Ehrlich II –2nd World Conference on Magic Bullets

Pharmacodynamic Monitoring of Calcineurin Inhibition Therapy VAN ROSSUM HH^1, DE FIJTER JW^2, VAN PELT J^1 Departments of Clinical Chemistry^1 and Nephrology^2, Leiden University Medical Center, Leiden, Netherlands Introduction: Calcineurin inhibiter (CNI) therapy by cyclosporine or tacrolimus is still considered the backbone of immune suppression after allograft transplantation, especially early after transplantation. Since both CNIs show unpredictable pharmacokinetic profiles, TDM is used to control drug exposure. Unfortunately, CNI therapy is still associated with severe side effects such as nephrotoxicity and unfavorable cardiovascular risk profile. To optimize CNI therapy, more accurate monitoring strategies are required and a pharmacodynamic strategy constitutes a novel and innovative approach. Content: Several pharmacodynamic monitoring strategies have been developed that all showed inverse relations between CNI concentration and pharmacodynamic markers, such as calcineurin (CN) activity and T cell activation transcripts. These markers have been investigated for allograft recipients treated with CNIs and although the first signs of their usefulness have been reported, they are not suitable for routine monitoring so far. A feature of many pharmacodynamic monitoring strategies is that the large inter-individual variation observed does not necessarily explain clinical and immunological observations. We have investigated whether sample choice and composition could contribute to the observed large inter-individual variation of these markers. We have therefore monitored sample composition when either total leukocyte fraction or peripheral blood mononuclear cells (PBMCs) are chosen as sample in renal allograft recipients. Next cellular CN activities and CN inhibition profiles for cyclosporine were determined. We found that based on the differences in cellular CN activity, the large variation in sample composition could directly confound measurement outcome. Cell-specific CN activity and drug sensitivity should therefore be considered for sample validation. Conclusions: The call for more accurate monitoring strategies for immunosuppressive therapy has led to the development of a large variety of pharmacodynamic monitoring strategies. The first indications of their clinical relevance are available, but further understanding of the analytical and clinical variables involved seems to be required before these markers can be tested in a clinical setting.	RNA-modified dendritic cells as therapeutic cancer vaccines: Dressed for success ? VAN TENDELOO VF, VAN DRIESSCHE A, VAN DE VELDE A, STEIN B, NIJS G, VERMEULEN K, PIETERS K, SCHROYENS WA, COOLS N, BERNEMAN ZN University of Antwerp – Vaccine & Infectious Desease Institute Messenger RNA (mRNA)-based gene transfer has gained an enormous interest over the last decade, especially in the field of immuno-gene therapy of cancer. In this area, most researchers have exploited low voltage electrical pulses (electroporation) as a means to introduce coding RNA into the cells. mRNA electroporation has become a method of choice for transfecting dendritic cells given its superior cytoplasmic expression efficiency, its simplicity over viral transduction protocols and its clinical safety profile because of a strictly transient expression profile and the inability to integrate into the host genome. Furthermore, it allows the simultaneous introduction of antigens and imunostimulatory proteins into dendritic cells through co-electroporation of multiple RNAs. Recently, optimized strategies to produce highly translatable mRNA and more insights into the immunostimulatory properties of RNA structures further advocates the use of RNA for vaccination purposes. Here, I will discuss our own experience with RNA-electroporated DC both in cancer and HIV. I will also present latest data on our recently performed phase I clinical trial using RNA-electroporated DC in leukemia patients. In conclusion, RNA loading by electroporation provides a versatile gene therapy tool for the design of DC-based therapeutic vaccines.
A functional steroid-binding element in an ATP-binding cassette multidrug transporter VAN VEEN HW Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, United Kingdom ABSTRACT: The human breast cancer resistance protein is an ATP-binding cassette (ABC) multidrug transporter that affects the bioavailability of chemotherapeutic drugs, and can confer drug resistance on cancer cells. It is the second member of the ABCG subfamily, other members of which are associated with human steroid disorders such as hypercholesterolemia, sitosterolemia, and atherosclerosis. The molecular bases of protein-steroid interactions in ABC transporters are unknown. Here, we identify a steroid-binding element in the membrane domain of ABCG2 with a similarity to steroid hormone/nuclear receptors. The element facilitates steroid hormone binding, and mediates modulation of ABCG2 activity. The identification of this element might provide an opportunity for the development of new therapeutic ligands for ABCG2.	The natural steroidal withanolide Withaferin A is a novel promising chemosensitising compound in B-cell chronic lymphocytic leukemia and metastatic breast cancer" Vanden Berghe W^1, Lust S^1, Kaileh M^1,4, Gerlo S^1, Boelens J^1, Offner F^1, Philippé J^1, Ganeff C^2, Dejardin E^2, Hillaert U^1, Van Calenbergh S^1, Essawi T^3, Haegeman G^1 ^1UGent, Gent, Belgium, ^2ULG, Liège, Belgium ^3University Birzeit, Palestine, ^4National Institute on Aging, Baltimore, USA Background: B-chronic lymphocytic leukemia (B-CLL), the most prevalent leukemia in adults in Western countries, still remains incurable. In aggressive metastatic breast cancer cells, loss of hormone receptors frequently leads to hormone therapy resistance. Aims: We wanted to investigate whether Withaferin A (WA), a natural steroidal withanolide purified from Withania Somnifera (Ashwagandha), is able to induce apoptosis in B-CLL and hormone refractory metastatic breast cancer cells and we explored the molecular mechanisms involved. Methods: We determined IC50 values of WA in B-CLL samples and metastatic breast cancer cells and evaluated various markers of apoptosis. Furthermore, we studied effects of WA on NFB/Stat3 signaling pathways involved in B-CLL and breast cancer survival. Results: WA induced apoptosis in B-CLL and metastatic breast cancer cells with IC50-values of 0.60±0.28 and 50±25 µg/ml respectively. Cell death was associated with impaired NFB activation and increased protein ubiquitination levels, independently of 20S proteasomal inhibition. This correlated with selective downregulation of various B-cell growth factors and anti-apoptotic gene products, including IL-6, IL-8, XIAP, A1/bfl1, Bcl-2. In metastatic breast cancer cells, we found that anti-invasive activity of WA results from cumulative inhibition of constitutive and inducible regulatory steps in the canonical and noncanonical NFB-signalling pathways. This allows WA to potently repress expression of interleukin (IL6, IL8) and metalloprotease (MMP3, MMP9) survival factors. Conclusions: These data illustrate the potential of WA as a multifaceted NFB/Stat3 inhibitor and novel promising chemosensitizing compound in B-CLL- and breast cancer therapy.
The Role of the Dopamine Transporter Gene in Smoking and Other Addictive Behaviors VANDENBERGH DJ^1, VASILOPOULOS T^1, VOGLER GP^1, GREENBERG MT^1, BIERMAN KL^1, COIE JD^2, DODGE KA^2, LOCHMAN JE^3, MCMAHON RJ^4, PINDERHUGHES EE^5 ^1Pennsylvania State Univ. XYZ, Univ. Park, PA, USA; ^2Duke University, Durham, NC, USA; ^3Univ of Alabama, Tuscaloosa, AL, USA; ^4Univ. of Washington, Seattle WA, USA; ^5Tufts Univ, Boston MA, USA Background: Genetic analysis of the human dopamine transporter (HDAT or SLC6A3) has been a central research focus since the cloning of the gene and identification of sequence variants in 1992. The most frequently assessed site is a Variable Number of Tandem Repeats (VNTR) present in the 3’-untranslated region of the gene. Assessment of smoking and addiction-related traits that have a genetic component is made difficult due to the complex nature of these phenotypes. A scan of the literature suggests that new approaches to genetic analysis of smoking, which is the focus of this presentation are needed. Methods: We analyzed gene-by-environment (G X E) interactions in an intervention program. Children selected at 5 or 6 years old based on ratings of difficult behavior in the classroom were given interventions including training for individual behaviors, interactions with peers, and parent-child interactions. A control group from the same schools, but without specific interventions, was used for comparison. Assessment of smoking and alcohol use in school grades 9, 10, and 11 was used as the outcome variables. There were 1199 participants of which 695 were successfully genotyped, resulting in a group that was 63% male, 48% European-American, 48.6% African-American for this study. The VNTR genotype was assigned as either 9-copy present or absent. Logistic regression was used to assess the effect of genotype by intervention status on frequency of smoking and alcohol use. Results: The present G X E study showed significant effects of the combination of genotype and intervention on drug use, while little of significance was seen for the main effect of HDAT. Those individuals that possess the 9-copy allele and given the intervention had lower levels of smoking and alcohol use. Conclusions: Published results have been mixed as to any consensus for a role for variants of HDAT in smoking and other addictive behaviors. Our G X E study showed significant effects suggesting that interactions might have larger consequenses than main effects of the genes themselves.	New perspectives in the treatment of melanoma by a glycobiological approach: results in mouse experimental model VANNUCCI L, KULDOVA M, ROSSMANN P, KREN V, BEZOUSKA K, SAIEH M, ULLMANOVA-BENSON V, KLEMENSOVA K, GROBAROVA V, and FISEROVA A Institute of Microbiology, Czech Academy of Sciences v.v.i., Prague, CZ. Background: Glycosylation products contribute to the structure and function of proteins and lipids on the cell surface (cell-cell interactions and immunogenicity). Lectin-like receptors expressed on natural immunity cells (especially NK cells), can recognize transformed cells by their aberrant glycosylation products, as well as tumor microenvironment (TME) degradation products of carbohydrate structures. Multivalent glyconjugates terminated with GlcNAc (GN) and high affinity for NKR-P1 receptor were synthesized. Aim: To test biological and immunological effects of GD in mouse melanoma model. Methods: Melanoma B16F10 in vivo (C57BL/6 mice) and in vitro;GN: PAMAM-GlcNAc8, PAMAM-GlcNAc4; evaluation of tumors growth, animal survival; FACS analysis and cell sorting of immune cell subpopulations; cytotoxicity; ELISA; western blotting; light, fluorescence, and confocal microscopy. Results: GN modulated both immunity and cancer cells: 1) B16F10 cells changed their glycophenotype and immunogenicity; 2) immune cells generated a Th1 response (increased IL-2, IFN-γ); 3) cancer growth was slowed; 3) animal survival increased (33% than control). GN administration after temporary inhibition of the disregulated TME immunity, triggered specific-target antitumor response. CD69 activation marker expression and IFN-γ production accompanied recovery of cytotoxic cell activity. In vitro tests showed functional interactions between GN, NK1.1 and NKG2D receptors. GN are internalized by both immune and melanoma cells with different cellular localization and effect (NK cells showed colocalization of the GN with the NK1.1 receptor). Recently, either new GlcNAc-terminated multivalent molecules, or Pleurotus ostreatus mushroom derivatives (containing glucans and chitin) were also found to modulate the melanoma growth and immunity. Conclusions: Data indicate: 1) the possibility of tumor-immunity modulation by GlcNAc-rich multivalent molecules inside the cancer microenvironment; 2) perspective suitability for immunotherapeutic interventions. Acknowledgements: grants IAA 500200509, IAA 500200510, IAA 500200620 and 310/08/H077;IRC AV0Z50200510(CZ); ARPA Foundation, Pisa (IT). E-mail: [e-mail] Concize statement on the research: Multivalent glycoconjugates targeting NK activation lectin-like receptors modulates mouse anticancer immunity in vivo
Preliminary results of the Semmelweis Budapest Mastocytosis Center VARKONYI J ^1, CSOMOR J ^2, FULE T ^2, SHÖNLEBER J ^2, FARKAS H ^1, VARGA L ^1, SRETER L^1 , MARSCHALKO M ^3, HIDVEGI B ^3, DARVAS ZS ^4, FALUS A ^4 1. 3^rd Department of Internal Medicine (Hematology Division) 2. Department of Pathology and Cancer Research 3. Department of Dermatology 4. Department of Genetics, Cell- and Immunbiology, Semmelweis University, Budapest, Hungary Background: The Semmelweis Budapest Mastocytosis Center has been started to be organized from the end of 2006, in Semmelweis University of Medicine. There was a wish to join European Competence Network on Mastocytosis (ECNM). Mastocytosis is a rare group of disorders has not been registered so far in Hungary. There were anecdotal case reports only in the literature. Joining ECNM the first task was to set up our network at Semmelweis University. Methods: Patients with characteristic skin manifestations were directed for hematological workup or patients who had bone marrow analysis for other reasons- and mast cell disease was found had also been registered. Laboratory methods in use are as follows: flow cytometry analysis; immuno- histochemistry (CD2, CD5, CD117) and DNA sequence analysis of cKIT gene on exon 17 to determine c-kit mutation, “nested” PCR for FIP1L1-PDGFR alpha mutation analysis. A longitudinal patient follow up with complement panel detection [ CH50/ml, concentration of C1q, C3,C4, C1 INH, C1 INH activity, anti-C1 INH (IGG, IGA, IGM) and anti- C1q (IgG) determination] had been introduced as well in the wish to find correlation- if there are any- to general symptoms or to the therapy had been applied. Results: 27 patients had been registered so far (10 M and 17 F): 8 cases of Urticaria Pigmentosa, Mastocyte Activation Syndrome: 2, Systemic Mastocytosis localized to Bone Marrow (SM-BMM): 7, SM-associated to hematological non mast cell disease (AHNMD): 10. Conclusions: 1. Authors consider setting up the network itself as significant result. In the next years by raising the number of registered patients - by widening the network to the whole country- and/or by establishing European collaborations - we might found the basis of scientific work as well. 2. This year in November 7-8 our center received the possibility to organize the annual meeting of ECNM.	Cotrimoxazole, an old “magic bullet” finds a new target and benefit in advanced fibrotic lung disease VARNEY VA, PARNELL H, SALISBURY DT, RATNATHEEPAN S, TAYAR R St Helier Hospital, Wrythe Lane,Carshalton,Surrey,UK Background: Advanced fibrotic lung disease has a prevalence of 175 cases per 100 000 population, but no effective treatment. Despite costly studies across the US & Europe, expensive immune modulating drugs demonstrated no benefit. The observation of a dramatic improvement in an individual given co-trimoxazole, was reproduced in a further 14 end-stage patients and led to a pilot study. Method: A double-blind randomised placebo-controlled study of 20 patients treated with oral cotrimoxazole for 3 months. Results: Clinical & statistically significant improvements in forced vital capacity +21% and walking distance +355 meters (shuttle walking test) at 3 months (p=0.002) compared with placebo. Oxygen desaturation on walking was reduced (p=0.003). MRC 5 point dypsnoea score improved significantly (p=0.05) at 3 months. The SGHRQ showed a reduction in symptoms scores (p=0.05) at 12months. Vascular endothelial growth factor showed a 50% reduction in the active group, and peripheral blood markers of oxidative stress (monocytes ,MCV & GGT )were significantly reduced p=0.0001. Repeat C.T. chest scans after 12 months of co-trimoxazole treatment showed reductions in ground glass change (p= 0.05),suggesting effects in the alveolar region of the lung. On going monitoring of all patients demonstrated equal responses in the placebo group with stability of lung function in all patients at 12 months. Conclusion: This drug is the first to fulfil the ATS/ERS 2000 criteria of a beneficial response to treatment. The time course of benefit (2-4weeks) is consistent with its effects as an antibiotic with relapse upon cessation & subsequent resistance. We are now investigating possible immune defects, to assess whether an infective agent could initiated the disease.
A new “quasi-adaptive” response to alkylating agents in E. coli cells due to posttranslational modification in S- nitrosylated Ada protein. Igor VASILIEVA SV Institute of Biochemical Physics, Russian Academy of Sciences, Moscow, Russia Background: An experimental verification of the original hypothesis - a “quasi-adaptive response (quasi-Ada) to alkylating agent in E. coli. Assessment perspectives of a new phenomenon application in medicine since structure and functional relationship of E. coli Ada-genes and their mammalian counterparts. Aims: 1)To study if S-nitrosylation (instead of S-alkylation in “true Ada”) of the key targets in the Ada protein might serve the signal for the quasi-Ada activation and contribute to the adapted cell resistance to challenge nitrosomethylure (NMU). 2)To answer the question if NO donor structure will affect the level quasi-Ada induction 3)To establish the structure of NO signal molecule. 4)To develop optimized protocol for NO donor application as pseudo substrate in experimental chemotherapy. Methods: A collection of E. coli mutants bearing (alkA::lacZ; alkB::lacZ and aidB::lacZ) operon fusion [Volkert, 1998] was used. Dinitrosyl iron complex (DNICglu) [Vanin, 1999] and a new stable tetranitrosyl iron complex with thiosulfate (TNIC thio) [Sanina, Aldoshin, 2000] were studied as NO donating agents. EPR-resonance spectroscopy was applied to detect a formation inside the cells the typical anisotropic EPR signal with g-factor 2,03. To measure the concentration of NO molecules generated we used an ami-NO-700 sensory electrode from in NO Nitric Oxide Measuring System. Results: Quasi-Ada induction by low toxic DNICglu could be quantified by 3-5 fold increasing in the levels of alkA and alkB expression and 1,5-2,5 fold decreasing in the rate of mutations and lethal lesions, induced by NMU. TNICthio was selectively effective in the alkA gene activation and in inhibition of the alkB and aidB genes expression. Intracellular iron was indispensable for NO-signaling: o-phenanthroline (OP) already prevented the phenomenon. Treatment of the cells with NO donors led to formation the EPR signal with g 2,03, which disappeared after OP cell pretreatment. It appeared that namely [Fe^+(NO^+)2 ] - mediated signaling cascade extends to the level of the Ada-gene transcription. In NMU responsive human tumour models of different genesis TNICthio increased their NMU sensitivity up to 36-92%. The promising results were obtained with some new NO-donors as the “independent” drugs, as well. Conclusions: The new quasi-Ada phenomenon extends NO functions in genetic signal transduction within the Ada response system. E. coli can be used as a valid model in identification of the new promising regulators of adaptive processes in mammals. This phenomenon is currently under study. This work was supported by Russian Foundation for Basic Research (08-04-00228).	Effect of caffeine on quinidine transport to the central nervous system in rats VASOVIC V, JAKOVLJEVIC V, BANIC B, SABO A, VUKMIROVIC S, MILIJASEVIC B Medical faculty of Novi Sad, Novi Sad, Serbia Background: Aim of research was to study the effect of caffeine on quinidine transition through the blood-brain barrier (BBB). Methods: Study was done on Wistar rats. Control group received sc physiological solution 30 min before quinidine administration; test group received sc caffeine 25 mg/kg bw. Quinidine 25 mg/kg bw was administrated into the right a. axillaris. Blood samples were taken from v. jugularis 30, 60, 90, 120, and 240 s after quinidine. After blood sampling, CNS was rinsed with 5 ml of distilled water injected to the left heart ventricle. Rats were sacrificed in same time intervals as for blood sampling. Brain tissue was divided into the brainstem, cerebellum, cerebral hemispheres. Qunidine concentrations were determined by standard spectrophotofluorimetric method. Results: At all time points caffeine decreased quinidine transport to brainstem compared to control (except in the 60th s, significant in 90th s). Decrease in quinidine transport to cerebellum was observed in all time points, except in 240th s (statistically significant in 60th, 90th and 120th s). In test group decrease in quinidine transition to left cerebral hemisphere compared to control, was observed in all time points except in 60th s (statistically significant in 90th s). Similar was observed for the right cerebral hemisphere but without statistical significance. Conslusions: 1) Observed decrease in quinidine transition to brainstem and cerebellum (1st CNS compartment) in presence of caffeine may be explained by effect of caffeine (also a cation) on quinidine transport through the BBB. 2) The hindered passage of quinidine to 2nd CNS compartment (cerebral hemispheres) under the influence of caffeine was less pronounced than in 1st compartment. This may be explained by local conditions of blood circulation, and regional properties of the BBB. 3) The quinidine concentration was higher in brain than in blood. Increased level in brain was registered with a latency compared to blood levels what could be explained by the involvement of active transport of quinidine, as well as by the fact that at higher physiological pH values, quinidine is mainly present in undissociated form. 4) Regional differences observed in quinidine transition to the brain indicate that there are some local factors influencing active transport.
The Prognostic Value of Genetic and Phenotypic Markers of Drug Metabolism and Host and Exposure Factors for Antituberculous Drug Induced Hepatotoxicity Vavilin VA^1, Makarova SI^1, KolpakovaTA^2, KudryashovAV^4, Mutaikhan J^2, Nikishina MV^1, Kojanova LA^3, Polyanskaya LV^3, Krasnov VA^2, Lyakhovich VV^1 ^1Institute of Molecular Biology and Biophysics, Novosibirsk, Russia, ² Novosibirsk Institute of Tuberculosis, Novosibirsk, Russia, ^3Close comany Institute Chromatograhy "EcoNova", Novosibirsk, Russia, ^4 Novosibirsk state University, Novosibirsk, Russia Background: The intensive antituberculous treatment regimens have as a concomitant result the high frequency of adverse reactions including hepatotoxicity. Estimation of patient individual genetic features in antituberculous drugs metabolism assume as a perspective approach to decreasing risk of these reactions. However discordance of genotype and phenotype in drug metabolism is possible. Methods: We investigated thepossibilityof the hepatotoxicity prediction in patients receiving isoniazid, rifampicin, pyrazinamide and ethambutol on the basis of isoniazid pharmacokinetics and genetic polymorphism of arylamine N-acetyltransferase 2 estimation. It was detected 282C>T, 481C>T, 590 G>A, 803G>A and 857G>A SNP’s in 75 patients with lung tuberculosis and in 52 patients was detected pharmacokinetics of isoniazid in first days of therapy. Results: Distribution of pharmacokinetic parameters of INH elimination (constant of elimination, Kel, clearance total (Cl) and time of half-elimination (t1/2) was bimodal with antimodes 0,2 h^-1; 3,5 ml/min/kg and 3,2 h, respectively. Median values of t1/2 were equal 6,44 h for slow and 1,8 h for rapid acetylators. Frequencies of 481T, 590A and 857A alleles were 0,36, 0,29 and 0,06, respectively. In group of examined patients were 13 cases (26%) of inconsistency between genetically predicted and pharmacokinetically determined acetylation phenotype, 11 of them were dislocation of genetically fast acetylators into phenotypically slow acetylators. Level of alanine aminotransferase (ALT) was elewated during first month of therapy in genetically as well as pharmacokinetically determined slow acetylators. However level of statistical significance of differences between initial values of ALT with those after 1, 2 and 3 month of therapy was higher in analysis with pharmacokinetic data. Conclusions: The pharmacokinetic estimations are more preferable for individual prognosis of drug-induced hepatotoxicity.	Can Cardiovascular Drugs Influence Clinical outcome in CABG patients VAVLUKIS M1, BOROZANOV V1, GEORGIEVSKA-ISMAIL Lj1, POP-GORCEVA D2, MAJSTOROV V2, KOSTOVA N1, ZDRAVKOVSKA M2 1Clinic for cardiology, Skopje, MK, 2 Institute for Patophysiology and Nuclear Medicine, Skopje, MK Background: The aim of our study was to analyze the effect of cardiovascular therapy on clinical outcome in Coronary Artery Bypass Graft surgery (CABG) population. Methods: A total of 494 CAD patients, treated with CABG surgery were analyzed. Pre and postoperative usage of cardiovascular (CV) therapy: digitalis, diuretics, aldosteron antagonists, ACE/ATRB inhibitors, beta blockers, Ca channel blockers, ASA alone or combined with oral anticoagulants, statins, nitrates and several other drugs were analyzed. Early (hospital morbidity and mortality) and late clinical outcome was followed during the period of 8,5 ± 4,6 y. Results: ASA alone or combined with oral anticoagulants applied early before CABG was found to be an independent predictor of reduced hospital mortality (OR 0,660; beta -2,818; p=0,000), acute heart and renal failure (AHF/ARF) and infections. In long term follow up, it reduces the risk of heart failure (HF) and myocardial infarction (OR 0,259; beta -1,692; p=0,048). Alone or together with oral anticoagulants, ASA is an independent predictor of reduced CAD progression (OR 0,137; beta -1,987; p=0,000) and need for re-revascularization (OR 0,169; beta -1,780; p=0,000). Postoperative ASA continuation reduces the risk for cardiac death (CD) (OR 0,360; beta -1,022; p=0,082), HF (OR 0,555; beta -0,588; p=0,019), CAD progression and re-revascularization. Surprisingly, statins increased the risk of arrhythmias early postoperativly (OR 2,920; beta 1,072; p=0,005), but they reduced the risk of AHF/ARF and late HF. They were also found to be independent predictors of reduced CAD progression (OR 0,378; beta -0,972; p=0,010), re-revascularization (OR 0,257; beta 1,276; p=0,022) and CD (OR 0,783; beta -1,180; p=0,034). Ca channel blockers reduce the risk of CAD progression and need of re-revascularization. Beta blockers given pre/postoperatively reduce the risk of HF (OR 0,455; beta -0,788; p=0,001 and OR 0,393; beta -0,934; p=0,000), but insignificantly increase the risk of CAD progression and re-revascularization. ACE/ATRB inhibitors gave us the most disappointing results. They were found to increase the risk of ARF/AHF, HF, myocardial infarction, CD, CAD progression and re-revascularization. Conclusions: 1) ASA and statin therapy should be started as early as possible after the diagnosis of CAD. 2) Ca channel blockers had benefitial effects in the reduction of CAD progression. 3) Beta blockers had benefitial effects in the reduction of HF, but no effect was found regarding to CAD progression. 4) We don't know where is the problem with ACE/ATRB inhibitors in our study?
Physico-Chemical Insights in Biological Conversions; the Role of H2O as driving Force in Cytochrome P450 catalysis VEEGER C^1, RIETJENS IMCM^1, ZAKHARIEVA O^2 ^1Dept. of Biochemistry, Wageningen Univ. Research Centre, Dreijenlaan 3, 6703 HA Wageningen, the Netherlands ^2Inst. für Physik, Univ. of Lübeck, Ratzeburger Allee 169, D-23538, Lübeck, Germany Background: For many years the involvement of the oxenoid iron form (Por^+Fe^IV=0) as active component in cytochrome P450 catalysis was generally accepted, despite many open questions. Method and Conclusions: An alternative approach was made by the study of microperoxidase-8 (MP-8). MP-8 is a peptide of eight amino acids bound by two cysteines to the pyrrole rings of the heme cofactor and ligated to a histidin of the peptide chain. MP-8 acts as a peroxidase in the presence of H2O2 that is fully inhibited in the presence of ascorbate, but remains active in different P450-activities. In the peroxidase mode it catalyses rather complicated polymerization processes, with one exception the nitration of phenols. In the P450 mode MP-8 either catalyses electrophilic substitution reactions like hydroxylation reactions or it catalyses nucleophilic substitution reactions like N-dealkylation and in dehalogenation. When the dehalogenation reaction is carried out in either methanol or ethanol, the halogen atom is replaced by the corresponding alkoxy-group. MP-8 catalyses O-exchange between H2O2 and H2O. Oxygen from water is incorporated in two compounds without any hetero atom, naphtalene and anthracene. This observation indicates that 55.5 M water is the driving force in MP-8 and most probably also in P450 catalysis. Insight into the actual involvement of different heme-species was obtained by electronic structure calculations in local spin-density approximation along the reaction pathway of the model compound hydroxylating its substrate. Only under specific geometry the peroxo-iron species (PorFe^III-OO^-) passes in the hydroxylating reaction pathway with the substrate a maximum around 190-200 pm followed by a minimum at approximately 130-140 pm, the length of an aromatic C-O bond, as well as region-selective substitution.. In Summary: Peroxo-iron can act as the single species in both nucleophilic and electrophilic P450-type of substitution reactions; Since MP-8 has limited stability under turnover conditions design of small and stable heme-iron catalysts is necessary for applying in clean chemical reactions.	Unprecedented Antitumor Effect of Irradiation Generated by 5-Trimethylsilyl-2-Trifluoroacetylfuran Oxime in Fibrosarcoma Cells VEINBERG G, DOMRACHEVA I, SHESTAKOVA I, VORONA M, IGNATOVICH L, LUKEVICS E, KALVINSH I Latvian Institute of Organic Synthesis, Riga, Latvia Background: Numerous experiments evidence that spontaneous or artificially induced signalling photon emission (SPE) produced by unicelllular and multicellular organisms serves not only for the regulation of metabolism but also for their biocommunication with surrounding biological systems. Emission, trapping, amplification and other aspects of SPE are mainly confined in the boundaries of appropriate biological system. However its small part break loose in outer space and could be detected both qualitatively and quantitatively using appropriate physical equipment or detectors of biological origin. During antitumor screening of organic substances in vitro using monolayer cells, we unexpectedly found that SPE with strong cytotoxic effect could be induced by 5-trimethylsilyl-2-trifluoroacetylfuran oxime (IOS-8596). Methods: The solution of IOS-8596 was added only in 3 rows of conventional polystyrene 96-multiwell plate containing monolayer human fibrosarcoma cells (HT-1080). Thus prepared plate was cultivated at 37^oC for 72 hours. A quantitative assay for the cell population treated and untreated with IOS-8596 was performed by staining with crystal violet (CV) or Coomassie Brilliant Blue R-250 (CB). Results: It was found, that IOS-8596 killed the cells, not only in the well-emitters in which it was added, but also in adjacent well-detectors containing cell culture alone. The intensity of cytotoxic effect observed in well-detectors directly depended on the amount of IOS-8596 added in well-emitters. The addition of -carotene in well-detectors at a concentration of 10 g/ml resulted in the twofold reduction in cytotoxic effect in well-detectors proving the participation of singlet molecular oxygen in metabolic processes induced by SPE. It was found that the intensity of SPE could be effectively regulated by optical fibres by the putting their one end in the well-emitter or in well-detector like antenna. For example the placement of one end of optical fibre with 400-2400 nm diapason of wavelength transmission in well-emitter resulted in 2-fold increase of cytotoxic effect in well-detectors compared with the control. Conclusions: Thus obtained experimental data evidence about the development of a simple and reproducible method of SPE generation, its detection and investigation in 96-multiwell plate the same as its amplification using optical fibers providing good perspective for non-invasive treatment of cancer.
Dopaminergic Receptors: Potential Therapeutic Applications VELASCO M^1, ISRAILI ZH^2 ^1Clinical Pharmacology Program, Vargas Medical School, Central University of Venezuela, Caracas, Venezuela; ^2Department of Medicine, Emory University, Atlanta, GA USA Background: Dopamine (DA) induces a variety of physiological responses in the cardiovascular (CV) and renal systems, which result from its interaction with DA receptors. DA and its agonists have a role in the treatment of high blood pressure (BP), other CV and renal dysfunction. Methods/Results/Discussion: The neurotransmitter DA, the precursor of noradrenaline, induces a variety of CV and renal physiological responses, including an increase in myocardial contractility and cardiac output without changes in heart rate, passive and active vasodilatation, diuresis and natriuresis. These responses result from its interaction with the DA receptors D1, D2, D3, D4 and D5, and recently discovered D6 and D7 receptors. The expression and functions of the DA receptors are tissue specific. The interaction of DA and dopaminergic compounds with the DA receptors involves cyclic AMP, intracellular calcium, potassium channels, Na+/H+ exchanger, Na+/H+/ATPase pump, etc. The CV, renal and hormonal actions of DA and dopaminergic compounds are dose-dependent. In some types of hypertension. DA is known to influence the control of arterial BP by influencing the central and peripheral nervous system and target organs such as the kidneys and adrenal glands. DA and DA-agonists, including inhaled DA have a role in the treatment of high BP, other CV diseases and renal dysfunction. Hence, it is important to review the physiological and pharmacological aspects of DA and its receptors, and the potential clinical utility of DA and its derivatives in the therapy of hypertension, other CV diseases, and renal dysfunction. Conclusions: DA is an important neurotransmitter with varied physiological action in the CV and renal systems. DA and DA-agonists, including inhaled DA have a role in the treatment of high BP, other CV diseases and renal dysfunction.	Preoperative chemoradiation with concurrent Capecitabine for Locally Advanced Resectable Rectal Cance (LARC): a Phase II study VELENIK V¹, ANDERLUH F¹, OBLAK I¹, SEGEDIN B¹, ZAKOTNIK B¹, STROJAN P¹ ¹ Institute of Oncology, Ljubljana, Slovenia Background: A prospective one-arm phase II study was performed between 6/2004-1/2005 to assess efficacy and toxicity of preoperative radiotherapy (RT) and concurrent oral capecitabine in patients with LARC. Methods: Patients were irradiated to 45 Gy in 25 fractions over 5 weeks to the pelvis concomitantly with oral capecitabine 825 mg/m2 bid including weekends. Surgery was scheduled 4-6 weeks after completion of the chemoradiotherapy. Four courses of chemotherapy were planned postoperatively. The primary endpoint of the study was complete response rate; secondary endpoints included toxicity, survival and long-term rectal and urogenital morbidity. Patients still alive and without recurrence of the disease, with a minimum follow up of 1 year, were questioned with LENT/SOMA late-effect scale for rectum, bladder and sexual function. Results: Fifty-seven patients entered the study (median age 67 years, 43 males and 14 females). During preoperative part, one female died after receiving 27 Gy (pulmonary embolism) and the most frequent grade 3 toxicity was dermatitis (33.9% of patients). Radical operation was performed in 55/57 of patients. The complete pathological response rate was 9.1%. T-, N- and overall downstaging rates were 40%, 52.9% and 49.1%, respectively. A total sphincter preservation rate was 65.5%. During the early perioperative period, one patient died due to sepsis. Five patients (10%) were reoperated due to anastomotic leakage, intraabdominal abscess, ileus, enterocutaneous fistula and stomal occlusion. At 1 year of follow-up, the rate of patients with severe late (SOMA grade 3 and 4) rectal, bladder and sexual toxicity was 40%, 19.2% and 31%, respectively. The local relapse has occured in 1 (1.8%) patient and disemination in 13 (24%) out of 54 patients with median time to progression 23 months (range 3-23 months). Second primary malignancy has occured in 2 patients. The actuarial 2-year (median follow-up) OS, DFS and DSS rates were 84.2%, 72.5% and 92.4%, respectively, and local control was 98.2%. Conclusions: Preoperative chemoradiotherapy with oral capecitabine is well tolerated, safe and convenient treatment regiment of LARC. It results in excellent local control. The rate of distant relapse and late functional morbidity is still of concern and asks for new treatment approaches
Vulvovaginal Colonization by Aspergillus Species in Nonimmunocompromised Women BAGGISH MS , VENTOLINI G Department of Obstetrics and Gynecology, Good Samaritan Hospital, Cincinnati, OH. Department of Obstetrics and Gynecology, Wright State University, Boonshoft School of Medicine, Dayton, OH This study was undertaken to determine the prevalence of female genital Aspergillus infections. Additionally, the study explored whether genital Aspergillus infections were associated with Aspergillus infections elsewhere (e.g., pulmonary, cardiac, orthopedic, or ophthalmologic). Between October 2005 and October 2007 vulvovaginal fungal cultures were performed for all patients seen in the Vulvar Disorders Center at the Good Samaritan Hospital (Cincinnati, OH) and similarly at Wright State University Boonshoft School of medicine Department of Obstetrics and Gynecology (Dayton, OH). Prospectively, any cultures showing Aspergillus species were segregated and a running list totaling 16 patients was maintained. The prevalence for Aspergillus in the vaginal culture was 6 per 1,000. The patients were all followed, examined and cultured at each subsequent visit for genital and extragenital disease. The patients responded to therapy with a culture-proven elimination of the fungus. Sporanox^® (itraconozole; Janssen Pharmaceuticals, Titusville, NJ) 200 mg daily for 30 days proved to be the most objectively effective agent for fungus elimination. No systemic Aspergillus infections were observed during the follow-up period. Disclosure: The Authors have no connection to any companies or products mentioned in this study.	Hyaluronan Mixed Esters of Butyric and Retinoic Acids: Multicomponent-Multitarget Drugs with Differentiating and Paracrine Logics for Cardiovascular Repair with Human Mesenchymal Stem Cells VENTURA C, CAVALLINI C, BIANCHI F, LIONETTI V, SCARLATA I, CANTONI S Laboratory of Molecular Biology and Stem Cell Engineering, National Institute of Biostructures and Biosystems at the Cardiovascular Department, University of Bologna, Italy Cell lineage specification is fashioned at multiple interconnected levels and is controlled by a complex interplay between cell signaling, nucleosomal assembly, the establishment of multifaceted transcriptional motifs and the temporal and spatial organization of chromatin in loops and domains. Stem cells are pluripotent elements that possess the unique capability to self-renew, grow indefinitely and develop into multiple types of cells and tissues and may both offer a clue for the identification of specific differentiation patterning and hold a promise for rescuing damaged tissues. The demonstration of a multineage potential in human mesenchymal stem cells has prompted such a perspective even in humans. Within this context, the development of strategies affording high-throughput of targeted commitment from pluripotent cells would have obvious therapeutic potential. However, overexpression of genes coaxing stem cells to a specific lineage by vector-mediated gene transfer is a cumbersome approach that may perturb normal homeostasis in both stem cells and recipient tissues, and is not readily envisionable in humans. It is now evident that the identification and/or the development of molecules which induce selected commitment in pluripotent cells would help to elucidate the molecular mechanisms underlying complex differentiating processes and may ultimately allow us to regenerate tissues in vivo. Recent developments in the area of stem cell research have been boosted by an increasing understanding of transcriptional regulation and epigenetic modifications, including histone acetylation, DNA methylation and chromatin remodeling. Within this context, we have developed hyaluronan mixed esters of butyric and retinoic acids (HBR), eliciting a remarkable increase in the transcription of cardiac lineage-promoting genes, and cardiac differentiation in mouse embryonic stem (mES) cells, ensuing in a high yield of spontaneously beating mES-derived cardiomyocytes [1]. On the whole, these results demonstrate the potential for chemically modifying the gene program of cardiac differentiation in stem cells without the aid of gene transfer technologies and may pave the way for novel approaches in tissue engineering and myocardial regeneration [1]. Recently, we have used HBR to successfully coax human mesenchymal stem cells from bone marrow (BMhMSCs) and alternative sources, including the dental pulp (DPhMSCs) and fetal membranes of term placenta (FMhMSCs), towards a cardiovascular decision in vitro [2]. In each cell population, HBR increased the transcription of cardiogenic and vasculogenic genes, leading to the appearance of cardiac marker-expressing cells and endothelial cells. Interestingly, HBR primed a remarkable and sustained increase in the secretion of both “Vascular Endothelial Growth Factor (VEGF” and “Hepatocyte Growth Factor (HGF)”, acting in a paracrine fashion as trophic mediators that not only possess angiogenic but also cardioprotective effects, including antiapoptotic, mitogenic and antifibrotic activities [3,4]. Under our experimental conditions, the differentiating and paracrine effects primed by HBR in vitro were considerably more pronounced in FMhMSCs than in BMhMSCs or DPhMSCs. Transplantation of FMhMSCs preconditioned ex vivo with HBR into the hearts of rats subjected to acute myocardial infarction by coronary ligation led to complete normalization of myocardial performance and dramatic reduction in scar formation [2]. The injection of HBR-treated cells was followed by a significant increase in density of capillaries negative for anti-human von-Willebrand Factor (vWF) (due to vasculogenic responses from the recipient tissue), indicating that enhanced in vivo release of VEGF and HGF by HBR-pretreated FMhMSCs may have contributed to the observed cardiac repair. Besides this, in the hearts injected with HBR-exposed FMhMSCs, the yield of cells positively stained with a human-specific anti-vWF antibody remarkably exceeded the number of vWF-positive cells detected in samples from the untreated group [2]. A consistent organization of human vWF positive cells into erythrocyte containing capillary vessels was only observed in hearts transplanted with HBR-treated FMhMSCs. Hence, the vascular lineage commitment primed by HBR in vitro was retained within the transplanted cells in the infarcted myocardium, suggesting that HBR-treated cells may also contribute to neovascularization and heart rescue through their ability to generate capillary-like structures. Some of the transplanted cells that had been pretreated with HBR also expressed cardiac-specific markers, although the physiological properties of this cardiac-like cells and their contribution to cardiac rescue remain to be assessed.Noteworthy, xenogeneic FMhMSC transplantation was devoid of immune rejection and did not require immunosuppressant procedures. This observation and the finding that FMhMSCs exhibited larger differentiating and paracrine responses to HBR compared to DPhMSCs or BMhMSCs may be relevant for future development of new chemistry designed for targeting stem cell fate and therapy. In fact, recent evidence suggests that age and disease states affect the collection of sufficient healthy autologous bone marrow-derived stem cells, which will decrease the ability of autologous cells to rescue infarcted hearts [5]. FMhMSCs do not induce allogeneic or xenogeneic lymphocyte proliferation and actively suppress lymphocyte responsiveness [6]. Moreover, FMhMSC transplantation in neonatal swine and rats resulted in human microchimerism in various organs and tissues [6].In conclusion, novel chemical agents with differentiating and paracrine logics may be used in combination with tolerogenic human mesenchymal stem cells to afford unprecedented strategies of stem cell therapy and regenerative medicine.