Establishment and characterization of four malignant pleural mesothelioma cell lines from Japanese patients

Malignant pleural mesothelioma (MPM) is an asbestos-related malignancy that is highly resistant to current therapeutic modalities. We established four MPM cell lines (ACC-MESO-1, ACC-MESO-4, Y-MESO-8A and Y-MESO-8D) from Japanese patients, with the latter two from the same patient with biphasic-like characteristics of MPM, showing epithelial and sarcomatous phenotypes, respectively, in cell culture. These cells grew well in RPMI-1640 medium supplemented with 10% fetal bovine serum under 5% CO2. Mutation and expression analyses demonstrated that the tumor suppressor gene NF2, which is known to be one of the most frequently mutated in MPM, is mutated in ACC-MESO-1. We detected homozygous deletion of p16INK4A/p14ARF in all four MPM cell lines. However, mutations of other tumor suppressor genes, including TP53, and protooncogenes, including KRAS, NRAS, BRAF, EGFR and HER2, were not found in these cell lines. Polymerase chain reaction amplification of the simian virus 40 sequence did not detect any products. We also analyzed genetic alterations of six other MPM cell lines and confirmed frequent mutations of NF2 and p16INK4A/p14ARF. To characterize the biological differences between Y-MESO-8A and Y-MESO-8D, we carried out cDNA microarray analysis and detected genes that were differentially expressed in these two cell lines. Thus, our new MPM cell lines seem to be useful as new models for studying various aspects of the biology of human MPM as well as materials for the development of future therapies.     

Age and sex-related differences in dose-dependent hemodynamic response to landiolol hydrochloride during general anesthesia

Objective Landiolol hydrochloride is a new ultra-short-acting β₁-adrenergic receptor blocking agent that is used for patients with tachycardia during general anesthesia. The hemodynamic response to a β-adrenergic receptor blocking agent is generally dependent on the subject. In the present investigation we investigated the effects of age and sex on the hemodynamic response to different doses of landiolol. Methods Following a persistence of tachycardia for more than 1 min, landiolol was infused at 0.03125, 0.0625 and 0.125 mg kg⁻¹ min⁻¹ for 1 min followed by 0.01, 0.02 and 0.04 mg kg⁻¹min⁻¹ for 10 min in groups L, M and H, respectively. Heart rate (HR), systolic (sBP) and diastolic blood pressure (dBP) were recorded each minute during the procedure. The respective changes were evaluated using logistic analysis with the equation , where p, q, m and k indicate the upper asymptote, lower asymptote, maximum slope and time at the maximum slope. Results Parameters q and k for HR in group H were smaller than those in group L, whereas the parameters for sBP and dBP were unchanged among the three groups. Parameter q for HR in group H decreased with age of the patient. There was no significant difference in the landiolol-induced change in HR between males and females. Conclusion The hemodynamic response to landiolol is reliably modeled by the logistic function, especially in terms of HR. Landiolol causes a rapid and dose-dependent decrease in HR, whereas landiolol-induced changes in sBP and dBP are independent of dose. The landiolol-induced decrease in HR becomes larger with aging, but shows no sex difference. The logistic model may be useful for studying hemodynamic responses to landiolol based on age and sex differences, and may allow development of an improved monitoring system. Implication statement The logistic function reliably represents the hemodynamic responses to landiolol. Landiolol reduces HR rapidly and in a dose-dependentl manner whereas landiolol-induced changes in sBP and dBP are independent of dose. A larger decrease in HR is caused by landiolol in elderly patients, but there is no sex difference in the landiolol-induced change in HR.     

Reliability of cardiac output measurements using LiDCOrapid™ and FloTrac/Vigileo™ across broad ranges of cardiac output values

Knowing a patient’s cardiac output (CO) could contribute to a safe, optimized hemodynamic control during surgery. Precise CO measurements can serve as a guide for resuscitation therapy, catecholamine use, differential diagnosis, and intervention during a hemodynamic crisis. Despite its invasiveness and intermittent nature, the thermodilution technique via a pulmonary artery catheter (PAC) remains the clinical gold standard for CO measurements. LiDCOrapid? (LiDCO, London, UK) and FloTrac/Vigileo? (Edwards Lifesciences, Irvine, CA) are less invasive continuous CO monitors that use arterial waveform analysis. Their calculations are based on arterial waveform characteristics and do not require calibration. Here, we evaluated LiDCOrapid? and FloTrac/Vigileo? during off-pump coronary artery bypass graft (OPCAB) and living-donor liver transplantation (LDLT) surgery. This observational, single-center study included 21 patients (11 OPCAB and 10 LDLT). We performed simultaneous measurements of CO at fixed sampling points during surgery using both devices (LiDCOrapid? version 1.04-b222 and FloTrac/Vigileo? version 3.02). The thermodilution technique via a PAC was used to obtain the benchmark data. LiDCOrapid? and FloTrac/Vigileo? were used in an uncalibrated fashion. We analyzed the measured cardiac index using a Bland–Altman analysis (the method of variance estimates recovery), a polar plot method (half-moon method), a 4-quadrant plot and compared the widths of the limits of agreement (LOA) using an F test. One OPCAB patient was excluded because of the use of an intra-aortic balloon pumping during surgery, and 20 patients (10 OPCAB and 10 LDLT) were ultimately analyzed. We obtained 149 triplet measurements with a wide range of cardiac index. For the FloTrac/Vigileo?, the bias and percentage error were ?0.44 L/min/m² and 74.4 %. For the LiDCOrapid?, the bias and percentage error were ?0.38 L/min/m² and 53.5 %. The polar plot method showed an angular bias (FloTrac/Vigileo? vs. LiDCOrapid?: 6.6° vs. 5.8°, respectively) and radial limits of agreement (?63.9 to 77.1 vs. ?41.6 to 53.1). A 4-quadrant plot was used to obtain concordance rates (FloTrac/Vigileo? vs. PAC and LiDCOrapid? vs. PAC: 84.0 and 92.4 %, respectively). We could compare CO measurement devices across broad ranges of CO and SVR using LDLT and OPCAB surgical patients. An F test revealed no significant difference in the widths of the LoA for both devices when sample sizes capable of detecting a more than two-fold difference were used. We found that both devices tended to underestimate the calculated CIs when the CIs were relatively high. These proportional bias produced large percentage errors in the present study.     

Surgical-site infection surveillance at a small-scale community hospital

Surveillance of surgical-site infection (SSI) is becoming more important given the current situation of increasing antibiotic resistance by microorganisms. It may be difficult to carry out SSI surveillance at small-scale community hospitals because of small staff numbers. We examined whether SSI surveillance could be carried out with a system we devised. Furthermore, we investigated the SSI rateat our small-scale community hospital (179 beds) in aJapanese city (populations, 330 000). Between June andDecember 2003, operations were performed on 210patients. Procedures were identified as clean (n = 85),clean-contaminated (n = 108), contaminated (n = 14), or dirty-infected (n = 3). A 7-month prospective survey ofSSI was conducted. SSIs were classified according to the Centers for Disease Control and Prevention criteria and identified using bedside surveillance and post-discharge follow-up. SSI developed following 16 procedures (7.6%). All patients who developed SSI had received antibiotic prophylaxis. Among the 16 patients with SSI, operations were clean (n = 1), clean-contaminated (n = 8), contaminated(n = 5), or dirty-infected (n = 2). Enterobacteriaceae were the most frequently isolated microorganisms, followed by Pseudomonas aeruginosa. SSI surveillance is just as important at small community hospitals as it is at larger hospitals, and SSI surveillance is relatively simple to institute at small-scale community hospitals with the selective use of investigation items.     

Effects of short interfering RNA against methicillin-resistant Staphylococcus aureus coagulase in vitro and in vivo

OBJECTIVES: The emergence of antibiotic-resistant bacteria such as Staphylococcus aureus calls for inventive research and development strategies. Inhibition of bacterial pathogenesis may be a promising therapeutic approach in this regard. The gene-silencing effect of short interfering RNA (siRNA) is useful for this strategy. We investigated the efficacy of siRNA on the expression of coagulase because it is the one of the most important enzymes in the pathogenesis of methicillin-resistant S. aureus (MRSA) infection. METHODS: We designed and synthesized 21 bp siRNA duplexes against staphylococcal coagulase. RT-PCR was performed to determine whether the siRNAs inhibit the expression of the coagulase mRNA and radio-labelled siRNA was used to confirm transfection to bacteria in vitro. The efficacy of siRNA was determined in a murine model of haematogenous pulmonary infection. RESULTS: RT-PCR showed that siRNAs significantly inhibited the expression of the coagulase mRNA. The coagulase titres in the siRNA and control groups were 8 and 32, respectively. Measurement of incorporated radioactivity indicated that the siRNAs were delivered into the bacteria. In the murine infection model, in control and siRNA groups, 7.64 +/- 0.42 and 6.29 +/- 0.23 log cfu/mL (mean +/- SEM) MRSA were detected, respectively, showing that there was a significant decrease in the number of viable bacteria in the siRNA group (P < 0.05). CONCLUSIONS: The results show that siRNA inhibited both mRNA expression and the activity of MRSA coagulase in vitro. The in vivo results revealed that the siRNA was effective in reducing the bacterial load in a murine model of haematogenous pulmonary infection. Targeting of coagulase with siRNA appears to be a novel strategy for treating MRSA infections.     

Detailed discussion on evidence for the further prevention of traffic fatalities in Japan: a comparison of trends in three countries

The total number of traffic fatalities has decreased in recent years. To further reduce the number of traffic fatalities, details regarding traffic fatalities and various aspects of traffic accidents must be studied. Therefore, this research examined the number of traffic fatalities by age group and the number of traffic fatalities by situation in Japan from 2003 to 2007, and the percentage of traffic fatalities by age group and the percentage of traffic fatalities by situation during the same period were calculated. In addition, aspects of traffic accidents in Japan were compared with those in Germany and France, and additional measures to counter traffic accidents that need to be taken in Japan in order to further reduce the number of traffic fatalities were studied. Results indicated that in each year of the period studied the highest percentage of traffic fatalities involved the 25-64 age group in Germany and France and the 65-or-over age group in recent years in Japan. In addition, additional measures to counter traffic accidents involving the elderly are needed in Japan, Germany and France. The percentage of traffic fatalities by situation indicated that attention should be paid to 'fatalities of pedestrians' in Japan and to 'fatalities of motor vehicle occupants' in all the three countries studied. In particular, measures to counter traffic accidents leading to 'fatalities of pedestrians' must be studied further in Japan. With the above findings in mind, bodies involved in implementing measures to counter traffic accidents must formulate specific measures.     

In vivo efficacies and pharmacokinetics of DX-619, a novel des-fluoro(6) quinolone, against Streptococcus pneumoniae in a mouse lung infection model

DX-619 is a novel des-fluoro(6) quinolone with potent activity against gram-positive pathogens. The in vivo activity of DX-619 against Streptococcus pneumoniae was compared with those of fluoro(6) quinolones, sitafloxacin, and ciprofloxacin in a mouse model. Two strains of S. pneumoniae were used: a penicillin-sensitive S. pneumoniae (PSSP) strain and a penicillin-resistant S. pneumoniae (PRSP) strain. Furthermore, these strains showed intermediate susceptibilities to ciprofloxacin. In murine lung infections caused by PSSP, the 50% effective doses (ED50s) of DX-619, sitafloxacin, and ciprofloxacin were 9.15, 11.1, and 127.6 mg/kg of body weight, respectively. Against PRSP-mediated pneumonia in mice, the ED50s of DX-619, sitafloxacin, and ciprofloxacin were 0.69, 4.84, and 38.75 mg/kg, respectively. The mean +/- standard error of the mean viable bacterial counts in murine lungs infected with PSSP and treated with DX-619, sitafloxacin, ciprofloxacin (10 mg/kg twice daily), and saline (twice daily) were 1.75 +/- 0.06, 1.92 +/- 0.23, 6.48 +/- 0.28, and 7.57 +/- 0.13 log10 CFU/ml, respectively. Furthermore, the numbers of viable bacteria in lungs infected with PRSP and treated with the three agents and not treated (control) were 1.73 +/- 0.04, 2.28 +/- 0.17, 4.61 +/- 0.59, and 5.54 +/- 0.72 log10 CFU/ml, respectively. DX-619 and sitafloxacin significantly decreased the numbers of viable bacteria in the lungs compared to the numbers in the lungs of ciprofloxacin-treated and untreated mice. The pharmacokinetic parameter of the area under the concentration-time curve (AUC)/MIC ratio in the lungs for DX-619, sitafloxacin, and ciprofloxacin were 171.0, 21.92, and 1.22, respectively. The AUC/MIC ratio in the lungs was significantly higher for DX-619 than for sitafloxacin and ciprofloxacin. Our results suggest that DX-619 and sitafloxacin are potent against both PSSP and PRSP in our mouse pneumonia model.     

Increased nitric oxide in proportion to the severity of heart failure in patients with dilated cardiomyopathy: close correlation of tumor necrosis factor-alpha with systemic and local production of nitric oxide.

Recent studies have demonstrated that proinflammatory cytokines induce large amounts of nitric oxide (NO) and that the amount increases in patients with congestive heart failure (CHF). There are, however, few reports regarding the relationships between NO production, cytokines and the severity of heart failure, so the plasma concentrations of nitrite and nitrate (NOx), tumor necrosis factor-alpha (TNF-alpha) and brain natriuretic peptide (BNP) were measured in 43 patients with CHF caused by dilated cardiomyopathy and 26 age- and sex-matched normal control subjects. Forearm blood flow (FBF) was measured using plethysmography during infusions of acetylcholine and nitroglycerin and after the administration of the NO synthesis inhibitor L-NMMA (N(G)-monomethyl-L-arginine). Plasma concentrations of both NOx and TNF-alpha were significantly higher in the patient group than in the control group (p<0.001) and correlated closely with BNP concentrations (p<0.001). There was a positive relationship between NOx and TNF-alpha concentrations (r=0.80, p<0.001). Administration of L-NMMA significantly reduced FBF in both groups, and the percent change in FBF from baseline correlated significantly with TNF-alpha concentrations (r=0.63, p<0.001). The FBF response to acetylcholine was depressed in the patient group and correlated inversely with TNF-alpha concentrations. The FBF response to nitroglycerin did not correlate with TNF-alpha concentrations. The findings indicate that the concentrations of NO and TNF-alpha in patients with CHF increase in proportion to the severity of heart failure, and that TNF-alpha plays a role in the enhanced systemic and local production of NO.