9 months of delamanid, linezolid, levofloxacin, and pyrazinamide versus conventional therapy for treatment of fluoroquinolone-sensitive multidrug-resistant tuberculosis (MDR-END): a multicentre, randomised, open-label phase 2/3 non-inferiority trial in South Korea.

BACKGROUND: With the introduction of new anti-tuberculosis drugs, all-oral regimens with shorter treatment durations for multidrug-resistant tuberculosis have been anticipated. We aimed to investigate whether a new all-oral regimen was non-inferior to the conventional regimen including second-line anti-tuberculosis drugs for 20-24 months in the treatment of fluoroquinolone-sensitive multidrug-resistant tuberculosis. METHODS: In this multicentre, randomised, open-label phase 2/3 non-inferiority trial, we enrolled men and women aged 19-85 years with multidrug-resistant tuberculosis confirmed by phenotypic or genotypic drug susceptibility tests or rifampicin-resistant tuberculosis by genotypic tests at 12 participating hospitals throughout South Korea. Participants with fluoroquinolone-resistant multidrug-resistant tuberculosis were excluded. Participants were randomly assigned (1:1) to two groups using a block randomisation, stratified by the presence of diabetes and cavitation on baseline chest radiographs. The investigational group received delamanid, linezolid, levofloxacin, and pyrazinamide for 9 months, and the control group received a conventional 20-24-month regimen, according to the 2014 WHO guidelines. The primary outcome was the treatment success rate at 24 months after treatment initiation in the modified intention-to-treat population and the per-protocol population. Participants who were "cured" and "treatment completed" were defined as treatment success following the 2014 WHO guidelines. Non-inferiority was confirmed if the lower limit of a 97·5% one-sided CI of the difference between the groups was greater than -10%. Safety data were collected for 24 months in participants who received a predefined regimen at least once. This study is registered with ClinicalTrials.gov, NCT02619994. FINDINGS: Between March 4, 2016, and Sept 14, 2019, 214 participants were enrolled, 168 (78·5%) of whom were included in the modified intention-to-treat population. At 24 months after treatment initiation, 60 (70·6%) of 85 participants in the control group had treatment success, as did 54 (75·0%) of 72 participants in the shorter-regimen group (between-group difference 4·4% [97·5% one-sided CI -9·5% to ∞]), satisfying the predefined non-inferiority margin. No difference in safety outcomes was identified between the control group and the shorter-regimen group. INTERPRETATION: 9-month treatment with oral delamanid, linezolid, levofloxacin, and pyrazinamide could represent a new treatment option for participants with fluoroquinolone-sensitive multidrug-resistant tuberculosis. FUNDING: Korea Disease Control and Prevention Agency, South Korea.

Fibroblast Growth Factor 12 Is a Novel Regulator of Vascular Smooth Muscle Cell Plasticity and Fate.

OBJECTIVE: Vascular smooth muscle cells (VSMCs) modulate their phenotype between synthetic and contractile states in response to environmental changes; this modulation plays a crucial role in the pathogenesis of restenosis and atherosclerosis. Here, we identified fibroblast growth factor 12 (FGF12) as a novel key regulator of the VSMC phenotype switch. APPROACH AND RESULTS: Using murine models and human specimens, we found that FGF12 was highly expressed in contractile VSMCs of normal vessel walls but was downregulated in synthetic VSMCs from injured and atherosclerotic vessels. In human VSMCs, FGF12 expression was inhibited at the transcriptional level by platelet-derived growth factor-BB. Gain- and loss-of-function experiments showed that FGF12 was both necessary and sufficient for inducing and maintaining the quiescent and contractile phenotypes of VSMCs. FGF12 inhibited cell proliferation through the p53 pathway and upregulated the key factors involved in VSMC lineage differentiation, such as myocardin and serum response factor. Such FGF12-induced phenotypic change was mediated by the p38 MAPK (mitogen-activated protein kinase) pathway. Moreover, FGF12 promoted the differentiation of mouse embryonic stem cells and the transdifferentiation of human dermal fibroblasts into SMC-like cells. Furthermore, adenoviral infection of FGF12 substantially decreased neointima hyperplasia in a rat carotid artery injury model. CONCLUSIONS: In general, FGF family members induce a synthetic VSMC phenotype. Interestingly, the present study showed the unanticipated finding that FGF12 belonging to FGF family, strongly induced the quiescent and contractile VSMC phenotypes and directly promoted VSMC lineage differentiation. These novel findings suggested that FGF12 could be a new therapeutic target for treating restenosis and atherosclerosis.

Stem cell factor is a potent endothelial permeability factor.

OBJECTIVE: Although stem cell factor (SCF) has been shown to play a critical role in hematopoiesis, gametogenesis, and melanogenesis, the function of SCF in the regulation of vascular integrity has not been studied. APPROACH AND RESULTS: We demonstrated that SCF binds to and activates the cKit receptor in endothelial cells, thereby increasing the internalization of vascular endothelial-cadherin and enhancing extravasation of dyes to a similar extent as vascular endothelial growth factor. SCF-mediated cKit activation in endothelial cells enhanced the phosphorylation of endothelial nitric oxide (NO) synthase via the phosphoinositide 3-kinase/Akt signaling pathway and subsequently increased the production of NO. Inhibition of endothelial NO synthase expression and NO synthesis using small interfering RNA knockdown and chemical inhibitors substantially diminished the ability of SCF to increase the internalization of vascular endothelial-cadherin and in vitro endothelial permeability. SCF-induced increase in extravasation of the dyes was abrogated in endothelial NO synthase knockout mice, which indicates that endothelial NO synthase-mediated NO production was responsible for the SCF-induced vascular leakage. Furthermore, we demonstrated that the expression of SCF and cKit was significantly higher in the retina of streptozotocin-injected diabetic mice than in the nondiabetic control animals. Depletion of SCF by intravitreous injection of anti-SCF-neutralizing immunoglobulin G significantly prevented vascular hyperpermeability in the retinas of streptozotocin-injected diabetic mice. CONCLUSIONS: Our data reveal that SCF disrupts the endothelial adherens junction and enhances vascular leakage, as well as suggest that anti-SCF/cKit therapy may hold promise as a potential therapy for the treatment of hyperpermeable vascular diseases.

Histone deacetylase 3 mediates allergic skin inflammation by regulating expression of MCP1 protein.

We have shown the induction of histone deacetylase 3 (HDAC3) in antigen-stimulated rat basophilic leukemia cells via NF-κB. We investigated the role of HDAC3 in allergic skin inflammation. We used a BALB/c mouse model of triphasic cutaneous anaphylaxis (triphasic cutaneous reaction; TpCR) and passive cutaneous anaphylaxis (PCA) to examine the role of HDAC3 in allergic skin inflammation. Triphasic cutaneous reaction involved induction of HDAC3 and was mediated by HDAC3. HDAC3 showed an interaction with FcεRIß. Trichostatin A (TSA), an inhibitor of HDAC(s), disrupted this interaction. Cytokine array analysis showed that the down-regulation of HDAC3 led to the decreased secretion of monocyte chemoattractant protein 1 (MCP1). FcεRI was necessary for induction of HDAC3 and MCP1. ChIP assays showed that HDAC3, in association with Sp1 and c-Jun, was responsible for induction of MCP1 expression. TSA exerted a negative effect on induction of MCP1. HDAC3 exerted a negative regulation on expression of HDAC2 via interaction with Rac1. The down-regulation of HDAC3 or inactivation of Rac1 induced binding of HDAC2 to MCP1 promoter sequences. TSA exerted a negative effect on HDAC3-mediated TpCR. The BALB/c mouse model of PCA involved induction of HDAC3 and MCP1. HDAC3 and MCP1 were necessary for PCA that involved ear swelling, enhanced vascular permeability, and angiogenesis. Recombinant MCP1 enhanced ß-hexosaminidase activity and histamine release and also showed angiogenic potential. TSA exerted a negative effect on PCA. Our data show HDAC3 as a valuable target for the development of allergic skin inflammation therapeutics.

Tuberculosis: A persistent unpleasant neighbour of humans.

Mycobacterium tuberculosis, the bacterium that causes tuberculosis, has long been an unpleasant neighbour of humans. Following transmission of the bacterium from patients with active infection, new hosts do not immediately develop symptoms, as M. tuberculosis initially remains quiescent. However, it is eventually triggered, leading to the infection of other individuals. Humans are the exclusive host, and the rapid proliferation of the human population worldwide along with increasing globalisation have contributed to the pathogen's persistence, as have the survival strategies employed by M. tuberculosis, especially its resistance to several antimicrobials. Defeating this enemy will require novel approaches.

Investigation of false-positive Mycobacterium tuberculosis culture tests using whole genome sequencing.

Although accurate identification of Mycobacterium tuberculosis is the gold standard for tuberculosis (TB) diagnosis, there have been several reports of false-positive results. After identifying a sudden increase in extensively drug-resistant TB, false-positive mycobacterial culture tests were suspected, and we contacted the supranational reference center for molecular typing. In silico genotyping tests showed that isolates from all five patients had an identical genotype pattern, and all harbored the same Beijing strain based on sequence-based phylogenic analysis and drug-resistant single nucleotide polymorphisms (SNPs) analysis. We also used whole genome sequencing (WGS) to compare the SNPs of all isolates with a reference genome, and all were identical. We adapted WGS to efficiently detect false-positive MTB culture tests.

Inhibitory mechanism of anti-allergic peptides in RBL2H3 cells.

Here we report the identification and functional characterization of several anti-allergic peptides identified through biopanning of pooled sera of patients with various allergies. Several peptides, including LSYLLWRSRLP (LSY), LVAHVGAGGVL (LVA), RVSSCRGRNHIV (RVS), ETIGARWVRIE (ETI), TDGVTYTNDCL (TDG), RVVRYDADFWI (RVV), GFWCRRSGLVGV (GFW), were further characterized. These peptides inhibited the release of histamine from antigen-stimulated mast cells isolated from lung tissues of guinea pigs. Furthermore, the peptides inhibited calcium influx in ovalbumin-stimulated mast cells isolated from lung tissues of guinea pigs. Likewise, the peptides inhibited the release of beta-hexosaminidase from antigen-stimulated rat basophilic leukemia (RBL2H3) cells and decreased calcium influx and intracellular reactive oxygen species production as well. We found that the peptides significantly decreased phosphorylation of extracellular regulated kinase (ERK) and that this was responsible for the decreased calcium influx and beta-hexosaminidase in antigen-stimulated RBL2H3 cells, suggesting that ERK plays an important role in allergic reactions. The peptides identified in this study also affected upstream signaling of allergic inflammation. In other words, these peptides decreased phosphorylation of Lyn, PKCalpha, and -delta. Lyn and PKC are known to be responsible for the phosphorylation of FcepsilonRI, in response to receptor aggregation. The peptides inhibited interaction between IgE and FcepsilonRI, suggesting that these peptides exert anti-allergic effects by inhibiting receptor cross-linking. These peptides also inhibited interaction between FcepsilonRI and PKCdelta. Taken together, these data suggest that peptides exert anti-allergic effect through the inhibition of upstream signaling, involving receptor cross-linking, and downstream multiple signaling.

Significance of p-STAT3 expression in human colorectal adenocarcinoma.

To examine the relationship between p-STAT3 and the clinicopathological parameters of colorectal adenocarcinoma (CRA), we initially conducted immunohistochemical (IHC) analyses on formalin-fixed tissues. A total of 127 invasive CRA, 20 colorectal adenomas and 20 normal mucosae were obtained. To clarify the validity of p-STAT3 as determined by the IHC analysis, quantitative real-time PCR was performed on fresh samples from 51 CRA-4 carcinomas in situ, 47 invasive CRA and on 51 normal mucosae. IHC analyses were conducted after formalin fixation from 51 CRA for comparison. The statistically significant difference of immunoreactivity for p-STAT3 between the CRA and adenoma, and between the CRA and normal mucosae was identified. Among the 174 CRA, p-STAT3 immunoreactivity significantly correlated with the T- and clinical stage. Among the 47 invasive CRA, the expression of STAT3 as determined by real-time PCR significantly correlated with tumor size, M stage and clinical stage. The overall findings of the real-time PCR analyses correlated with the findings of the IHC analyses. These findings suggest that p-STAT3 expression has an important role related to the tumorigenesis and tumor progression of CRAs. Moreover, IHC analysis is a reliable and useful modality of assessing the status of p-STAT3 expression in formalin-fixed samples.

Epigenetic mechanisms involved in differential MDR1 mRNA expression between gastric and colon cancer cell lines and rationales for clinical chemotherapy.

BACKGROUND: The membrane transporters such as P-glycoprotein (Pgp), the MDR1 gene product, are one of causes of treatment failure in cancer patients. In this study, the epigenetic mechanisms involved in differential MDR1 mRNA expression were compared between 10 gastric and 9 colon cancer cell lines. METHODS: The MDR1 mRNA levels were determined using PCR and real-time PCR assays after reverse transcription. Cytotoxicity was performed using the MTT assay. Methylation status was explored by quantification PCR-based methylation and bisulfite DNA sequencing analyses. RESULTS: The MDR1 mRNA levels obtained by 35 cycles of RT-PCR in gastric cancer cells were just comparable to those obtained by 22 cycles of RT-PCR in colon cancer cells. Real-time RT-PCR analysis revealed that MDR1 mRNA was not detected in the 10 gastric cancer cell lines but variable MDR1 mRNA levels in 7 of 9 colon cancer cell lines except the SNU-C5 and HT-29 cells. MTT assay showed that Pgp inhibitors such as cyclosporine A, verapamil and PSC833 sensitized Colo320HSR (colon, highest MDR1 expression) but not SNU-668 (gastric, highest) and SNU-C5 (gastric, no expression) to paclitaxel. Quantification PCR-based methylation analysis revealed that 90% of gastric cancer cells, and 33% of colon cancer cells were methylated, which were completely matched with the results obtained by bisulfite DNA sequencing analysis. 5-aza-2'-deoxcytidine (5AC, a DNA methyltransferase inhibitor) increased the MDR1 mRNA levels in 60% of gastric cells, and in 11% of colon cancer cells. Trichostatin A (TSA, histone deacetylase inhibitor) increased the MDR1 mRNA levels in 70% of gastric cancer cells and 55% of colon cancer cells. The combined treatment of 5AC with TSA increased the MDR1 mRNA levels additively in 20% of gastric cancer cells, but synergistically in 40% of gastric and 11% of colon cancer cells. CONCLUSION: These results indicate that the MDR1 mRNA levels in gastric cancer cells are significantly lower than those in colon cancer cells, which is at least in part due to different epigenetic regulations such as DNA methylation and/or histone deacetylation. These results can provide a better understanding of the efficacy of combined chemotherapy as well as their oral bioavailability.

Detection of resistance to fluoroquinolones and injectable drugs among antituberculosis drugs by allele-specific primer extension on a microsphere-based platform.

Molecular drug susceptibility testing (DST) for antituberculosis drugs is important for improving the efficacy of multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) treatment. In this study, we developed a molecular high-throughput assay system based on allele-specific primer extension (ASPE) and MagPlex-TAG microspheres, referred to here as TAG-ASPE, which can detect mutations related to resistance to injectable second-line drugs and fluoroquinolones. Target genes were amplified by multiplex PCR using DNA from H37Rv and 190 clinical Mycobacterium tuberculosis strains and extended by ASPE using 22 ASPE primers. ASPE products were then sorted on the TAG-ASPE array and detected using a Luminex 200 system. The performance of the TAG-ASPE method was compared with that of sequencing and phenotypic DST. Comparison of the TAG-ASPE method with sequencing showed that the sensitivity and specificity of the TAG-ASPE method were 100% [95% confidence interval (CI), 96.38-100%] and 100% (95% CI, 95.70-100%) for the rrs gene and 100% (95% CI, 96.90-100%) and 100% (95% CI, 95.07-100%) for the gyrA gene, respectively. Compared with phenotypic DST, the sensitivity and specificity of the TAG-ASPE method for detecting drug-resistance mutations against injectable second-line drugs were 92.52% (95% CI, 85.8-96.72%) and 98.7% (95% CI, 92.98-99.97%), respectively. Additionally, the sensitivity and specificity for fluoroquinolone-resistance detection were 85.4% (95% CI, 78.36-90.85%) and 100% (95% CI, 92.38-100%), respectively. The results of this study demonstrate that the TAG-ASPE method can effectively detect mutations conferring resistance to second-line antituberculosis drugs in numerous clinical specimens.

Determining Genotypic Drug Resistance by Ion Semiconductor Sequencing With the Ion AmpliSeq™ TB Panel in Multidrug-Resistant Mycobacterium tuberculosis Isolates.

BACKGROUND: We examined the feasibility of a full-length gene analysis for the drug resistance-related genes inhA, katG, rpoB, pncA, rpsL, embB, eis, and gyrA using ion semiconductor next-generation sequencing (NGS) and compared the results with those obtained from conventional phenotypic drug susceptibility testing (DST) in multidrug-resistant Mycobacterium tuberculosis (MDR-TB) isolates. METHODS: We extracted genomic DNA from 30 pure MDR-TB isolates with antibiotic susceptibility profiles confirmed by phenotypic DST for isoniazid (INH), rifampin (RIF), ethambutol (EMB), pyrazinamide (PZA), amikacin (AMK), kanamycin (KM), streptomycin (SM), and fluoroquinolones (FQs) including ofloxacin, moxifloxacin, and levofloxacin. Enriched ion spheres were loaded onto Ion PI Chip v3, with 30 samples on a chip per sequencing run, and Ion Torrent sequencing was conducted using the Ion AmpliSeq TB panel (Life Technologies, USA). RESULTS: The genotypic DST results revealed good agreement with the phenotypic DST results for EMB (Kappa 0.8), PZA (0.734), SM (0.769), and FQ (0.783). Agreements for INH, RIF, and AMK+KM were not estimated because all isolates were phenotypically resistant to INH and RIF, and all isolates were phenotypically and genotypically susceptible to AMK+KM. Moreover, 17 novel variants were identified: six (p.Gly169Ser, p.Ala256Thr, p.Ser383Pro, p.Gln439Arg, p.Tyr597Cys, p.Thr625Ala) in katG, one (p.Tyr113Phe) in inhA, five (p.Val170Phe, p.Thr400Ala, p.Met434Val, p.Glu812Gly, p.Phe971Leu) in rpoB, two (p.Tyr319Asp and p.His1002Arg) in embB, and three (p.Cys14Gly, p.Asp63Ala, p.Gly162Ser) in pncA. CONCLUSIONS: Ion semiconductor NGS could detect reported and novel amino acid changes in full coding regions of eight drug resistance-related genes. However, genotypic DST should be complemented and validated by phenotypic DSTs.

Comparison of DNA extraction methods for drug susceptibility testing by allele-specific primer extension on a microsphere-based platform: Chelex-100 (in-house and commercialized) and MagPurix TB DNA Extraction Kit.

Tuberculosis (TB), caused by infections of the Mycobacterium tuberculosis (MTB) complex, is the ninth leading cause of death worldwide, and several molecular approaches for MTB species identification and the detection of mutations associated with drug resistance have been developed to date. We previously developed a diagnostic assay for drug susceptibility testing that can detect mutations conferring resistance to anti-TB drugs using allele-specific primer extension on a microsphere-based platform for multiplex polymerase chain reaction. The aim of the present study was to optimize this diagnostic assay based on the evaluation of three methods for extracting mycobacterial DNA from clinical samples. Mycobacterial DNA of 81 samples was digested and decontaminated by N-acetyl-l-cysteine-2% NaOH and then extracted using three methods: "in-house" 5% Chelex-100 chelating resin, InstaGene Matrix, and MagPurix TB DNA Extraction Kit. The former two methods are manual extraction methods, whereas the MagPurix TB DNA Extraction Kit is an automated extraction method used with the MagPurix 12 s automated nucleic acid purification system. The extracted DNA was then subjected to our diagnostic assay, and the results were compared among methods. The magnetic bead method exhibited a higher extraction efficiency and resulted in greater diagnostic efficacy than the two resin-based methods with respect to both target gene detection and acid-fast bacilli smear grades. Therefore, the MagPurix TB DNA Extraction Kit is the optimal MTB DNA extraction method for our diagnostic assay of TB drug susceptibility testing.

A molecular epidemiological analysis of tuberculosis trends in South Korea.

Molecular epidemiological data are needed to assess tuberculosis (TB)-management policy outcomes in South Korea. IS6110 restriction fragment-length polymorphism (IS6110-RFLP) and mycobacterial interspersed repetitive unit-variable-number tandem repeat (MIRU-VNTR) analyses are major molecular epidemiological tools for investigating the transmission or reactivation of active TB. Here, we determined trends in the clustering rate (i.e., the prevalence of Mycobacterium tuberculosis isolates with identical genotype patterns) of active TB and related differences between the 1990s and 2000s in Korea. M. tuberculosis isolates (1,007) of nationwide origins were analyzed by IS6110-RFLP and 24-locus standardized MIRU-VNTR genotyping. The clustering rate was measured by IS6110-RFLP, 24-locus MIRU-VNTR, and both analytical methods in combination. IS6110-RFLP, 24-locus MIRU-VNTR typing, and the combined method revealed 882, 754, and 983 distinct profiles; 809, 651, and 961 unique isolates; and 198, 356, and 46 clustered isolates grouped into 73, 103, and 22 clusters, respectively. In addition, we confirmed that the clustering rates in the 2000s decreased by 11.2%, 2.1%, and 3.1% relative to that in the 1990s using the three methods, respectively. Furthermore, in multivariate analysis, the younger-age group (<30) clustered more frequently than the older-age group (>50), based on all the three methods. Our study is the first report to provide nationwide molecular epidemiological information on TB in Korea.

Effect of betulinic acid on anticancer drug-resistant colon cancer cells.

Primary or acquired resistance of tumours to established chemotherapeutic regimens is a major concern in oncology. Attempts to improve the survival of cancer patients largely depend on strategies to prevent tumour cell resistance. 5-Fluorouracil (5-FU)-based chemotherapy with a combination of other drugs such as irinotecan (IRT) and oxaliplatin (OXT) has been reported to be effective, even though an optimal regimen has yet to be defined due to the relatively high toxicity of the procedure. The aim of this study was to examine the effect of betulinic acid (BetA) as a chemosensitizer for anticancer drug treatment in chemoresistant colon cancer cell lines. A chemoresistant cell line to 5-fluorouracil (SNU-C5/5FU-R), irinotecan (SNU-C5/IRT-R) and oxaliplatin (SNU-C5/OXT-R) treatment were derived from the wild-type colon adenocarcinoma cell line (SNU-C5/WT). The effect of BetA or a combination of anticancer drugs and BetA on the multidrug resistance-related genes, caspases, Bcl-2, Bad and cell death in the SNU-C5/WT and SNU-C5/R cell lines was analysed. BetA alone was an effective chemotherapeutic drug for the SNU-C5/WT, SNU-C5/5FU-R and SNU-C5/OXT-R cells. The combination of BetA with IRT or OXT was effective against SNU-C5/5FU-R cells, and the combination of BetA with 5-fluorouracil, IRT or OXT was effective against SNU-C5/OXT-R cells. BetA induced cancer cell death by apoptosis through the mitochondrial pathway. These findings indicate that the use of BetA as a chemosensitizer may be a new strategy to enhance the efficacy of chemotherapy. However, further studies will be needed for confirmation.

Expression of cyclooxygenase-2 and its relationship to p53 accumulation in colorectal cancers.

PURPOSE: Cyclooxygenase (COX)-2 is an inducible isoform responsive to cytokines, mitogens, and growth factors, and is believed to be an important enzyme related to colorectal cancer (CRC). Existing evidence suggests that COX-2 expression is normally suppressed by wild-type p53 but not mutant p53, suggesting that loss of p53 function may result in the induction of COX-2 expression. The aim of this study was to determine the relationship between COX-2 expression and p53 levels in CRC. MATERIALS AND METHODS: Patients with sporadic colorectal adenocarcinoma (n=161) who underwent curative surgery in Chosun University Hospital were enrolled in this study. Expression of COX-2 and p53 proteins was examined by immunohistochemistry in paraffin-embedded cancer tissue blocks, and the relationship between COX-2 and/or p53 expression with clinicopathologic parameters was analyzed. RESULTS: Expression of COX- 2 was positive in 47.8% of colorectal cancers, and significantly associated with the depth of tumor invasion (p= 0.042). In contrast, p53 was positive in 50.3% of the cases, and was associated with both age (p=0.025) and the depth of tumor invasion (p=0.014). There was no correlation between COX-2 expression and p53 expression (p=0.118). CONCLUSION: These results suggest that COX-2 expression might play an important role in the progression of colorectal cancer. However, COX-2 expression was not associated with mutational p53. Further studies are needed to clarify the regulatory mechanisms governing COX-2 overexpression in colorectal cancers.

Molecular mechanisms of heptaplatin effective against cisplatin-resistant cancer cell lines: less involvement of metallothionein.

BACKGROUND: Heptaplatin is a new platinum derivative with anticancer activity against various cancer cell lines, including cisplatin-resistant cancer cell lines (Cancer Chemother Pharmacol 1995; 35: 441). METHODS: Molecular mechanisms of heptaplatin effective against cisplatin-resistant cancer cell lines has been investigated in connection with metallothionein (MT). Cytotoxicity was determined by an MTT assay. MT mRNA, was determined by RT-PCR assay. Transfection study was carried out to examine the function of MT. RESULTS: Of various gastric cancer cell lines, SNU-638 and SNU-601 showed the highest and lowest levels of MT mRNA, respectively, showing 80-fold difference. The IC50 values of SNU-638 to cisplatin, carboplatin and heptaplatin were 11.2-fold, 5.1-fold and 2.0-fold greater than those of SNU-601, respectively. Heptaplatin was more effective against cisplatin-resistant and MT-transfected gastric cancer sublines than cisplatin or carboplatin was. In addition, heptaplatin attenuated cadmium, but not zinc, induction of MT. CONCLUSION: These results indicate that molecular mechanisms of heptaplatin effective against cisplatin-resistant gastric cancer sublines is at least in part due to the less involvement of MT in heptaplatin resistance as well as its attenuation of MT induction.

Differential induction of Mn-containing superoxide dismutase by paraquat in peripheral lymphocytes of normal subjects and gastric cancer patients.

Reactive oxygen species (ROS) cause macromolecular damage and may play an important role in tumor development. Superoxide dismutase (SOD) and metallothionein (MT) serve as initial and final defense mechanisms, respectively, against ROS. We hypothesized that the inducibility of Mn-SOD and MT mRNA by paraquat, an intracellular superoxide generator, might be altered in lymphocytes of gastric cancer patients. The inducibility of Mn-SOD mRNA by paraquat in lymphocytes of 19 normal subjects and the 14 gastric cancer patients was 162.4 +/- 16.7% and 87.9 +/- 9.5%, respectively (P = 0.001). The inducibility of MT mRNA by paraquat in the normal subjects and the gastric cancer patients was 126.7 +/- 15.8% and 115.4 +/- 12.9%, respectively. This suggests that the failure of Mn-SOD mRNA induction by oxidative stress in peripheral lymphocytes may be involved in the development of gastric cancer and may be of value in predicting the future occurrence of gastric cancer. In addition, the wide variation in Mn-SOD and MT mRNA levels among normal subjects may reflect different susceptibilities to diseases including cancer.

Reactive oxygen species regulate the quiescence of CD34-positive cells derived from human embryonic stem cells.

AIMS: Reactive oxygen species (ROS) are involved in a wide range of cellular processes. However, few studies have examined the generation and function of ROS in human embryonic vascular development. In this study, the sources of ROS and their roles in the vascular differentiation of human embryonic stem cells (hESCs) were investigated. METHODS AND RESULTS: During vascular differentiation of hESCs, CD34(+) cells had quiescence-related gene expression profiles and a large fraction of these cells were in G0 phase. In addition, levels of ROS, which were primarily generated through NOX4, were substantially higher in hESC-derived CD34(+) cells than in hESC-derived CD34(-) cells. To determine whether excess levels of ROS induce quiescence of hESC-derived CD34(+) cells, ROS levels were moderately reduced using selenium to enhance antioxidant activities of thioredoxin reductase and glutathione peroxidase. In comparison to untreated CD34(+) cells, selenium-treated CD34(+) cells exhibited changes in gene expression that favoured cell cycle progression, and had a greater proliferation and a smaller fraction of cells in G0 phase. Thus, selenium treatment increased the number of hESC-derived CD34(+) cells, thereby enhancing the efficiency with which hESCs differentiated into vascular endothelial and smooth muscle cells. CONCLUSION: This study reveals that NOX4 produces ROS in CD34(+) cells during vascular differentiation of hESCs, and shows that modulation of ROS levels using antioxidants such as selenium may be a novel approach to increase the vascular differentiation efficiency of hESCs.

DNA methyl transferase I acts as a negative regulator of allergic skin inflammation.

The role of DNA methyl transferase I (DNMT1) in allergic inflammation was investigated. Antigen stimulation decreased expression of DNMT1 in rat basophilic leukemia cells (RBL2H3). The down regulation of DNMT1 induced expression of histone deacetylase 3 (HDAC3). HDAC3 was necessary for allergic skin inflammation, such as such as triphasic cutaneous reaction and passive cutaneous anaphylaxis. The down regulation of DNMT1 resulted from activation of PKC and rac1 which were necessary for proteasome-dependent ubiquitination of DNMT1 by antigen stimulation. N-acetyl-L-cysteine, an inhibitor of reactive oxygen species production, exerted negative effects on allergic skin inflammation. Antigen stimulation led to increased expression of Tip60, a histone acetyl transferase. Wild type, but not mutant form, Tip60 decreased expression of DNMT1 while increasing expression of HDAC3, suggesting role for acetylation in ubiquitin-dependent proteasomal degradation of DNMT1. In vivo down regulation of DNMT1 increased ear thickness, typical of allergic skin inflammation, induced vascular leakage and promoted angiogenesis in BALB/c mouse. The down regulation of DNMT1 enhanced angiogenic potential of rat aortic endothelial cells (RAEC) accompanied by activation of VEGR-2 and induced interaction between VEGR-2 and syk in RAEC. The enhanced angiogenic potential of RAEC was associated with the induction of VEGF by down regulation of DNMT1 in RBL2H3 cells. The down regulation of DNMT1 induced leukocytes-endothelial cell interaction and expression of various adhesion molecules. Aspirin exerted a negative effect on allergic skin inflammation by indirect regulation on DNMT1 via Tip60. Taken together, these results suggest novel role for DNMT1 in allergic skin inflammation.

Reducing the pain of microemulsion propofol injections: a double-blind, randomized study of three methods of tourniquet and lidocaine.

BACKGROUND: Although the new formulation of lipid-free microemulsion propofol (MP) has some advantages over the lipid emulsion, it reportedly produces more injection pain than lipid-based propofol. Intravenous lidocaine with application of a rubber tourniquet before administration of propofol is considered to be the best method for reducing injection pain; however, this technique is not perfect. OBJECTIVE: The goal of this study was to evaluate the effect of different methods of tourniquet application and lidocaine administration on MP injection pain. METHODS: This single-center, randomized controlled clinical trial was conducted in 140 patients aged 18 to 65 years. Patients were randomly divided into 4 groups (n = 35 each). Group A received MP (2 mg/kg) after lidocaine (0.6 mg/kg) with a tourniquet with arm down (venous engorgement); group B received MP after lidocaine with a tourniquet with arm up (venous gravity drainage); group C received MP with a tourniquet with arm down; and group D (control group) received MP only (with no tourniquet). In groups A and C, the tourniquet was released after MP; in group B, the tourniquet was released before MP. Injection pain was evaluated by using a verbal pain score (VPS). The bispectral index, the time from the beginning of drug injection to the loss of eyelash reflex, and time to the lowest bispectral index value were recorded. RESULTS: Group A showed significantly less incidence of pain than the control group when MP was injected. The mean VPS was significantly lower in groups A, B, and C than in group D (the control group). The VPS of group A was significantly lower than that in group B. Other observed values were not significantly different. CONCLUSIONS: We concluded that intravenous retention of lidocaine with the application of a rubber tourniquet under venous engorgement of the arm reduces the incidence and intensity of MP injection pain. CLINICAL TRIAL REGISTRY: UMIN000010725.