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Previously, we reported that glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a binding partner of prolyl oligopeptidase (POP) in neuroblastoma NB-1 cells and that the POP inhibitor, SUAM-14746, inhibits cytosine arabinoside (Ara-C)-induced nuclear translocation of GAPDH and protects against Ara-C cytotoxicity. To carry out a more in-depth analysis of the interaction between POP and GAPDH, we generated POP-KO NB-1 cells and compared the nuclear translocation of GAPDH after Ara-C with or without SUAM-14746 treatment to wild-type NB-1 cells by western blotting and fluorescence immunostaining. Ara-C did not induce the nuclear translocation of GAPDH and SUAM-14746 did not protect against Ara-C cytotoxicity in POP-KO cells. These results indicate that the anticancer effects of Ara-C not only include the commonly known antimetabolic effects, but also the induction of cell death by nuclear transfer of GAPDH through interaction with POP.

Background: Undergraduate clinical training is a useful program for dental students to learn clinical skills. The aim of this study was to investigate whether dental students who had undergone clinical training understood the clinical significance of dental treatment and its connection to systemic health.Methods: An awareness survey was conducted in dentistry students both before and after undergraduate clinical training. A total of 42 dental students were recruited before clinical training, and 32 dental students responded to the survey. In addition, all of the 42 dental students responded to the survey after clinical training. A total of 53 medical students were recruited as control subjects. Differences between the 2 groups were analyzed using Fisher's exact test.Results: Before clinical training, the percentage of dental students who recognized that periodontal treatment has positive effects on systemic diseases was higher than that of the control. After clinical training, a higher percentage of dental students recognized the positive effects of periodontal treatment on systemic health. A higher percentage of dental students before clinical training recognized the positive effects of prosthetic treatment on geriatric conditions; however, after clinical training no significant differences were found between the dental students and the control when surveyed about the positive effects of periodontal and prosthetic treatment on geriatric conditions.Conclusion: Undergraduate clinical training is useful for improving the students’ understanding of the clinical significance of dental treatment. The results of these surveys suggest that dental education supported by experience contributes to an appreciation of the role of dental treatment.

Aim: To improve the prognosis of patients with heart failure, risk stratification in their early stage is important. We assessed whether the change in transmitral flow (TMF) velocity pattern during preload augmentation can predict future hemodynamic worsening in early-stage heart failure patients with impaired relaxation TMF pattern. Methods: We designed a prospective cohort study that included 155 consecutive patients with impaired relaxation (IR) pattern at rest. Preload stress echocardiography was achieved using leg positive pressure (LPP), and changes of TMF pattern during the LPP was observed during baseline echocardiographic examination. The patients whose TMF pattern developed to pseudonormal (PN) pattern throughout the study period were classified into the change to PN group, and patients whose TMF pattern stayed in IR pattern were classified into the stay in IR group. Results: The median follow-up period was 17 months. The average age was 68 ± 11 years old, and 97 patients (63%) were male. Among 155 patients, 27 were classified into the change to PN group. A Cox proportional hazard analysis confirmed that the change in the peak atrial systolic TMF velocity during the LPP (ΔA, hazard ratio = 0.58 per 1SD; 95% CI = 0.39 - 0.88, P = 0.010) was the powerful independent predictor of change into PN pattern. Kaplan-Meier analysis revealed that the patients with ΔA ≤ -7 cm/s had more likely to develop into PN pattern than patients with ΔA > -7 cm/s (P = 0.001). Conclusions: Evaluation of a response in TMF during the LPP might provide an incremental diagnostic value to detect future overt heart failure in patients with early-stage heart failure.

The culprit lesion of acute myocardial infarction could be predicted by electrocardiogram findings. However, we experienced some cases with coronary angiographic finding in the area of ST-T elevation that was different from that predicted. The lambda-like J wave could be caused by ischemia although the mechanism has not been fully elucidated. We report a case of acute myocardial infarction that showed discrepancy between ST-T elevation with lambda-like ischemic J wave in a broad area and coronary angiographical finding of diagonal branch occlusion. J. Med. Invest. 66 : 185-187, February, 2019.

Polymerase chain reaction (PCR) is an essential diagnostic method for highly sensitive detection of Plasmodium-infected erythrocytes in patients with malaria. This study compared the performance of filter papers used for the preparation of dried blood spots (DBS) in detecting Plasmodium by PCR. Whole blood spiked with P. falciparum-infected erythrocytes to obtain samples with various levels of parasitemia were applied to Whatman 3MM Chr papers, FTA Cards, or FTA Elute Cards to prepare the DBS. DNA was purified from the DBS using a DNA purification kit and used as the template for nested PCR. In probit analysis, the estimated limit of detection (LoD) was 5.5 parasites/μL blood for Whatman 3MM Chr papers and FTA Cards and 1.6 parasites/μL blood for the FTA Elute Card. This result suggested that the DBS prepared on an FTA Elute Card yield the best template DNA for subsequent high-sensitivity PCR-based detection of P. falciparum-infected erythrocytes. This finding can help improve the accuracy of malarial diagnostic tests.

Gan-Lu-Yin (GLY), a traditional Chinese herbal medicine, shows therapeutic effects on periodontitis, but that mechanism is not well known. This study aims to clarify the precise mechanism by investigating the inhibitory effects of GLY extracts on osteoclastogenesis in vitro and on bone resorption in periodontitis in vivo. RAW264.7 cells are cultured with soluble receptor activator of nuclear factor-kappa B (sRANKL) and GLY extracts (0.01&ndash, 1.0 mg/mL), and stained for tartrate-resistant acid phosphatase (TRAP) to evaluate osteoclast differentiation. Experimental periodontitis is induced by placing a nylon ligature around the second maxillary molar in rats, and rats are administered GLY extracts (60 mg/kg) daily for 20 days. Their maxillae are collected on day 4 and 20, and the levels of alveolar bone resorption and osteoclast differentiation are estimated using micro-computed tomography (CT) and histological analysis, respectively. In RAW264.7 cells, GLY extracts significantly inhibit sRANKL-induced osteoclast differentiation at a concentration of more than 0.05 mg/mL. In experimental periodontitis, administering GLY extracts significantly decreases the number of TRAP-positive osteoclasts in the alveolar bone on day 4, and significantly inhibits the ligature-induced bone resorption on day 20. These results show that GLY extracts suppress bone resorption by inhibiting osteoclast differentiation in experimental periodontitis, suggesting that GLY extracts are potentially useful for oral care in periodontitis.

The mechanisms for supplying octanoic acid for ghrelin acylation in X/A-like cells are incompletely understood. We found that long-chain fatty acids were incorporated at a higher rate in the ghrelin-producing cell line MGN3-1 than in MIN6 cells, in part due to higher expression level of long-chain fatty acyl-CoA synthetase family member 1 (Acsl1). Inhibition of ACSLs by triacsin C profoundly suppressed acylated ghrelin production. These results suggest that high incorporation of long-chain fatty acids into the ghrelin-producing cells plays a role in the supply of octanoic acid for ghrelin acylation.

Background The aim of this study was to assess the role of clinically available vascular function tests as predictors of cardiovascular events and decline in kidney function. Methods and results One hundred and fourteen patients who had at least 2 cardiovascular risk factors were recruited for vascular function assessment including ankle-brachial blood pressure index (ABI), brachial-ankle pulse wave velocity (baPWV), cardio-ankle vascular index (CAVI) and flow-mediated vasodilatation (%FMD). During a median period of 51 months, 35 patients reached the primary endpoint (29 cardiovascular events and 6 cardiac deaths), and 30 patients reached the secondary endpoint (decline in kidney function: defined as a 5% per year decline of estimated glomerular filtration rate). In sequential Cox models, a model on the basis of the Framingham risk score, hemoglobin, and high-sensitivity C-reactive protein (chi-squared, 16.6) was improved by the ABI (chi-squared: 21.5; P=0.047). The baPWV (hazard ratio: 1.42 per 1 SD increase; P=0.025) and the CAVI (hazard ratio: 1.52 per 1 SD increase; P=0.040) were associated with the secondary endpoint. The %FMD was only slightly associated with the primary and secondary endpoints. Conclusions Both ABI and baPWV are significantly associated with future cardiovascular events in high-risk patients with cardiovascular disease. The predictive capabilities of these parameters are greater than that of other parameters in this cohort.

Hollow titania microparticles were prepared using the bio-templating method with wet yeast cells as templates. Wet yeast cells were used to eliminate fixation or drying of as-cultured cells, and titania shells were formed on the cells via the sol–gel method with tetraisopropyl titanate (TTIP). When the citric acid was used to stabilize TTIP and catalyze TTIP hydrolysis, hollow titania microparticles with well-defined yeast cell morphologies were successfully synthesized. The diameter of the obtained particles was approximately 1.3 μm, and the crystal structure was anatase. The specific surface area and average pore diameter were 41.5 m2/g and 9.6 nm, respectively. Titania nanoparticles generated by homogeneous nucleation were rarely observed over a wide range of water concentrations, indicating that reaction on the biological cell surface, rather than monomer diffusion toward the surface, was rate-limiting. A significant advantage of our method is that the use of dry cultured cells is not required, in contrast to methods using powdered biological cell templates.

Objective YKL-40 is a chitin-binding glycoprotein, the level of which increases in inflammatory diseases, diabetes mellitus (DM), cardiovascular diseases, and tumors. Gingival crevicular fluid (GCF) contains many proteins and markers of periodontitis. The purpose of this study was to investigate YKL-40 level in GCF from patients with periodontitis and DM and the association between YKL-40 level and chronic periodontitis (CP) or DM. Subjects and Methods The subjects were 121 patients with DM, CP, DM and periodontitis (DM-P), and healthy subjects (H). GCF was collected using paper strips after the sites for GCF collection were clinically evaluated for probing depth (PD), gingival index (GI), and bleeding on probing (BOP). YKL-40 in GCF was identified by Western blotting, and its level was determined by ELISA. Results YKL-40 was contained in GCF samples from H, DM, CP, and DM-P sites, and its levels (amount and concentration) in CP and DM-P were significantly higher than those in H and DM. GCF YKL-40 level significantly correlated with PD and GI, and its level in BOP-positive sites was significantly higher than that in BOP-negative ones. Conclusions GCF YKL-40 level was elevated in periodontitis, but not DM. YKL-40 in GCF may be an inflammatory marker for periodontitis.

Oral epithelial cells produce antimicrobial peptides (AMPs) to prevent microbial infection. Calprotectin (S100A8/S100A9) is one of these AMPs in oral epithelial cells, the expression of which is up-regulated by interleukin-1α (IL-1α). Hangeshashinto (HST) is a traditional Japanese herbal medicine that has anti-inflammatory effects. The purpose of this study was to investigate the effect of HST on the expression of calprotectin through the regulation of IL-1α in oral epithelial cells. Human oral epithelial cells (TR146) were cultured with HST in the presence or absence of anti-IL-1α antibody or IL-1 receptor antagonist, or with six major components of HST (3,4-dihydroxybenzaldehyde, baicalin, ginsenoside Rb1, glycyrrhizin, oleanolic acid and berberine). The expression of S100A8, S100A9, other AMPs and cytokine mRNAs was examined by RT-PCR and quantitative real-time PCR. Calprotectin expression and IL-1α secretion were investigated by ELISA. HST (6 μg/ml) increased the expression of S100A8/S100A9 mRNAs and calprotectin protein, and also up-regulated β-defensin 2 (DEFB4) and S100A7 expression. The expression of IL-1α mRNA and its protein was slightly but significantly increased by HST. A neutralizing antibody against IL-1α and IL-1 receptor antagonist inhibited HST-up-regulated S100A8/S100A9 mRNA expression. Although 3,4-dihydroxybenzaldehyde, baicalin and ginsenoside Rb1 as HST components increased S100A8/S100A9 expression, oleanolic acid and berberine decreased their expression. These results suggest that HST increases the expression of calprotectin, DEFB4 and S100A7 in oral epithelial cells. In response to HST, up-regulation of calprotectin expression may be partially induced via IL-1α.

Diabetes mellitus (DM) is a risk factor for periodontal diseases and may exacerbate the progression of the pathogenesis of periodontitis. Advanced glycation end-products (AGEs) cause DM complications relative to levels of glycemic control and larger amounts accumulate in the periodontal tissues of patients with periodontitis and DM. In the present study, we investigated the effects of AGEs on the expression of inflammation-related factors in human gingival fibroblasts (HGFs) to elucidate the impact of AGEs on DM-associated periodontitis.HGFs were cultured with or without AGEs. Cell viability was examined, and RNA and protein fractions were isolated from AGE-treated cells. The expression of interleukin (IL)-6, intercellular adhesion molecule-1 (ICAM-1), and the receptor for AGE (RAGE) was investigated using reverse transcription-polymerase chain reaction, quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay, and reactive oxygen species activity was measured using a kit with 2',7'-dichlorofluorescin diacetate. Human monocytic cells (THP-1) labeled with a fluorescent reagent were co-cultured with HGFs treated with AGEs and IL-6 siRNA, and the adhesive activity of THP-1 cells to HGFs was assessed. The expression of IL-6 and ICAM-1 was examined when HGFs were pretreated with recombinant human IL-6, the siRNAs of RAGE and IL-6, and inhibitors of MAPK and NF-κB, and then cultured with and without AGEs. The phosphorylation of MAPK and NF-κB was assessed using western blotting.AGEs increased the mRNA and protein expressions of RAGE, IL-6, ICAM-1 and reactive oxygen species activity in HGFs, and promoted the adhesion of THP-1 cells to HGFs, but had no effect on cell viability until 72 hours. Recombinant human IL-6 increased ICAM-1 expression in HGFs, while the siRNAs of RAGE and IL-6 inhibited AGE-induced IL6 and ICAM1 mRNA expression, and IL-6 siRNA depressed AGE-induced THP-1 cell adhesion. AGEs increased the phosphorylation of p38 and ERK MAPKs, p65 NF-κB and IκBα, while inhibitors of p38, ERK MAPKs and NF-κB significantly decreased AGE-induced IL-6 and ICAM-1 expression.AGEs increase IL-6 and ICAM-1 expression via the RAGE, MAPK and NF-κB pathways in HGFs and may exacerbate the progression of the pathogenesis of periodontal diseases.

In the current study, we examined the effects of LPS and inflammatory cytokines including IL-1β, TNF-α, and IL-6 on the expression of ghrelin in MGN3-1 cells. We found that IL-1β, and TNF-α with lesser extent, significantly suppressed ghrelin mRNA expression in the cells. MGN3-1 cells expressed IL-1β receptor and IL-1β significantly stimulated NF-κB, p38, JNK, and ERK pathways. Knockdown of IKK2 by siRNA significantly attenuated the suppression of ghrelin mRNA by IL-1β. These results indicate that IL-1β directly suppressed ghrelin mRNA via NF-κB pathway at least partially, which may have a role in the regulation of appetite during inflammation.

ObjectivesThis study sought to introduce and confirm the efficacy of pre-load stress echocardiography with leg-positive pressure (LPP) for improving risk stratification of patients with mild stable heart failure.BackgroundHeart failure patients with mild symptoms and a poor prognosis should be identified and treated aggressively to improve clinical outcome.MethodsWe performed transthoracic echocardiography with LPP in 202 patients with chronic cardiac disease. Twenty-two of these patients also underwent cardiac catheterization, and left ventricular pressure was measured during LPP along with simultaneous Doppler recordings. Patients were classified into 3 groups on the basis of their left ventricular (LV) diastolic dysfunction as assessed by transmitral flow velocity: restrictive or pseudonormal (PN) at rest, impaired relaxation (IR) at rest and during LPP (stable IR), and IR at rest and PN during LPP (unstable IR). Clinical outcome was compared among these groups.ResultsThe LPP increased LV end-diastolic pressure from 15.8 ± 4.7 mm Hg to 20.5 ± 5.0 mm Hg in the unstable IR group and from 10.5 ± 2.6 mm Hg to 14.7 ± 3.8 mm Hg in the stable IR group (both p < 0.001). During an average follow-up of 548 ± 407 days, 5 patients had cardiac death, 37 had acute heart failure, 4 had an acute myocardial infarction, and 7 had a stroke. The all-cause cardiac event rate in unstable IR was higher than in stable IR (p < 0.001), and was similar in the PN group (p = 0.81). Event-free survival was significantly lower in unstable IR than in stable IR (p = 0.003). In a Cox proportional hazards model, unstable IR was an independent predictor of all-cause cardiac events (hazard ratio: 8.0; p

Background: Augmentation index (AI) has been used as a clinical index of arterial stiffness and has been reported to be an independent predictor of cardiovascular events, but some investigators have reported that AI is not a useful marker to identify coronary artery disease (CAD) in elderly patients. The majority of CAD patients are elderly people, therefore the aim of this study was to examine whether AI is a useful marker to identify the risk of CAD. Methods and Results: A total of 120 patients (69±10 years of age; 83 male) who underwent cardiac catheterization for suspected CAD were enrolled. Invasive central blood pressure (BP) was measured using a fluid-filled catheter. Non-invasive AI was calculated by the SphygmoCor (AtCor Medical) system at the end of catheterization. Subjects consisted of 99 patients with CAD and 21 patients without CAD. There was no significant difference in AI between the CAD and the non-CAD groups (24±10 vs. 24±14%). Non-invasive systolic central BP was lower than the invasive systolic central BP (115±18 vs. 130±23 mmHg, P

In animal models, ghrelin production is suppressed by LPS administration. To elucidate the detailed molecular mechanisms involved in the phenomenon, we investigated the effects of LPS and LPS-inducible cytokines, including TNF-α, IL-1β, and IL-6, on the expression of ghrelin in the ghrelin-producing cell line MGN3-1. These cells expressed IL-1R, and IL-1β significantly suppressed ghrelin mRNA levels. The suppressive effects of IL-1β were attenuated by knockdown of IKKβ, suggesting the involvement of the NF-κB pathway. These results suggested that IL-1β is a major regulator of ghrelin expression during inflammatory processes.

Periodontitis is a multifactorial disease associated with genetic and environmental factors. Single-nucleotide polymorphisms (SNPs) are associated with susceptibility to common diseases such as diabetes and periodontitis. Although the oral cavity is exposed to various organisms, the conditions are well controlled by innate and acquired immune systems. Antimicrobial peptides (AMPs) play an important role in the innate immune system; however, the association of AMP-SNPs with periodontitis has not been fully elucidated. This study investigated the relationship between AMP-SNPs and periodontitis in Japanese. One hundred and five Japanese subjects were recruited, which included patients with aggressive, severe, moderate and mild periodontitis, and age-matched healthy controls. Genomic DNA was isolated from peripheral blood and genotypes of SNPs of β-defensin-1 and lactoferrin genes (DEFB1: rs1799946, rs1800972 and rs11362; and LTF: rs1126478) were investigated using the PCR-Invader assay. Protein level of AMPs in gingival crevicular fluid (GCF) was quantified by ELISA. Case-control studies revealed that the -44 CC genotype of DEFB1 (rs1800972) was associated with periodontitis (OR 2.51), particularly with severe chronic periodontitis (OR 4.15) and with combined severe and moderate chronic periodontitis (OR 4.04). No statistical differences were found in other genotypes. The β-defensin-1 concentrations in GCF were significantly lower in subjects with the -44 CC genotype of DEFB1 than in those without this genotype. No significant differences between GCF concentrations of AMPs and other genotypes were detected. The -44 CC genotype of the β-defensin-1 gene (DEFB1 rs1800972) may be associated with susceptibility to chronic periodontitis in Japanese.

A 69-year-old female with polymyositis was referred to our hospital with a chief complaint of dyspnea. Transthoracic echocardiography showed right ventricular overloading. In addition to two-dimensional echocardiography, observation of the abnormal free-floating string-like mass by three-dimensional echocardiography provided superior visualization of the features of the mass which protruded into the right ventricle across the tricuspid valve during diastole. These findings enabled us to confirm the diagnosis of venous thrombus. The thrombus disappeared during the echocardiographic examination. Multidetector-row computed tomography showed a string-like thrombus across the bifurcation of the main pulmonary artery. Anticoagulation therapy was initiated with heparin and warfarin, and fondaparinux was started on the fourth day. Three-dimensional echocardiography was useful in characterizing the motion and extent of the thrombus.

S100A9 is a calcium-binding protein and subunit of antimicrobial calprotectin complex (S100A8/A9). Produced by neutrophils, monocytes/macrophages and keratinocytes, S100A9 expression increases in response to inflammation. For example, IL-1α produced by epithelial cells acts autonomously on the same cells to induce the expression of S100A8/A9 and cellular differentiation. Whereas it is well known that IL-1α and members of the IL-10 family of cytokines upregulate S100A8 and S100A9 in several cell lineages, the pathway and mechanism of IL-1α-dependent transcriptional control of S100A9 in epithelial cells are not established. Modeled using human epidermal keratinocytes (HaCaT cells), IL-1α stimulated the phosphorylation of p38 MAPK and induced S100A9 expression, which was blocked by IL-1 receptor antagonist, RNAi suppression of p38, or a p38 MAPK inhibitor. Transcription of S100A9 in HaCaT cells depended on nucleotides -94 to -53 in the upstream promoter region, based upon the use of deletion constructs and luciferase reporter activity. Within the responsive promoter region, IL-1α increased the binding activity of CCAAT/enhancer binding protein β (C/EBPβ). Mutated C/EBPβ binding sequences or C/EBPβ-specific siRNA inhibited the S100A9 transcriptional response. Hence, IL-1α is strongly suggested to increase S100A9 expression in a human epidermal keratinocyte cell line by signaling through the IL-1 receptor and p38 MAPK, increasing C/EBPβ-dependent transcriptional activity.

The renin-angiotensin-aldosterone system (RAAS) and arginine vasopressin (AVP) regulate body fluids. Although conventional diuretics have been used for treating heart failure, they activate RAAS and exacerbate renal function. Tolvaptan, a newly developed vasopressin-2 receptor antagonist, elicits aquaresis and improves volume overload in heart failure patients, however, the predictors of tolvaptan effectiveness and the influence on the RAAS and renal function according to tolvaptan therapy are not established. We evaluated 26 chronic heart failure patients receiving therapy with 15 mg/day tolvaptan and examined their laboratory and urinary data before and after tolvaptan therapy. A response to tolvaptan was defined as a body weight decrease by more than 2 kg in a week and a urine volume increase by 500 mL/ day compared with that before tolvaptan administration. Body weight, urine volume, and brain natriuretic peptide levels significantly improved (P < 0.05), without any worsening of renal function represented by serum creatinine, sodium, and potassium. Moreover, no significant changes were observed in the plasma renin activity and plasma aldosterone concentration (PAC). In the responder group, urine osmolality before tolvaptan administration was significantly higher (P < 0.05) but declined significantly after tolvaptan administration (P < 0.05). The AVP/PAC ratio before administration was positively correlated with the efficacy of tolvaptan. Tolvaptan treatment could prevent RAAS activation in chronic heart failure patients. Moreover, monitoring the AVP/PAC ratio may be useful in predicting the tolvaptan response.

Recent studies have demonstrated that chronic kidney disease (CKD) may be associated with the progression of periodontal disease. Diabetes mellitus (DM) is a major risk factor for CKD. The objective of this study was to clarify the relationship between periodontal condition and kidney dysfunction in patients who had kidney failure with or without DM. One hundred sixty‐four patients with kidney dysfunction were enrolled (male: N = 105; female: N = 59), and the relationship between periodontal condition and kidney dysfunction was analyzed in a cross‐sectional study. The subjects were divided into three groups: (a) patients with DM, (b) dialysis patients with nephropathy due to various kidney diseases, and (c) dialysis patient with nephropathy due to DM (diabetic nephropathy). Then, the effect of DM on the periodontal condition was analyzed. The patients were also stratified by CKD stage (into G1–G5) using the estimated glomerular filtration rate (eGFR), and the G5 group was divided in patients with or without DM. Correlations between eGFR and parameters of periodontal condition were calculated in patients from G1 to G4. The number of missing teeth was significantly higher in dialysis patients with diabetic nephropathy than in patients with DM, whereas alveolar bone loss did not show a significant difference among the three groups. In addition, the G5 patients with DM had a significantly higher number of missing teeth than the other CKD groups, whereas alveolar bone loss did not show a significant difference. In G5 patients with DM, Community Periodontal Index and Oral Hygiene Index scores were significantly higher than in G1‐4 patients with DM. There was a significant negative correlation between eGFR and the number of missing teeth. Patients with diabetic nephropathy have a higher rate of periodontal problems such as missing teeth in Japanese adults.

Objectives Gingival crevicular fluid (GCF) contains calprotectin, which appears to be a useful biomarker for periodontal diseases because of its high level in GCF from periodontally diseased pockets. To determine calprotectin in GCF that has a very small volume, sandwich enzyme-linked immunosorbent assay (ELISA) on a microchip was performed and its utility was estimated. Design and methods Anti-calprotectin primary antibody was discharged on a microchip using a piezoelectric inkjet printing system. Calprotectin standard and calprotectin in GCF samples from eleven subjects were determined by the ELISA method with the prepared microchip and their values were compared with those obtained by conventional ELISA. Results Using the ELISA on a microchip, a reasonable standard curve of calprotectin protein (1.56–100 ng/mL) was obtained. Calprotectin in GCF samples was quantified and showed reasonable values in accordance with the condition of periodontal diseases. The values determined by the microchip method and conventional ELISA showed a significant linear relationship ( R 2 = 0.981). Conclusions Calprotectin in GCF was determined using the ELISA on a microchip with high efficiency and this ELISA method for calprotectin determination may become a useful method for diagnosing periodontal diseases.

Background Accurate assessment of left ventricular (LV) function in patients with atrial fibrillation (AF) remains difficult, mainly because of the beat-to-beat variability of many echocardiographic parameters. The aim of this study was to assess the hypothesis that LV function can be estimated from an index-beat echocardiographic assessment in patients with AF using myocardial strain and strain rate. Methods A prospective study was conducted to assess 25 patients with AF (mean age, 66 ± 10 years). Peak systolic longitudinal strain (LS) and peak diastolic longitudinal strain rate (dSR) were measured using two different methods: (1) mean LS and dSR, the averages of instantaneous LS and dSR over 10 sec, and (2) index-beat LS and dSR, calculated when the ratio of the preceding (RR1) to the pre-preceding (RR2) interval was 1 (range, 0.96–1.04). These variables were compared with simultaneously measured LV pressure parameters using Millar catheters. Results There was a positive linear relationship between mean LS and index-beat LS at RR1/RR2 = 1 ( r = 0.94, P r = 0.69, P r = −0.73, P r = −0.63, P Conclusions Index-beat parameters accurately reflect the mean values of parameters in patients with AF. These noninvasively obtained index-beat parameters are useful to assess surrogate LV function even in patients with AF.

Whereas ghrelin is produced primarily in the stomach, a small amount of it is produced in pancreatic islets. Although exogenous administration of ghrelin suppresses insulin secretion in vitro or in vivo, the role of intraislet ghrelin in the regulation of insulin secretion in vivo remains unclear. To understand the physiological role of intraislet ghrelin in insulin secretion and glucose metabolism, we developed a transgenic (Tg) mouse model, rat insulin II promoter ghrelin-internal ribosomal entry site-ghrelin O-acyl transferase (RIP-GG) Tg mice, in which mouse ghrelin cDNA and ghrelin O-acyltransferase are overexpressed under the control of the rat insulin II promoter. Although pancreatic desacyl ghrelin levels were elevated in RIP-GG Tg mice, pancreatic ghrelin levels were not altered in animals on a standard diet. However, when Tg mice were fed a medium-chain triglyceride-rich diet (MCTD), pancreatic ghrelin levels were elevated to ∼16 times that seen in control animals. It seems likely that the gastric ghrelin cells possess specific machinery to provide the octanoyl acid necessary for ghrelin acylation but that this machinery is absent from pancreatic β-cells. Despite the overexpression of ghrelin, plasma ghrelin levels in the portal veins of RIP-GG Tg mice were unchanged from control levels. Glucose tolerance, insulin secretion, and islet architecture in RIP-GG Tg mice were not significantly different even when the mice were fed a MCTD. These results indicate that intraislet ghrelin does not play a major role in the regulation of insulin secretion in vivo.

Hiroshima Y, Bando M, Inagaki Y, Mihara C, Kataoka M, Murata H, Shinohara Y, Nagata T, Kido J. Resistin in gingival crevicular fluid and induction of resistin release by Porphyromonas gingivalis lipopolysaccharide in human neutrophils. J Periodont Res 2012; 47: 554–562. © 2012 John Wiley & Sons A/S Background and Objective: Resistin is an adipocytokine that induces insulin resistance and is predominantly expressed in adipocytes and peripheral blood mononuclear cells. Resistin expression increases in inflammatory diseases as well as diabetes mellitus, and is upregulated by bacterial pathogens and proinflammatory cytokines. The aim of this study was to identify resistin in human gingival crevicular fluid, to compare the resistin levels in gingival crevicular fluid between subjects with and without periodontitis and diabetes mellitus and to investigate the regulation of resistin release from human neutrophils by Porphyromonas gingivalis lipopolysaccharide (P-LPS). Material and Methods: Gingival crevicular fluid samples were collected from patients with chronic periodontitis (n = 24), patients with diabetes mellitus-related periodontitis (n = 18) and healthy subjects (n = 21). Resistin in gingival crevicular fluid was determined using western blot analysis and an ELISA kit. The glycated hemoglobin (HbA1c) value was obtained from patients with diabetes mellitus-related periodontitis by a medical interview. Human neutrophils were cultured with P-LPS (0–1000 ng/mL), or incubated with inhibitors of actin or microtubule polymerization in the absence or presence of P-LPS. The medium and cellular fractions were used for determination of resistin by ELISA. Results: The resistin level in gingival crevicular fluid from patients with periodontitis or diabetes mellitus-related periodontitis was significantly higher than that of healthy subjects. The resistin level in gingival crevicular fluid was correlated with gingival index score, but not blood HbA1c value. The P-LPS increased resistin release from human neutrophils, and its induction was decreased by actin polymerization inhibitors. Conclusion: We show, for the first time, the presence of resistin in gingival crevicular fluid. A high resistin level in gingival crevicular fluid samples from periodontitis patients may to some extent be related to P-LPS-induced resistin release from neutrophils.

Kido J, Bando M, Hiroshima Y, Iwasaka H, Yamada K, Ohgami N, Nambu T, Kataoka M, Yamamoto T, Shinohara Y, Sagawa I, Nagata T. Analysis of proteins in human gingival crevicular fluid by mass spectrometry. J Periodont Res 2012; 47: 488–499. © 2012 John Wiley & Sons A/S Background and Objective: Gingival crevicular fluid is a bodily fluid transuded from periodontal tissues into the gingival crevice and periodontal pocket, and contains many species of components. Proteins in gingival crevicular fluid have been studied as markers for periodontal diseases. Mass spectrometric analysis is used for the analyses of proteins, lipids, saccharides and metals, and expected as an approach for disease diagnosis. For better analysis of the protein components in gingival crevicular fluid, we investigated proteins in gingival crevicular fluid samples from the healthy gingival crevice and periodontal pocket using mass spectrometry. Material and Methods: Gingival crevicular fluid samples were collected from subjects who gave their informed consent and were periodontally healthy or had diseased pockets. These samples were electrophoretically separated, and each fraction on the gels was analysed by nano liquid chromatography coupled with tandem mass spectrometry. Antimicrobial peptides detected in gingival crevicular fluid were confirmed by western blotting. Results: One hundred and four proteins were detected in gingival crevicular fluid samples from both healthy sites and sites of periodontitis; 64 proteins were contained only in gingival crevicular fluid from healthy sites and 63 proteins were observed only in gingival crevicular fluid from periodontitis sites. These proteins were blood-, cytoskeleton-, immunity-, inflammation- and lipid-related proteins and enzymes. Some proteins, including ceruloplasmin, glycogen phosphorylase, glutathione S-transferase, phosphoglycerate mutase, psoriasin, S100A11 and resistin, were identified for the first time in gingival crevicular fluid. Antimicrobial peptides, such as lactoferrin, α1-antitrypsin, lipocalin, S100A7, S100A8, S100A9 and cathelicidin, were observed by mass spectrometry and western blotting. Conclusion: Multiple protein components in gingival crevicular fluid were analysed at the same time using mass spectrometry, and this approach may be useful for the diagnosis of periodontal diseases.

To determine the comprehensive G protein-coupled receptor (GPCR) expression profile in ghrelin-producing cells and to elucidate the role of GPCR-mediated signaling in the regulation of ghrelin secretion, we determined GPCR expression profiles by RNA sequencing in the ghrelin-producing cell line MGN3-1 and analyzed the effects of ligands for highly expressed receptors on intracellular signaling and ghrelin secretion. Expression of selected GPCRs was confirmed in fluorescence-activated cell-sorted fluorescently tagged ghrelin-producing cells from ghrelin-promoter CreERT2/Rosa-CAG-LSL-ZsGreen1 mice. Expression levels of GPCRs previously suggested to regulate ghrelin secretion including adrenergic-β1 receptor, GPR81, oxytocin receptor, GPR120, and somatostatin receptor 2 were high in MGN3-1 cells. Consistent with previous reports, isoproterenol and oxytocin stimulated the Gs and Gq pathways, respectively, whereas lactate, palmitate, and somatostatin stimulated the Gi pathway, confirming the reliability of current assays. Among other highly expressed GPCRs, prostaglandin E receptor 4 agonist prostaglandin E2 significantly stimulated the Gs pathway and ghrelin secretion. Muscarine, the canonical agonist of cholinergic receptor muscarinic 4, stimulated both the Gq and Gi pathways. Although muscarine treatment alone did not affect ghrelin secretion, it did suppress forskolin-induced ghrelin secretion, suggesting that the cholinergic pathway may play a role in counterbalancing the stimulation of ghrelin by Gs (eg, by adrenaline). In addition, GPR142 ligand tryptophan stimulated ghrelin secretion. In conclusion, we determined the comprehensive expression profile of GPCRs in ghrelin-producing cells and identified two novel ghrelin regulators, prostaglandin E2 and tryptophan. These results will lead to a greater understanding of the physiology of ghrelin and facilitate the development of ghrelin-modulating drugs.

Introduction: Epicardial adipose tissue(EAT), which is thought to be an ectopic adipose tissue, has been paid attention in association with coronary artery disease (CAD). Hypothesis: We hypothesized that EAT in anterior interventricular groove (AIG) obtained by echocardiography can be an additional marker over classical risk factors for prediction of CAD. Methods: We enrolled 311 patients (mean age 67±11 yrs, 208 men) who underwent coronary angiography between December 2011 and December 2013 at our hospital. We measured EAT thickness on the AIG and right ventricular free wall (EAT-RV) using high-frequency linear probe. Subjects were divided into 2 groups with and without significant coronary stenosis (≧75%) from coronary angiography. The performance of clinical risk factors (including age, male gender, body mass index (BMI), diabetes mellitus, hypertension, dyslipidemia, and smoking) plus various combinations of EAT thickness measurements for predicting CAD was assessed using the area under the curve (AUC) in ROC analysis. Results: The EAT-AIG thickness was significantly greater in the CAD group than that in the non-CAD group (8.3±3.0 vs. 6.3±2.5 mm, p Conclusions: Echocardiographic EAT-AIG thickness was greater in the CAD group than the non-CAD group. This non-invasive index may have clinical potential as a maker for predicting coronary atherosclerosis.

Introduction: The 6-minute walk distance (6MWD) as a surrogate of exercise capacity is often shown to be the best predictor of mortality in pulmonary hypertension (PH). Recent studies have described that mean pulmonary artery pressure (mPAP) - cardiac output (CO) relationships have a potential to assess the detail of pulmonary circulation. Hypothesis: We hypothesized that pressure-flow relationships of the pulmonary circulation (mPAP/CO) would be associated with 6MWD in connective tissue disease (CTD). Methods: We prospectively performed 6-minute walk test and echocardiographic studies in 202 CTD patients (56±14 years; 11% male) with measurements of cardiac output using electric cardiometry. We have calculated the pulmonary artery pressure-cardiac output relationships as mPAP divided by CO (mPAP/CO). Results: Firstly, we have checked the accuracy of CO by electric cardiometry. In our cohort with invasive data, there is a good correlation between right heart catheter and electric cardiometry values of cardiac output (r=0.85; p Conclusions: Elevated mPAP/CO was associated with reduced 6MWD independent from LV and RV diastolic function. These results may recommend that we would assess not only the pressure but also the pressure-flow relationships of the pulmonary circulation in high risk group of PH.

We report a case of a 78-year-old woman with cardiac sarcoidosis with a history of syncope and palpitation. Further assessment with echocardiography, gadolinium-enhanced cardiovascular magnetic resonance (CMR) and histology led to a diagnosis of cardiac sarcoidosis. As the patient suffered from ventricular tachycardia (VT) despite active corticosteroid therapy, an implantable cardioverter-defibrillator (ICD) was positioned. She was also administered a beta blocker, but an electrical storm appeared every several days requiring ICD therapy. The drug-refractory VT was finally controlled with a catheter ablation session, during which we could detect the VT focus in the right ventricular outflow tract next to the aneurysm by using an electroanatomic mapping system (CARTO). Referring to echocardiographic and CMR images proved very useful in detecting the aneurysm using the CARTO system.

Tryptophan is reportedly the most potent agonist for GPR142. Glucose-stimulated insulin secretion (GSIS) from pancreatic beta cells are enhanced by GPR142-mediated signal. It is not clear, however, if GPR142-mediated signals is solely attributable to GSIS enhancement after tryptophan load in various pathophysiological settings. This study aims to reveal the significance of GPR142 signaling in tryptophan-mediated GSIS enhancement in normal and obese mice. Tryptophan significantly improved glucose tolerance in both lean and DIO mice, but the extent of improvement was bigger in DIO mice with augmented glucose-stimulated insulin secretion (GSIS) enhancement. The same results were obtained in ob/ob mice. GPR142 deletion almost completely blocked tryptophan actions in lean mice, suggesting that GPR142 signaling was solely responsible for the GSIS enhancement. In obese GPR142KO mice, however, a significant amount of tryptophan effects were still observed. Calcium-sensing receptors (CaSR) are also known to recognize tryptophan as ligand. Expression levels of CaSR were significantly elevated in the pancreas of DIO mice, and CaSR antagonist further blocked tryptophan's actions in DIO mice with GPR142 deletion. Although GPR142 signaling had a major role in tryptophan recognition for the enhancement of GSIS in lean mice, other pathways including CaSR signaling also had a significant role in obese mice, which seemed to contribute to the augmented enhancement of GSIS by tryptophan in these animals.

To establish a tool to study ghrelin production and secretion in vitro, we developed a novel ghrelin-producing cell line, MGN3–1 (mouse ghrelinoma 3-1) cells from a gastric ghrelin-producing cell tumor derived from ghrelin-promoter Simian virus 40-T-antigen transgenic mice. MGN3-1 cells preserve three essential characteristics required for the in vitro tool for ghrelin research. First, MGN3-1 cells produce a substantial amount of ghrelin at levels approximately 5000 times higher than that observed in TT cells. Second, MGN3-1 cell expressed two key enzymes for acyl modification and maturation of ghrelin, namely ghrelin O-acyltransferase for acylation and prohormone convertase 1/3 for maturation and the physiological acyl modification and maturation of ghrelin were confirmed. Third, MGN3-1 cells retain physiological regulation of ghrelin secretion, at least in regard to the suppression by somatostatin and insulin, which is well established in in vivo studies. Thus, MGN3-1 cells are the first cell line derived from a gastric ghrelin-producing cell preserving secretion of substantial amounts of ghrelin under physiological regulation. This cell line will be a useful tool for both studying the production and secretion of ghrelin and screening of ghrelin-modulating drugs.

Ghrelin was initially identified as an endogenous ligand for the GH secretagogue receptor. When administrated exogenously, ghrelin stimulates GH release and food intake. Previous reports in ghrelin-null mice, which do not exhibit impaired growth nor appetite, question the physiologic role of ghrelin in the regulation of the GH/IGF-I axis. In this study, we generated a transgenic mouse that expresses human diphtheria toxin (DT) receptor (DTR) cDNA in ghrelin-secretion cells [ghrelin-promoter DTR-transgenic (GPDTR-Tg) mice]. Administration of DT to this mouse ablates ghrelin-secretion cells in a controlled manner. After injection of DT into GPDTR-Tg mice, ghrelin-secreting cells were ablated, and plasma levels of ghrelin were markedly decreased [nontransgenic littermates, 70.6 ± 10.2 fmol/ml vs. GPDTR-Tg, 5.3 ± 2.3 fmol/ml]. To elucidate the physiological roles of circulating ghrelin on GH secretion and somatic growth, 3-wk-old GPDTR-Tg mice were treated with DT twice a week for 5 wk. The GH responses to GHRH in male GPDTR-Tg mice were significantly lower than those in wild-type mice at 5 wk of age. However, those were normalized at 8 wk of age. In contrast, in female mice, there was no difference in GH response to GHRH between GPDTR-Tg mice and controls at 5 or 8 wk of age. The gender-dependent differences in response to GHRH were observed in ghrelin-ablated mice. However, GPDTR-Tg mice did not display any decreases in IGF-I levels or any growth retardation. Our results strongly suggest that circulating ghrelin does not play a crucial role in somatic growth.

Hiroshima Y, Bando M, Kataoka M, Shinohara Y, Herzberg MC, Ross KF, Inagaki Y, Nagata T, Kido J. Shosaikoto increases calprotectin expression in human oral epithelial cells. J Periodont Res 2009; doi: 10.1111/j.1600-0765.2009.01203.x. © 2009 John Wiley & Sons A/S Background and Objective: Oral epithelial cells help to prevent against bacterial infection in the oral cavity by producing antimicrobial peptides (AMPs). A broad-spectrum AMP, calprotectin (a complex of S100A8 and S100A9 proteins), is expressed by oral epithelial cells and is up-regulated by interleukin-1α (IL-1α). Shosaikoto (SST) is a traditional Japanese herbal medicine that has immunomodulatory effects and is reported to enhance the levels of IL-1α in epithelial cells. The purpose of this study was to investigate the effect of SST on the expression of calprotectin and other AMPs through the regulation of IL-1α in oral epithelial cells. Material and Methods: Human oral epithelial cells (TR146) were cultured with SST (at concentrations ranging from 10 to 250 μg/mL) in the presence or absence of anti-IL-1α or IL-1 receptor antagonist. The expression of S100A8- and S100A9-specific mRNAs was examined by northern blotting. Calprotectin expression and IL-1α secretion were investigated by immunofluorescent staining or ELISA. The expression of other AMPs and IL-1α was analyzed by RT-PCR and by quantitative real-time PCR. Results: Shosaikoto (25 μg/mL) significantly increased the expression of S100A8- and S100A9-specific mRNAs and calprotectin protein. Shosaikoto increased S100A7 expression, but had no effect on the expression of other AMPs. The expression of IL-1α-specific mRNA and its protein were slightly increased by SST. A neutralizing antibody against IL-1α or IL-1 receptor antagonist inhibited SST up-regulated S100A8/S100A9 mRNA expression. Conclusion: These results suggest that SST increases the expression of calprotectin and S100A7 in oral epithelial cells. In response to SST, up-regulation of calprotectin may be partially induced via IL-1α.

Calprotectin is an antimicrobial complex composed of the S100A8 and S100A9 protein family subunits. Contributing to innate immunity, calprotectin expression is increased by interleukin-1alpha (IL-1alpha), which modulates keratinocyte differentiation. Keratinocyte growth factor (KGF) is produced by mesenchymal cells and has a mitogenic activity for epithelial cells. In this study, we investigated the effect of KGF on calprotectin expression in keratinocytes and modulation by IL-1alpha. Human keratinocytes were cultured with KGF in the presence or absence of a KGF receptor (KGFR) inhibitor or mitogen-activated protein kinase (MAPK) inhibitors. Calprotectin (S100A8/S100A9) expression was determined by northern blotting and enzyme-linked immunosorbent assay, respectively, whereas MAPK phosphorylation was analyzed by western blot analysis. KGF significantly decreased the expression of S100A8/S100A9-specific mRNAs and calprotectin protein. In the presence of KGF, KGFR inhibitor or extracellular-regulated kinase inhibitor restored KGF-downregulated expression of S100A8/S100A9. KGF increased IL-1alpha expression in keratinocytes, whereas IL-1alpha increased KGF expression in fibroblasts. Cocultured fibroblast and keratinocytes showed lower S100A8/S100A9 mRNA expression than keratinocytes alone in the presence or absence of IL-1alpha or KGF. These results suggest that fibroblast-derived KGF reduces or restricts calprotectin expression in keratinocytes, which supports our hypothesis that calprotectin expression in keratinocytes is modulated by factors associated with epithelial-mesenchymal interactions.

Many middle-aged and elderly people suffer from periodontal diseases that mainly cause tooth loss and result in some systemic diseases. The prevention of periodontal diseases is very important for oral and systemic health, but the present diagnostic examination is not fully objective or suitable. To diagnose periodontal diseases exactly, some biomarkers that represent inflammation, tissue degradation, and bone resorption in gingival crevicular fluid (GCF) and saliva are known. We demonstrated that the GCF levels of calprotectin, an inflammation-related protein, and carboxy-terminal propeptide of type I procollagen, a bone metabolism-related protein, were associated with the clinical condition of periodontal diseases, and suggested that these proteins might be useful biomarkers for periodontal diseases. Recently, determinations of genes and proteins by using microdevices have been studied for the diagnosis of some diseases. We detected calprotectin protein by chemiluminescent immunoassay on a microchip and showed the possibility of specific and quantitative detection of calprotectin in a very small amount of GCF. The determination of multiple markers in GCF by using microdevices will contribute to the development of accurate and objective diagnostic systems for periodontal diseases. © 2012 Wiley Periodicals, Inc. Electr Eng Jpn, 179(1): 40–45, 2012; Published online in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/eej.21150

Calprotectin, a heterodimeric complex of S100A8 and S100A9, is an antimicrobial peptide (AMP) expressed in healthy or inflamed epithelium and epidermis. The expression of calprotectin and other AMPs in keratinocytes is regulated by proinflammatory cytokines, bacterial products and keratinocyte differentiation modulators. Interleukin-1α (IL-1α), an autonomous cytokine in keratinocytes, induces keratinocyte differentiation and up-regulates calprotectin expression. Since keratinocyte differentiation is also specifiedby interactions with the basement membrane and connective tissue proteins, we sought to learn whether calprotectin expression is affected by extracellular matrix (ECM) proteins in the presence or absence of IL-1α. Human immortalized epidermal keratinocytes (HaCaT cells) were grown in vitro on different ECM substrates including type I collagen (Col I), type IV collagen (Col IV), fibronectin (FN) and laminin (LM) with or without IL-1α and were then cultured in basement membrane extract (Matrigel)-coated dishes or on a feeder layer of fibroblasts. Calprotectin expression in the cultured cells was investigated using immunohistochemical and northern blot analyses, and an enzyme-linked immunosorbent assay. The four ECM proteins (Col I, Col IV, FN and LM) had little effect on calprotectin expression. IL-1α significantly increased the expression of S100A8/ S100A9 mRNAs and calprotecin protein in the cells cultured on each of the ECM(Col I, Col IV, FN and LM)-coated dishes,but these ECM protein substrates did not show a further effect on calprotectin expression in the presence of IL-1α. On the other hand, Matrigel and the fibroblast-feeder layer slightly decreased the expression of S100A8/S100A9 mRNAs in HaCaT cells. These results suggest that calprotectin expression in keratinocytes is regulated by cytokines during epithelial-mesenchymal interactions.Nihon Shishubyo Gakkai Kaishi (J Jpn Periodontol) 49 : 224-232,2007.

Echocardiography now plays a central guiding role in the management of patients with atrial fibrillation (AF). However, the current guidelines mention little about the presence AF during the assessment of echocardiographic variables in the clinical setting. AF itself may impact on tricuspid annular plane systolic excursion (TAPSE) as a right ventricular systolic function compared with sinus rhythm (SR). The aim of this study was to compare and assess the echocardiographic parameters including TAPSE in patients with AF and SR. From January 1, 2013, to September 30, 2014, patients with AF without any cardiovascular disease were retrospectively evaluated using echocardiography. Age-, gender-, and left ventricular ejection fraction-matched patients with SR were selected from our database on the basis of a comprehensive history, physical examination, and echocardiographic findings. During the study period, we identified 239 patients with AF (74 ± 9 years; 65% men) and without any cardiac disease who underwent echocardiography. We also included 281 patients in the SR group (74 ± 8 years; 67% men). In all study subjects, TAPSE in AF was smaller than in SR regardless of age (17 ± 3 vs 20 ± 3 mm, p0.001). In the stepwise multiple regression model, TAPSE was strongly associated with the presence of AF (standardized β = -0.362, p0.001) and stroke volume index (standardized β = 0.173, p0.001) after adjustment for age, gender, heart rate, left ventricular ejection fraction, and tricuspid regurgitant grade. In conclusions, patients with AF had lower TAPSE than those with SR regardless of age. When we assess TAPSE in the clinical setting, we must pay attention to the presence of AF.