enhancer region. MZF1 overexpression significantly increased CLDND1 expression, whereas overexpression of SP1 had no effect. Moreover, the identified enhancer region exhibited stronger transcriptional and binding capacity than the first intron. Thus, CLDND1 expression is more strongly regulated by competitive action of MZF1 and SP1 binding to the promoter-enhancer region than the first intron silencer region. These results provide novel insights for the development of potential therapies and preventive strategies for stroke in the future.

Moesin is a member of the ezrin, radixin and moesin proteins that are involved in the formation and/or maintenance of cortical actin organization through their cross-linking activity between actin filaments and proteins located on the plasma membranes as well as through regulation of small GTPase activities. Microglia are immune cells in the central nervous system. They show dynamic reorganization of the actin cytoskeleton in their process elongation and retraction as well as phagocytosis and migration. They also secrete proinflammatory cytokines such as tumor necrosis factor−α (TNF−α). Moesin is the predominant ezrin, radixin and moesin family protein in microglia. Recently, we found that moesin is involved in microglial activation accompanying morphological changes and reorganization of actin cytoskeleton by using moesin knockout mice in vivo and ex vivo. Here we studied the effects of a small molecule inhibitor specific for ezrin and moesin, NSC305787, on the moesin phosphorylation, phagocytosis, migration, and TNF−α secretion of the primary microglia. NSC305787 at the concentration of 10 μM inhibited the moesin phosphorylation, UDP-induced phagocytosis, ADP-induced migration and lipopolysaccharide-induced TNF−α secretion without effect on cell viability. These results in combination with the previous results using moesin knockout mice suggest the functional importance of moesin in microglial activities.

Lysophosphatidic acid (LPA), a small ubiquitous lipid found in vertebrate and non-vertebrate organisms, mediates diverse biological actions. LPA activates mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase, and low-molecular-weight G-proteins by binding to multiple LPA receptors (LPA_1–6, and GPR87). We previously demonstrated that colonies of A431 cells, a human epidermoid carcinoma cell line, were dispersed by LPA1 and GPR87 activation. This LPA-induced A431 cell dispersal is accompanied by epithelial-mesenchymal transition (EMT) and is believed to contribute to tumor progression. Endoplasmic reticulum (ER) stress has been implicated in tumor progression and growth. A recent study found that activation of the inositol-requiring enzyme 1α/X-Box binding protein 1 pathway promotes tumor progression and EMT in colorectal carcinoma. In addition, another report indicated that ER stress preconditioning using stress inducers promotes transforming growth factor β1-induced EMT and apoptosis in human peritoneal mesothelial cells. To investigate the effect of ER stress preconditioning on LPA-induced cell dispersal, we analyze the crosstalk between LPA-induced and ER stress-induced cellular responses using A431 cells. Interestingly, preconditioning via tunicamycin, an ER stress inducer, inhibited LPA-induced A431 cell dispersal, whereas thapsigargin, another inducer, promoted cell dispersal. Furthermore, western blot analysis illustrated that LPA-induced p38 MAPK phosphorylation was enhanced by thapsigargin pretreatment but not by tunicamycin. These results indicate that ER stress inducers differentially alter LPA-induced A431 cell dispersal by modifying LPA-related signals.

The importance of preventing infectious disease for public health continues to increase, and effective disinfectants are needed to inactive pathogenic microorganisms. Chlorine dioxide (ClO₂) is known as one of the most efficient disinfectants. We studied the inhibitory effect of a novel disinfectant, MA-T, for three species of bacteria (Escherichia coli, Staphylococcus aureus, and Aggregatibacter actinomycetemcomitans). We found that NADH:O₂ oxidoreductase activity (NADH oxidase activity) was markedly decreased in all three species, corresponding to the decrease in colony-forming units following treatment with MA-T. In E. coli, NADH:ubiquinone-1(Q₁) oxidoreductase (NADH-Q₁ dehydrogenase; NDH) activity was decreased following MA-T exposure, indicating that both the NDH-1 and NDH-2 enzymes were targets of this disinfectant. The activity of ubiquinol-1 (Q₁H₂): O₂ oxidoreductase (Q₁H₂ oxidase) also was decreased, indicating that cytochromes bo3 and bd were damaged by MA-T. In S. aureus, NADH-ferricyanide dehydrogenase activity and Q₁H₂ oxidase activity were strongly decreased, suggesting that NDH-2, cytochrome bd, and cytochrome aa3 were targets of MA-T in this species. In A. actinomycetemcomitans, only Q₁H₂ oxidase activity was decreased, indicating that in this species, only cytochrome bd was impaired by MA-T treatment. NADH oxidase activity in membrane vesicles prepared from untreated E. coli was not markedly affected by treatment with MA-T, suggesting that MA-T may attack components of the respiratory chain only in live bacteria (i.e., those possessing a membrane potential), because the membrane vesicles cannot produce the membrane potential.