Protective effects of imeglimin on the development of atherosclerosis in ApoE KO mice treated with STZ.

BACKGROUND: Imeglimin is a new anti-diabetic drug which promotes insulin secretion from pancreatic ß-cells and reduces insulin resistance in insulin target tissues. However, there have been no reports examining the possible anti-atherosclerotic effects of imeglimin. In this study, we investigated the possible anti-atherosclerotic effects of imeglimin using atherosclerosis model ApoE KO mice treated with streptozotocin (STZ). METHODS: ApoE KO mice were divided into three groups: the first group was a normoglycemic group without injecting STZ (non-DM group, n = 10). In the second group, mice were injected with STZ and treated with 0.5% carboxymethyl cellulose (CMC) (control group, n = 12). In the third group, mice were injected with STZ and treated with imeglimin (200 mg/kg, twice daily oral gavage, n = 12). We observed the mice in the three groups from 10 to 18 weeks of age. Plaque formation in aortic arch and expression levels of various vascular factors in abdominal aorta were evaluated for each group. RESULTS: Imeglimin showed favorable effects on the development of plaque formation in the aortic arch in STZ-induced hyperglycemic ApoE KO mice which was independent of glycemic and lipid control. Migration and proliferation of vascular smooth muscle cells and infiltration of macrophage were observed in atherosclerotic lesions in STZ-induced hyperglycemic ApoE KO mice, however, which were markedly reduced by imeglimin treatment. In addition, imeglimin reduced oxidative stress, inflammation and inflammasome in hyperglycemic ApoE KO mice. Expression levels of macrophage makers were also significantly reduced by imeglimin treatment. CONCLUSIONS: Imeglimin exerts favorable effects on the development of plaque formation and progression of atherosclerosis.

Intramolecular Cyclopropanation of Active Methylene Derivatives Based on FeCl2 or FeCl3-Promoted Radical-Polar Crossover Reactions.

Radical-polar crossover reactions were studied for the intramolecular cyclopropanation of active methylene derivatives. In the presence of FeCl3 as a stoichiometric oxidant and K2HPO4 as a base, the dehydrogenative cyclopropanation of active methylenes proceeded through the FeCl3-promoted oxidative radical cyclization followed by the ionic cyclization to give the bicyclic cyclopropanes. The use of α-chloro-active methylenes leads the subcatalytic cyclopropanation involving two redox pathways. In the presence of K2HPO4, the redox cyclopropanation proceeded by using FeCl2 (20 mol%) in combination with ligand (20 mol%).

Targeting ROR1 in combination with osimertinib in EGFR mutant lung cancer cells.

Lung cancer that exhibits epidermal growth factor receptor (EGFR) gene mutation is sensitive to EGFR-tyrosine kinase inhibitors (TKIs), such as osimertinib. Receptor tyrosine kinase-like orphan receptor 1 (ROR1) may be involved in overcoming EGFR-TKI resistance. Growth inhibition, colony formation, apoptosis, and mRNA/protein levels in four osimertinib-sensitive and resistant cell lines transfected with small interfering RNA (siRNA) targeting ROR1 (siROR1) were evaluated. Cell growth and colony formation were suppressed and apoptosis was increased in all cell lines treated with siROR1. Although EGFR, AKT, and ERK phosphorylation were not suppressed in all cell lines, TGF-ß2, AXL, CDH2, PARP1, PEG10, and TYMS mRNA expression levels were reduced. The combination of osimertinib with siROR1 was effective for the four cell lines, particularly in the two osimertinib-sensitive lines. In conclusion, targeting ROR1 in combination with osimertinib in EGFR mutant lung cancer may be a novel therapeutic option.

Lead discovery for mammalian elongation of long chain fatty acids family 6 using a combination of high-throughput fluorescent-based assay and RapidFire mass spectrometry assay.

A high-throughput RapidFire mass spectrometry assay is described for elongation of very long-chain fatty acids family 6 (Elovl6). Elovl6 is a microsomal enzyme that regulates the elongation of C12-16 saturated and monounsaturated fatty acids. Elovl6 may be a new therapeutic target for fat metabolism disorders such as obesity, type 2 diabetes, and nonalcoholic steatohepatitis. To identify new Elovl6 inhibitors, we developed a high-throughput fluorescence screening assay in 1536-well format. However, a number of false positives caused by fluorescent interference have been identified. To pick up the real active compounds among the primary hits from the fluorescence assay, we developed a RapidFire mass spectrometry assay and a conventional radioisotope assay. These assays have the advantage of detecting the main products directly without using fluorescent-labeled substrates. As a result, 276 compounds (30%) of the primary hits (921 compounds) in a fluorescence ultra-high-throughput screening method were identified as common active compounds in these two assays. It is concluded that both methods are very effective to eliminate false positives. Compared with the radioisotope method using an expensive 14C-labeled substrate, the RapidFire mass spectrometry method using unlabeled substrates is a high-accuracy, high-throughput method. In addition, some of the hit compounds selected from the screening inhibited cellular fatty acid elongation in HEK293 cells expressing Elovl6 transiently. This result suggests that these compounds may be promising lead candidates for therapeutic drugs. Ultra-high-throughput fluorescence screening followed by a RapidFire mass spectrometry assay was a suitable strategy for lead discovery against Elovl6.

Pharmacokinetics and disposition of CS-0777, a sphingosine 1-phosphate receptor modulator, in rats and monkeys.

1. Disposition and metabolism of CS-0777 (1-{5-[(3R)-3-amino-4-hydroxy-3- methylbutyl]-1-methyl-1H-pyrrol-2-yl}-4-(4-methylphenyl) butan-1-one), a selective sphingosine 1-phosphate receptor-1 modulator under development for autoimmune conditions was investigated following oral and/or i.v. bolus administration to rats and monkeys. 2. After oral administration of [14C]CS-0777, CS-0777 was well absorbed in rats and monkeys with total recoveries of over 90% of the dose, majorly in feces. CS-0777 and phosphorylated pharmacologically active metabolite of CS-0777 (M1) were highly bound to plasma proteins among rats, monkeys and humans (>93%). 3. The structures of 12 metabolites were identified and phosphorylation and two hydroxylation pathways were proposed as primary metabolism. In the blood of rats and monkeys, the major metabolite was M1 and a few phosphorylated metabolites were also detected. Meanwhile, in urine and feces of rats and monkeys, not phosphorylated, but oxidized CS-0777 metabolites and/or those various conjugated metabolites were observed. This suggests that CS-0777 and its oxidized metabolites would be phosphorylated in the body, but their phosphorylated metabolites would revert back to their dephosphorylated form again then be further metabolized and finally eliminated from the body. 4. Pharmacokinetic analysis using a reversible metabolism model revealed that the clearance of phosphorylation was larger than the clearance of dephosphorylation and elimination.

[The role and future task of the occupational health nurse].

The Industrial Safety and Health Act was enacted focusing on occupational disease prevention in 1972. It has been revised over the years to include consideration of work associated diseases, and the participation and cooperation of employer and employees. From now, positive participation of employer-and-employees in occupational health activity becomes important in order to achieve the expanded purpose of the law. It is necessary to empower all workers to be able to perform occupational health activity independently. Florence Nightingale defined nursing in the 1850's. "Nursing is to put the patient in the best condition by improvement of environment, including a population approach. The goal of nursing is to enable the patient to use his faculty fully." The Public Health Nurse is, "assistance to the process of solving one's health, identifying health issues based on a community, using systematic measures which lead to prevention, and aiming at public responsibility." The daily activity of Nurses including Occupational Health Nurses (OHNs) is based on the theory and technology of "empowerment". In promoting the employer-and-employees independent Occupational Safety and Health Activity, the OHN's professional specialty of "empowerment" can play an important role.

Comprehensive Search for GPCR Compounds which Can Enhance MafA and/or PDX-1 Expression Levels Using a Small Molecule Compound Library.

It has been shown that chronic hyperglycemia gradually decreases insulin biosynthesis and secretion which is accompanied by reduced expression of very important insulin gene transcription factors MafA and PDX-1. Such phenomena are well known as ß-cell glucose toxicity. It has been shown that the downregulation of MafA and/or PDX-1 expression considerably explains the molecular mechanism for glucose toxicity. However, it remained unknown which molecules can enhance MafA and/or PDX-1 expression levels. In this study, we comprehensively searched for G protein-coupled receptor (GPCR) compounds which can enhance MafA and/or PDX-1 expression levels using a small molecule compound library in pancreatic ß-cell line MIN6 cells and islets isolated from nondiabetic C57BL/6 J mice and obese type 2 diabetic C57BL/KsJ-db/db mice. We found that fulvestrant and dexmedetomidine hydrochloride increased MafA, PDX-1, or insulin expression levels in MIN6 cells. We confirmed that fulvestrant and dexmedetomidine hydrochloride increased MafA, PDX-1, or insulin expression levels in islets from nondiabetic mice as well. Furthermore, these reagents more clearly enhanced MafA, PDX-1, or insulin expression levels in islets from obese type 2 diabetic db/db mice in which MafA and PDX-1 expression levels are reduced due to glucose toxicity. In conclusion, fulvestrant and dexmedetomidine hydrochloride increased MafA, PDX-1, or insulin expression levels in MIN6 cells and islets from nondiabetic mice and obese type 2 diabetic db/db mice. To the best of our knowledge, this is the first report showing some molecule which can enhance MafA and/or PDX-1 expression levels. Therefore, although further extensive study is necessary, we think that the information in this study could be, at least in part, useful at some point such as in the development of new antidiabetes medicine based on the molecular mechanism of ß-cell glucose toxicity in the future.

The endoplasmic reticulum stress-inducible protein, Herp, is a potential triggering antigen for anti-DNA response.

Anti-dsDNA Abs are highly specific indicators of systemic lupus erythematosus (SLE) and play a pathogenic role in lupus nephritis. Human anti-dsDNA Abs are most likely generated by an Ag-driven mechanism. However, the Ag responsible for triggering anti-dsDNA Ab production has not been identified. To search for proteins that are cross-reactive with anti-dsDNA Abs, we screened a cDNA library from a patient with SLE with single-chain Fv of O-81 human anti-ss/dsDNA mAb by using a two-hybrid system. Homocysteine-induced ER protein (Herp), an endoplasmic reticulum (ER) stress-inducible ER membrane protein, was identified and shown to bind to original O-81 Ab and human lupus anti-dsDNA Abs. Some IgG purified from patients with active SLE by Herp-immobilized affinity chromatography bound to dsDNA. BALB/c mice immunized with Herp showed IgG anti-dsDNA Abs, IgG anti-nucleosome Abs, and glomerular IgG deposition. Herp reactivity was strongly positive in a proportion of PBLs from patients with active SLE, but undetectable in those from healthy controls. Moreover, activation of caspases was observed in the Herp-positive cells, implying that ER stress-induced apoptosis likely occurs in patients with active SLE. Herp is exposed on blebs of ER stress-induced apoptotic cells, suggesting that Herp can be recognized by immune cells. These results indicate that Herp mimics structural determinants of DNA immunologically and can be immunogenic in vivo. Thus, Herp represents a candidate autoantigen for anti-DNA Abs. This study may help explain how common environmental factors induce the production of anti-DNA Abs and contribute the development of SLE.

Discovery of Apararenone (MT-3995) as a Highly Selective, Potent, and Novel Nonsteroidal Mineralocorticoid Receptor Antagonist.

Overactivation of the mineralocorticoid receptor (MR) is involved in many diseases, such as hypertension, kidney disease, and heart failure. Thus, MR antagonists (MRAs) are expected to be beneficial to patients with these diseases. In order to identify novel nonsteroidal MRAs that overcome the issues of already marketed steroidal MRAs, we searched for new compounds guided by our hypothesis that T-shaped compounds with a hydrophobic core structure, two polar functional groups at both extremities able to interact with MR, and a bulky substituent that can interfere with the folding of the C-terminal helix 12 may exhibit antagonist activity toward MR. We discovered that the novel 1,4-benzoxazin-3-one derivative 19 (apararenone: MT-3995) acted as a highly selective and potent nonsteroidal MRA. Apararenone exhibited a more potent antihypertensive and organ-protective activity than steroidal MRA eplerenone in a primary aldosteronism rat model obtained by infusing aldosterone in uninephrectomized rats.

Imeglimin exerts favorable effects on pancreatic ß-cells by improving morphology in mitochondria and increasing the number of insulin granules.

Imeglimin is a new anti-diabetic drug commercialized in Japan (Twymeeg®) and has been drawing much attention in diabetes research area as well as in clinical practice. In this study, we evaluated the effect of imeglimin on pancreatic ß-cells. First, single-dose administration of imeglimin enhanced insulin secretion from ß-cells and decreased blood glucose levels in type 2 diabetic db/db mice. In addition, single-dose administration of imeglimin significantly augmented insulin secretion in response to glucose from islets isolated from non-diabetic db/m mice. Second, during an oral glucose tolerance test 4-week chronic treatment with imeglimin enhanced insulin secretion and ameliorated glycemic control in diabetic db/db mice. Furthermore, the examination with electron microscope image showed that imeglimin exerted favorable effects on morphology in ß-cell mitochondria and substantially increased the number of insulin granules in type 2 diabetic db/db and KK-Ay mice. Finally, imeglimin reduced the percentage of apoptotic ß-cell death which was accompanied by reduced expression levels of various genes related to apoptosis and inflammation in ß-cells. Taken together, imeglimin directly enhances insulin secretion in response to glucose from ß-cells, increases the number of insulin granules, exerts favorable effects on morphology in ß-cell mitochondria, and reduces apoptotic ß-cell death in type 2 diabetic mice, which finally leads to amelioration of glycemic control.

Early combination therapy of empagliflozin and linagliptin exerts beneficial effects on pancreatic ß cells in diabetic db/db mice.

Effects of combination therapy of dipeptidyl peptidase-4 (DPP-4) inhibitor and sodium-glucose co-transporter 2 (SGLT2) inhibitor on ß-cells are still unclear, although combination agent of these two drugs has become common in clinical practice. Therefore, we aimed to elucidate the effects of DPP-4 inhibitor and/or SGLT2 inhibitor on ß-cell mass and function and compared their effects between in an early and advanced phase of diabetes. We used 7-week-old db/db mice as an early phase and 16-week-old mice as an advanced phase and treated them for 2 weeks with oral administration of linagliptin, empagliflozin, linagliptin + empagliflozin (L + E group), and 0.5% carboxymethylcellulose (Cont group). Blood glucose levels in Empa and L + E group were significantly lower than Cont group after treatment. In addition, ß-cell mass in L + E group was significantly larger than Cont group only in an early phase, accompanied by increased Ki67-positive ß-cell ratio. In isolated islets, mRNA expression levels of insulin and its transcription factors were all significantly higher only in L + E group in an early phase. Furthermore, mRNA expression levels related to ß-cell differentiation and proliferation were significantly increased only in L + E group in an early phase. In conclusion, combination of DPP-4 inhibitor and SGLT2 inhibitor exerts more beneficial effects on ß-cell mass and function, especially in an early phase of diabetes rather than an advanced phase.

Glutamate-activated chloride channels: Unique fipronil targets present in insects but not in mammals.

Selectivity to insects over mammals is one of the important characteristics for a chemical to become a useful insecticide. Fipronil was found to block cockroach GABA receptors more potently than rat GABA(A) receptors. Furthermore, glutamate-activated chloride channels (GluCls), which are present in cockroaches but not in mammals, were very sensitive to the blocking action of fipronil. The IC(50)s of fipronil block were 30 nM in cockroach GABA receptors and 1600 nM in rat GABA(A) receptors. Moreover, GluCls of cockroach neurons had low IC(50)s for fipronil. Two types of glutamate-induced chloride current were obswerved: desensitizing and non-desensitizing, with fipronil IC(50)s of 800 and 10 nM, respectively. We have developed methods to separately record these two types of GluCls. The non-desensitizing and desensitizing currents were selectively inhibited by trypsin and polyvinylpyrrolidone, respectively. In conclusion, in addition to GABA receptors, GluCls play a crucial role in selectivity of fipronil to insects over mammals. GluCls form the basis for development of selective and safe insecticides.

[The relationship between the contents and the level of achievement of objectives in an occupational health nursing practicum].

The purpose of this study was to evaluate the program and teaching methods of the occupational health nursing practicum in order to enforce students' learning experience in the clinical practice. Self-evaluation sheets graded from levels 1 to 4 were from 63 students, and statistical analysis was performed in relation to their performance levels. The results of the analysis of 63 students' performance sheets indicated that the students achieved 3 points above average in all 14 course objectives. Scales analysis of the students' evaluation sheets also revealed that students' achievement levels were lower at the Industrial Health Organization in comparison with those at the industrial enterprises. To make students' practice more valuable, students' assessment skills of the workers and working environment should be emphasized in the classroom teaching and experience of learning at the laboratory. Moreover, the course objectives should be sufficiently linked to the practice areas in order to differentiate between the features of the Health organizations and enterprises.

Targeting ROR1 in combination with pemetrexed in malignant mesothelioma cells.

OBJECTIVE: Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is overexpressed in a subset of malignant cells. However, it remains unknown whether ROR1 is targetable in malignant mesothelioma (MM). Therefore, in this study, we investigated the effects of ROR1 inhibition in mesothelioma cells. MATERIALS AND METHODS: Growth inhibition, colony formation, apoptosis, and mRNA/protein levels using siRNA-transfected MM cells were evaluated. Cluster analysis using Gene Expression Omnibus repository of transcriptomic information was also performed. RESULTS: Our results indicated that in three (H2052, H2452, and MESO-1) among four MM cell lines, ROR1 inhibition had anti-proliferative and apoptotic effects and suppressed the activation of AKT and STAT3. Although growth inhibition by siROR1 was minimal in another mesothelioma cell line (H28), colony formation was significantly suppressed. Microarray, quantitative polymerase chain reaction, and Western blot analyses showed that there were differences in the suppression of mRNA and proteins between H2452 and H28 cells transfected with siROR1 compared with those transfected with control siRNA. Cluster analysis further showed that MM tumors had relatively high ROR1 expression, although the cluster in them was different from that in MM cell lines. Thymidylate synthase, a target of pemetrexed, was downregulated in H2452 cells transfected with siROR1. Accordingly, a combination of pemetrexed with siROR1 was found to be effective in the three MM cell lines we studied. CONCLUSION: Our findings may provide novel therapeutic insight into the treatment of advanced MM.

Rate of torque development and the risk of falls among community dwelling older adults in Japan.

BACKGROUND: Rate of torque development (RTD) is defined as the slope of the torque-time curve obtained during an isometric contraction. Several studies have shown that RTD is lower in fallers than in nonfallers. However, these studies had small sample size and was not adjusted confounding factors. RESEARCH QUESTION: Is RTD associated with falls history in healthy community dwelling older adults. METHODS: This was cross-sectional study. In total, 122 participants aged ≥65 (mean, 71.3 ± 4.4) years were recruited for this study. We assessed RTD, muscle strength, functional capacity, and physical activity. We assessed RTD over the first 200 ms of the maximal isometric contraction, whereby the onset of contraction was deemed as the point at which torque had risen 4 Nm above the baseline. Differences between the 3 groups (no fall group, single fall group and multiple falls group) were examined using one-way analysis of variance or Kruskal-Wallis test. A post-hoc Bonferroni or Games-Howell test was used to assess the differences between the individual groups. A multivariate multinomial logistic model was built using the factors associated with the fall category. RESULTS: RTD was significantly different between the no fall group and multiple falls group (P = 0.047). Similarly, RTD was significantly different between the single fall group and multiple falls group (P = 0.016). RTD was associated with both the no fall group and single fall group (odds ratio = 2.05, 95% confidence interval: 1.06-3.97, odds ratio = 2.45, 95% confidence interval: 1.20-4.98, respectively) in multinomial logistic regression. SIGNIFICANCE: This is the first study to investigate the relationship between RTD and falls history in community-dwelling older adults in multivariate analysis. RTD is more strongly associated with falls history than other performance measures in community-dwelling elderly.

Association of active and passive smoking with sleep disturbances and short sleep duration among japanese working population.

BACKGROUND: The relationship between passive smoking and sleep is uncertain. PURPOSE: To examine the association of passive/active smoking with sleep disturbances. METHOD: 732 women and 1,896 men, working in a suburb of Tokyo, were surveyed using a self-administered questionnaire. Information on smoking, passive smoking exposure, and sleep was elicited. Exposure levels to passive smoking were assessed separately at work and at home as no, occasional, or regular exposure. Risk of sleep disturbances according to smoking status was estimated using logistic regression with odds ratios (OR) and 95% confidence intervals (CIs) as measures of association. RESULTS: Compared to never smokers, odds of difficulty awakening in the morning (DAM) in current smokers were significantly higher for women (OR 1.95) and men (OR 1.50), while increased difficulty initiating sleep (OR 1.88) and decreased early morning awakening (OR 0.31) were found only in women. Never smoking men occasionally exposed to passive smoking at work but not at home had increased odds (OR 1.81) of short sleep duration (SSD, < 6 h) than unexposed counterparts. CONCLUSIONS: The analyses suggest that exposure to passive smoking at work is associated with SSD in men, while current smoking relates to various subtypes of sleep disturbances in both sexes.

Perceived job stress and sleep-related breathing disturbance in Japanese male workers.

To examine the association of job stress with sleep-related breathing disturbance (SBD), a cross-sectional sample of 1940 males aged 17-83 (mean 45) years in 292 small and medium-sized enterprises in Japan were surveyed by means of a self-administered questionnaire. Perceived job stress was evaluated by the Japanese version of the Generic Job Stress Questionnaire developed by the US National Institute for Occupational Safety and Health, which included 13 job stress variables. Participants were divided into thirds according to their job stress scores. SBD was assessed by the question "Have you ever felt difficulty breathing during sleep or has anyone in your family told you that you have such difficulty?" SBD was defined as presence of symptoms more than once a month. Risk of SBD through job stress was estimated using logistic regression with odds ratios (ORs) and 95% confidence intervals (CIs) as measures of association. Prevalence of study-defined SBD was 6.7%. Participants who perceived the lowest level of social support from supervisors, and highest levels of job future ambiguity, interpersonal conflict at the workplace, job dissatisfaction, variance in workload, and quantitative workload had significantly increased risk of SBD after adjusting for potential confounders. High depressive symptoms, as measured by Center for Epidemiologic Studies Depression scale scores of 16 or higher, were also significantly associated with increased SDB. Although the results should be considered preliminary because of the self-reporting and cross-sectional design, data suggest that exposure to high job stress could be a possible risk factor for developing or aggravating SBD. Results also indicate that job stress should be considered when evaluating SBD in occupational and clinical settings.

Development and process evaluation of the participatory and action-oriented empowerment model facilitated by occupational health nurses for workplace health promotion in small and medium-sized enterprises.

The objective of this study is to develop an available empowerment model for workplace health promotion (WHP) in small and medium-sized enterprises (SMEs) and to evaluate its applicability and feasibility. Semi-structured interviews with employers and workers in SMEs were conducted to assess their actual requirements for support. The structure of our new empowerment model was discussed and established through several rounds of focus group meetings with occupational safety and health researchers and practitioners on the basis of results of our interviews. We developed a new participatory and action-oriented empowerment model based on needs for support of employers and workers in SMEs. This new model consists of three originally developed tools: an action checklist, an information guidebook, and a book of good practices. As the facilitators, occupational health nurses (OHNs) from health insurance associations were trained to empower employers and workers using these tools. Approximately 80 SMEs (with less than 300 employees) were invited to participate in the model project. With these tools and continued empowerment by OHNs, employers and workers were able to smoothly work on WHP. This newly developed participatory and action-oriented empowerment model that was facilitated by trained OHNs appears to be both applicable and feasible for WHP in SMEs in Japan.

Bortezomib treatment induces a higher mortality rate in lupus model mice with a higher disease activity.

BACKGROUND: Bortezomib (Bz) is a proteasome inhibitor that directly targets antibody-producing plasma cells. We recently reported the first randomized control trial that evaluated the effects of Bz in patients with systemic lupus erythematosus (SLE). In that study, we demonstrated that Bz treatment is associated with many adverse reactions in patients with refractory disease. In the present study, we examine the therapeutic and toxic effects of Bz on MRL/MpJ-lpr/lpr (MRL/lpr) mice with severe disease activity. METHODS: Female MRL/lpr mice at 10 and 14 weeks of age were treated with phosphate buffered saline (PBS) (n = 19), Bz (750 µg/kg twice weekly) (n = 27), or cyclophosphamide (Cyc) (1 mg/body, once in 2 weeks) (n = 20). Cellular subsets, serum immunoglobulin, anti-double-stranded DNA (anti-dsDNA) antibody titer, and a pathological index of glomerulonephritis were then analyzed at 22 weeks of age. Survival curves of the 10-week-old and 14-week-old Bz-treated groups were compared. Blood counts, creatinine, liver enzymes, and serum cytokine levels were measured 1 week after Bz treatment. Gene expression profiling of spleens from Bz and Cyc treatment mice were compared with those from control mice. RESULTS: The anti-dsDNA antibody levels were significantly higher in 14-week-old than in 10-week-old mice, indicating a higher disease activity at 14 weeks. A significant decrease in the number of splenic cells and glomerulonephritis index was observed in Bz-treated and Cyc-treated mice. Bz, but not Cyc, significantly decreased serum immunoglobulin and anti-dsDNA antibody titer levels. Survival curve analysis revealed a significantly higher mortality rate in 14-week-old than in 10-week-old Bz-treated and control groups. Following two injections of Bz, serum IL-6 and TNF-α levels were significantly more elevated in 14-week-old than in 10-week-old mice. Potentially immunogenic molecules, such as heat shock proteins, were characteristically upregulated in spleens of Bz-treated but not Cyc-treated mice. CONCLUSIONS: In spite of its therapeutic effect, Bz treatment had more toxic effects associated with increased proinflammatory cytokine levels in mice with a higher disease activity. Understanding the mechanism of the toxicity and developing preventive strategies against it is important for the safe clinical application of Bz in human SLE.

Targeted silencing of SOX2 by an artificial transcription factor showed antitumor effect in lung and esophageal squamous cell carcinoma.

SOX2 is a transcription factor essential for early mammalian development and for the maintenance of stem cells. Recently, SOX2 was identified as a lineage specific oncogene, recurrently amplified and activated in lung and esophageal squamous cell carcinoma (SCC). In this study, we have developed a zinc finger-based artificial transcription factor (ATF) to selectively suppress SOX2 expression in cancer cells and termed the system ATF/SOX2. We engineered the ATF using six zinc finger arrays designed to target a 19 bp site in the SOX2 distal promoter and a KOX transcriptional repressor domain. A recombinant adenoviral vector Ad-ATF/SOX2 that expresses ATF/SOX2 suppressed SOX2 at the mRNA and protein levels in lung and esophageal SCC cells expressing SOX2. In these kinds of cells, Ad-ATF/SOX2 decreased cell proliferation and colony formation more effectively than the recombinant adenoviral vector Ad-shSOX2, which expresses SOX2 short hairpin RNA (shSOX2). Ad-ATF/SOX2 induced the cell cycle inhibitor CDKN1A more strongly than Ad-shSOX2. Importantly, the ATF did not suppress the cell viability of normal human cells. Moreover, Ad-ATF/SOX2 effectively inhibited tumor growth in a lung SCC xenograft mouse model. These results indicate that ATF/SOX2 would lead to the development of an effective molecular-targeted therapy for lung and esophageal SCC.