Ehrlich II –2nd World Conference on Magic Bullets

An Arabidopsis Transcription Factor, AtNFXL1 Gene Negatively Regulates Fusarium Phytotoxin Trichothecene-Induced Defense Response ASANO T^1, YASUDA M^2, NAKASHITA H^2, KIMURA M^2, YAMAGUCHI K^1, NISHIUCHI T^1 ^1Univ. KANAZAWA, Kanazawa, Japan; ^2RIKEN, Wako, Japan Background: Phytopathogenic Fusarium species produced trichothecene family of phytotoxins, which function as a virulence factor during infection of plants. Trichothecenes are known to act as translational inhibitors in eukaryotic ribosomes. Recently, we revealed that some trichothecenes such as T-2 toxin induce a programmed cell death. It is likely that trichothecene-induced cell death is contributing directly to virulence of necrotrophic fungi. Methods: Arabidopsis thaliana (ecotype Columbia; Col-0) plants were gown on MS agar medium with or without trichothecenes in a growth chamber (a 16-h light / 8-h dark cycle at 22 °C) after a 3-d vernalization period. A T-DNA insertion mutant (atnfxl1-1) of AtNFXL1 (CS24940) was obtained from the Arabidopsis Biological Resource Center. The microarray experiment was carried out using the Agilent Arabidopsis 1 Oligo Microarray SA and SAG levels were determined using HPLC. Results: In Arabidopsis, expression of AtNFXL1, a homologue of the putative human transcription repressor NF-X1, was significantly induced by application of type A trichothecenes, such as T-2 toxin. An atnfxl1 mutant growing on medium lacking trichothecenes showed no phenotype, whereas a hypersensitivity phenotype was observed in T-2 toxin-treated atnfxl1 mutant. Microarray analysis indicated that several defense-related genes (i.e. WRKYs, NBS-LRRs, EDS5, ICS1, etc.) were upregulated in T-2 toxin-treated atnfxl1 mutant compared to wild type. Enhanced salicylic acid (SA) accumulation was observed in T-2 toxin-treated atnfxl1 mutant, which suggests that AtNFXL1 functions as a negative regulator of these defense-related genes via an SA-dependent signaling pathway. We also found that the expression of AtNFXL1 was induced by SA. Moreover, the atnfxl1 mutant was less susceptible to a compatible phytopathogen, Pseudomonas syringae (Pst DC3000). Conclusions: Our results indicate that AtNFXL1 plays an important role in the trichothecene response, as well as general defense response in Arabidopsis. Authors’ disclosure statement: Furthermore, several components of the AtNF-XL1-containing complex will be discussed.

Brain’s Immune Cells And Anti-Inflammatory Effects Of Neuropeptides NODA M, IFUKU M, OKUNO Y, YAMAKAWA Y Laboratory of Pathophysiology, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, Japan Background: There has been an explosion of interest in microglia, the immune cells of the central nervous system (CNS), over the past few years. They express various receptors for neurotransmitter or neuropeptides. In responding to these neurotransmitters or peptides, microglia show chemotaxis, release different kinds of cytokines and trophic factors, or attenuating excess release of cytokines induced by bacterial toxins. Many neuropeptides are produced in the brain during trauma and stroke, and are regarded as mediators of inflammation. Since a number of receptors for neuropeptides are also expressed in glial cells, they are likely to play an important role in the brain via various neuropeptides. Methods: Primary glial cells were isolated from mixed cultures of cerebrocortical cells in postnatal day 1-3 Wistar rats or C57BL/6 mice (Kyudo, Kumamoto, Japan). Motility of microglia under the control of temperature and gas was monitored with time lapse video microscopy system (Nikon INSTEC, Fukuoka, Japan). Chemoaxis was tested using a 48-well microchemotaxis Boyden chamber (Neuroprobe, Bethesda, MD). Assay of tumor necrosis factor- (TNF- and prostaglandin E2 (PGE2) was done using ELISA and EIA kit. Expression of NGF was tested by SYBR green-based real-time quantitative RT-PCR. Results: Some of the neuropeptides, such as bradykinin, endothelin, vasopressin and galanin, increased microglial motility and chemotaxis. Among neuropeptides, bradykinin (BK), a mediator of pain and inflammation and is produced at the site of injury, had neuroprotective effect. They inhibited lipopolysaccharide (LPS)-induced TNF- release. As a mechanism of such negative feedback system, production of PGE2 was observed. In addition, BK and endothelin up-regulated the production of neurotrophic factors such as nerve growth factor (NGF) in astrocytes. Conclusion: These results suggest that some neuropeptides may have anti-inflammatory and neuroprotective effects through multiple functions on immune cells in the brain. These observations may help to understand the paradox on the role of neuropeptides in the central nervous system and may be useful for therapeutic strategy.

Combined Therapy With Pitavastatin And Eicosapentaenoic Acid Improve Platelet Activation Markers In Hyperlipidemic Patients With Type 2 Diabetes Mellitus NOMURA S^1, INAMI N^2, SHOUZU A^2, IWASAKA T^2 ^1Kishiwada City Hospital, Osaka, Japan; ^2Kansai Medical University, Osaka, Japan Background: Platelet-derived microparticles (PDMP) play an important role in the pathogenesis of diabetic vasculopathy, and statins or eicosapentaenoic acid (EPA) have been shown to have a beneficial effect on atherosclerosis in hyperlipidemic patients. However, the influence of EPA and statins on PDMP and adiponectin in atherosclerosis is poorly understood. We investigated the effect of pitavastatin and EPA on circulating levels of PDMP and adiponectin in hyperlipidemic patients with type 2 diabetes. Methods: One-hundred and ninety-one hyperlipidemic patients with type 2 diabetes were divided into three groups: group A received pitavastatin 2 mg once daily (n=64), group B received EPA 1,800 mg daily (n=55), and group C received both drugs (n=72). PDMP and adiponectin were measured by ELISA at baseline and after 3 and 6 months of drug treatment. Results: 30 normolipidemic patients were recruited as healthy controls. PDMP levels prior to treatment in hyperlipidemic patients with diabetes were higher than levels in healthy controls (10.4 ± 1.9 vs. 3.1 ± 0.4 U/ml, p<0.0001), and adiponectin levels were lower than controls (3.20 ± 0.49 vs. 5.98 ± 0.42 μg/ml, p<0.0001). PDMP decreased significantly in group B (before vs. 6M, 10.6 ± 2.0 vs. 8.0 ± 1.7 U/ml, p<0.01), but not in group A (before vs. 6M, 9.4 ± 1.9 vs. 9.6 ± 1.7 U/ml, not significant). In contrast, group A exhibited a significant increase in adiponectin levels after treatment (before vs. 6M, 3.29± 0.51 vs. 4.16 ± 0.60 μg/ml, p<0.001). Furthermore, group C exhibited significant improvement in both PDMP and adiponectin levels after treatment (PDMP, before vs. 6M, 11.2 ± 2.0 vs. 4.5 ± 2.7 U/ml, p<0.001; adiponectin, before vs. 6M, 3.24 ± 0.41 vs. 4.02 ± 0.70 μg/ml, p<0.001). Reductions of PDMP in combined therapy were significantly greater than those observed with EPA alone (p < 0.05 by ANOVA). In addition, soluble CD40 ligand exhibited almost the same change as PDMP in all therapy groups. Conclusions: These results suggest that pitavastatin possesses an adiponectin-dependent antiatherosclerotic effect, and this drug is able to enhance the anti-platelet effect of EPA. The combination therapy of pitavastatin and EPA may be beneficial for the prevention of vascular complication in hyperlipidemic patients with type 2 diabetes.

Serotonin Bridge Of 5-HT2C - 5-HT1B Receptor Over Appetite NONOGAKI K Tohoku Univ., Sendai, Japan Background: Serotonin (5-hydroxytryptamine; 5-HT) 2C receptors reportedly mediate the appetite-suppressing effects of 5-HT drugs such as m-chlorophenylpiperazine (mCPP) and fenfluramine. Fluvoxamine, a selective serotonin reuptake inhibitor, is used for the treatment of binge eating disorder. Aims: 1) To determine the mechanisms by which fluvoxamine exerts appetite suppressing effects. 2) To determine the pharmacologic interactions of 5-HT2C and 5-HT1B receptors. 3) To propose a pathogenesis of fluvoxamine-responsive binge eating syndrome. Methods: At first, male 5-wk-old C57BL6J mice were deprived of food for 23 h and were injected intraperitoneally (i.p.) with saline or SB 242084 (0.5-2.0 mg/kg), a selective 5-HT2C receptor antagonist. Thirty minutes later, animals were injected with saline or fluvoxamine (3–30 mg/kg) i.p., and after a further 30 min they were given chow pellets; intake of pellets was measured for the next 2 h. In the second experiment, animals were deprived of food for 23 h and were injected i.p. with saline or SB 242084 (2 mg/kg) plus SB 224289 (5 mg/kg), a selective 5-HT1B receptor antagonist. Thirty minutes later, animals were injected i.p. with saline or fluvoxamine (10 mg/kg), and after a further 30 min they were given chow pellets; intake of pellets was measured for the next 2 h. In the third experiment, animals were deprived of food for 23 h and were injected i.p. with saline or CP94253 (2.5-10 mg/kg), a selective 5-HT1B receptor agonist. Thirty minutes later, they were given chow pellets and intake of pellets was measured for next 2 h. Finally, animals were injected i.p. with saline or SB 242084 (2 mg/kg). Sixty minutes later, animals were decapitated and determined hypothalamic 5-HT1B receptor expression. Results: Fluvoxamine, in the presence of SB242084, exerted appetite-suppressing effects while fluvoxamine or SB242084 alone has no effect. The appetite-suppressing effects were attenuated in the presence of SB224289. CP94253 exerted appetite-suppressing effects. SB242084 increased the expression of hypothalamic 5-HT1B receptors. Conclusions: 1) Fluvoxamine and inactivation of 5-HT2C receptors exert feeding suppression through activation of 5-HT1B receptors. 2) Pharmacologic inactivation of 5-HT2C receptors increases the expression of hypothalamic 5-HT1B receptors. 3) Fluvoxamine-responsive binge eating syndrome may result from a perturbation of 5-HT2C receptor signaling.

LC-MS/(MS) Strategies For Increased Metabolite Coverage In Metabolomics NORDSTRÖM A^1, LEHTIÖ J^1, LEWENSOHN R^1; SIUZDAK G^2 ^1Karolinska Biomics Center, Department of Oncology-Pathology, Karolinska Institutet and Karolinska University Hospital, Solna, Stockholm Sweden ; ^2The Scripps Research Institute, Department of Molecular Biology, Scripps Center for Mass Spectrometry, North Torrey Pines Rd., CA, USA Background: Metabolomics is the global and untargeted measurement of as many metabolites as possible in a given biological system. There is no one single analytical technique available to measure all the metabolites in a complex system such as a cell, however, liquid chromatography mass spectrometry (LC-MS) is establishing it self as the preffered metabolomics analytical technique. We have explored the possibility to use ultra high pressure chromatography and multiple ionization strategies for the mass spectrometry analysis in order to expand the number of detectable metabolites. Methods: Methanolic extracts of human serum was used to investigate what the impact of various LC-MS strategies had on the number of detected metabolites. Evaluation of chromatography was performed through comparision of 1.7 and 3.5 uM particle size columns. Evaluation of the multiple ionization strategy was performed using an Agielnt MSD SL system with ESI and Multimode ionization sources. All LC-MS data was extracted and aligned using the freely available XCMS software (http://masspec.scripps.edu/xcms/xcms.php). Results: Ultra high pressure liquid chromatography (UPLC) resulted in 20% more ion features detected when compared to conventional HPLC. Performing MS analysis in both positive and negative ionmode doubled the number of unique ion features compared to positive mode only, and performing APCI (atmospheric pressure chemical ionization) on the same sample resulted in an additional 20% increase in unique ion features. Conclusions: To achieve truly non-targeted and global metabolite analysis of a complex biological system, multiple analytical techniques are needed. We have shown that sub 2uM particle size liquid chormatography paired with multiple ionization mass spectrometry can dramtically increase the number of measured metabolites.

Predicting Fluoroquinolones Ability To Kill Resistant Streptococcus Pneumoniae Isolates Expressing Different Genetic Mutations: Target Attainment Analysis Simulating Therapeutic Doses To Patients With Community Acquired Pneumonia NOREDDIN AM^1, HOBAN D^2 , ZHANEL GG^2 ^1University of Minnesota, Duluth, USA; ^2Univeristy of Manitoba, Winnipeg, Canada Background: Streptococcal pneumonia is a major cause of morbidity and mortality worldwide. Fluoroquinolones are one of the mainstay drugs for treatment of these infections. However emerging resistance poses a threat to the class’s future utility. This work aims to evaluate the probable efficacy of ciprofloxacin, levofloxacin, gemifloxacin, garenoxacin, and moxifloxacin in eradicating infections and preventing continued growth of resistance. Methods: Using patient data from strep pneumonia patients in hospitals and MIC data from the CROSS study; drug regimens were compared to see the likelihood of attaining fAUC0-24/ MICall ratios depicting goal clinical outcomes. Results: Probability of target attainment of the fluoroquinolones against all the genetically identified resistant isolates of Streptococcus pneuminiae is showed in the following table: Conclusions: Very few regimens are able to prevent further growth of resistant organisms when ParC mutations have occurred. Only garenoxacin and moxifloxacin were able to eradicate extremely resistant isolates in serum and ELF respectively.

Unexpected Effects On Angiogenesis By Type Of Low-Molecular-Weight Heparin (LMWH) And By Type Of Vehicle In Chemotherapy NORRBY K University of Gothenburg, Sweden Background: Tumor growth is angiogenesis dependent. The study of anti-angiogenic effects per se in tumors is not feasible, however. We have developed a mammalian non-tumor angiogenesis assay allowing the objective quantitative assessment of relevant variables following s.c./p.o./i.v. treatment with test agents. Aims: 1) To study the effect of (i) standard heparin (UFH), (ii) different mean molecular weight (MW) fractions of UFH, (iii) tinzaparins of different MWs (produced by heparinase digestion of UFH), and (iv) dalteparin (produced by nitrous acid depolymerization of UFH). 2) To examine how low-dose metronomic-like chemotherapy affects angiogenesis induced by VEGF-A, a key heparin-binding angiogenic factor in most tumors. Methods: The mesentery assay in adult rats was use: a pro-angiogenic agent was injected i.p. and the ensuing angiogenesis in the membranous mesentery was recorded following the s.c. injection of a heparin or the s.c. continuous-infusion chemotherapy. Heparins: UFH (MW ~15 kD), MW 2.5, 8, 15 and 22 kD fractions of UFH, MW 2.5 and 5.5 kD tinzaparins, and MW 6.0 kD dalteparin. Cytostatics: cyclophosphamide, paclitaxel, doxorubicin, cisplatin, and 5-FU. Conventional vehicles were used, some containing a radical oxygen species (ROS) scavenger. In other cases the ROS scavenger N-acetylcystein (NAC) was given concurrently with the chemotherapy. Results: Heparin MW influenced endotoxin-induced angiogenesis strictly (r = 0.97): low MW inhibited and high MW stimulated. In VEGF-A-mediated angiogenesis, the 2.5 kD tinzaparin fraction lacked effect while the 5.5 kD tinzaparin fraction inhibited angiogenesis. Conversely, dalteparin (MW 6.0 kD) stimulated angiogenesis. Cisplatin and 5-FU monotherapy surprisingly stimulated angiogenesis. NAC monotherapy was inert. 5-FU + NAC co-treatment reversed the significant pro-angiogenic effect of 5-FU into a significant anti-angiogenic effect. The anti-angiogenic effect of paclitaxel emerged only when the vehicle contained a ROS scavenger. Conclusions: 1) Heparins exert MW-related effects in angiogenesis. 2) Similarly sized LMWHs, produced by diverse methods, affect angiogenesis differently. 3) Metronomic-like chemotherapy drug-specifically influences angiogenesis: some drugs stimulate while other inhibit angiogenesis. 4) ROS play a major role in angiogenesis during low-dose chemotherapy

Apoptin Magics: Bullet Or Sensor? NOTEBORN MHM^1, PENG D^2, SUN J^2, QU S^2, BACKENDORF C^1 ^1University Leiden, Leiden, the Netherlands; ^2Huazhong University of Science and Technology, Wuhan, PR China Background: Apoptin, a protein derived from chicken anemia virus (CAV), induces selectively apoptosis in human tumor and transformed cells as compared to normal cells. Various gene-therapy approaches have shown the power of apoptin as a tumor-selective killer. Apoptin interaction with e.g. the anaphase promoting complex results in tumor-selective apoptosis. Furthermore, apoptin becomes phosphorylated selectively in tumor cells by a yet unknown kinase activity. These features make apoptin both a potential bullet for novel anti-cancer therapy and a sensor for identifying novel potential drug targets. Methods: We used the transforming ability of the SV40 large T (LT) and/or small T (ST) oncogenic proteins in normal human fibroblasts to identify tumor-related pathways sensed by apoptin by co-transfection experiments. Besides, we asked whether protein-transduction domain (PTD4)-mediated delivery of apoptin protein has anticancer potential. To that end, we established several subcutaneous tumors in nude mice and applied PTD4-apoptin and controls on the tumor bearing epidermis. Results: Systematic mutagenesis of selected protein domains in SV40 LT and ST established that both the p53- and the Rb-binding domains are not needed for activation of the tumor-selective apoptin kinase. However, both the J domain and the PP2A binding site were essential for activation of apoptin kinase. Inactivation of PP2A by means of the siRNA technology confirmed a role of this phosphatase in apoptin regulation. In-vivo, delivery of PTD4-apoptin protein resulted in a significant growth inhibition of human hepato -, gastric - , and cervix carcinoma. Apoptosis and disruption of the tumor integrity were apparent in PTD4-apoptin, but absent in the control-treated tumors. Despite the obvious presence of apoptin protein in normal tissues, no side effects could be monitored. Conclusion: Recent crystallographic data demonstrated that both J and PP2A-binding domains within ST are involved in its interaction with PP2A, which corroborates our data that specific suppression of PP2A results in a tumor-related kinase. PTD4-Apoptin protein delivery fulfills the requirements of a powerful and safe anticancer therapy. Overall, we conclude that apoptin is both a potential anti-cancer bullet and a valuable tumor-selective sensor, which deserves development towards the clinic.

Neurodevelopment Of Children Following Exposure To Psychotropic Medications During Gestation: A Novel Approach To Behavioral Teratology NULMAN I University of Toronto, Toronto, Canada; Division of Clinical Pharmacology, The Motherisk Program, The Hospital for Sick Children, Toronto, Canada Background: Maternal psychopathology should be balanced against the potential teratogenic effects of psychotropic medications (PTMs), higher levels of stress hormones, neonatal syndromes, higher fetal central nervous system (CNS) transmitter activity, and child’s long-term neurodevelopment. Assessing neurocognitive development of children exposed to medications in pregnancy is an inseparable part of drug safety studies in teratology, which are complicated by multiple confounders. Study design plays an integral role in controlling these factors. Objectives: 1.To review available literature and determine the possible risks of adverse fetal/child CNS development following exposure to PTMs in utero. 2. To present a novel approach to studying behavioural teratology, while controlling for genetic and environmental factors by testing unexposed siblings from the same families. Methods: A systematic review of studies on long-term child neurodevelopment was conducted. IQ scores of children exposed to Venlafaxine (VLF) (n=32) were compared to their unexposed VLF siblings and healthy controls. Participants were hierarchically matched for age, gender and order of delivery. The primary outcome measure is the children’s full scale IQ measured by the WPPSI-III Scales of Intelligence. Statistical analysis accounted for the clustering effect. Results: Full Scale IQ, Performance IQ and Verbal IQ were not different between the VLF-exposed children and their unexposed siblings (105+12vs100+8; 102+15vs105+7; 105+12vs95+10). Healthy controls scored significantly higher than the VLF group in all three IQ measures (P= 0.011; 0.041; and 0.028 respectively). There were no differences between the groups in maternal IQ, socioeconomic status or children’s physical characteristics. Conclusions: In utero exposure to PTMs was not associated with impaired long-term neurocognitive development. Assessment of siblings helps to separate the impact of antidepressant drugs from genetic and environmental factors and is strong evidence in psychotropic drug safety studies. Factors other than the antidepressant are strongly associated with children’s cognitive abilities. Supported by Wyeth Pharmaceuticals.