Ehrlich II –2nd World Conference on Magic Bullets

Results: The blood culture was positive in 31% and the pus culture was positive in 13%. The causal bacteria were, Staphylococcus aureus in 23%, Salmonella in 17 %, streptococcus pneumonia in 13%, Citrobacter freundii in 9%, others bacterias in 20%. The first line antibiotherapy was effective in 25% and antibiotic resistance was observed in 33%. The bacteria which produced betalactase with a large spectrum was observed in 15%, classic quinolone resistance was observed in 15% and the cross resistance to the quinolone in 50%. Staphylococcus aureus was methicillino-resistant in 25%, and negativ gram bacteria resitance in 40%.

Conclusion: The first antibotherapy line with oxacillin and thirst generation of cephalosporin indicated osteo-articular infection is less effective because of the bacteria resistance.

Anti-microbial, Anti-diarrhoeal and Toxicity Profile of Cylicodiscus gabunensis Stem Bark (Mimosaceae) KOUITCHEU MABEKU LAURE B^1, PENLAP BENG V^1, KOUAM J^2, ESSAME OYONO^3, ETOA FX^1 ^1:Laboratory of the Applied Microbiology and Molecular Pharmacology, Department of Biochemistry, Faculty of Science, P. O. Box 812, University of Yaoundé I, Cameroon. ^2: Phytochemistry Laboratory, Department of Chemistry Faculty of Science, P. O. Box 812, University of Yaoundé I, Cameroon. ^3: Anatomo-Pathology Laboratory, Department of Morphology and Pathological Anatomy of the Faculty of Medical and Biological Sciences. University of Yaoundé I, Cameroon. Background: Diarrhea is a major health problem for children worldwide, accounting for 5-8 million deaths each year. Cylicodiscus gabunensis (CG) is a plant of Cameroonian pharmacopeia. It is reputed for its beneficial effects in the treatment of diarrhoea related illnesses. But their use as medicament based simply on a traditional folk use that as been perpetuated along several generations with no scientific data on their efficacy. Since sub-acute or sub-chronic toxicity data are required to predict the safety associated to the use of medical products, it was necessary to provide this information in order to bridge the gap in knowledge about the toxicity profile of this plant. Therefore the present study has been planned to investigate the real efficacy and safety of this drug. Methods: In order to be sure of the therapeutic properties that this plant may have as anti-diarrhoeal, the ethyl acetate (EA) extract of the stem bark of (CG) was evaluated in vitro for its anti-microbial activities against 17 pathogenic microbial strains involved in diarrhoeal infection isolated from patient : Escherichia coli, Klebsiella pneumoniae, Shigella dysenteriae, Shigella flexneri, Morganella morganii, Proteus vulgaris, Proteus mirabilis , Salmonella typhi, Citrobacter freundii , Enterobacter cloacae, Enterobacter agglomerans , Staphylococcus aureus , Streptococcus feacalis , Pseudomonas aeruginosa, Bacillus cereus T, Candida albicans and Candida glabrata. Disc diffusion method was employed for the determination of anti-microbial activities and MICs (minimum inhibition concentration) against the test organisms by broth macrodilution method. The effect of the (EA) extract of (CG) was further investigated for anti-diarrheoal activity in vivo against castor oil-induced diarrhoea, charcoal-induced gut transit and castor oil-induced small intestine enteropooling. The electrolyte concentration of the small intestinal fluid was also evaluated. The toxicity profile of the (EA) extract of the stem bark of (CG) was studied on wistar rats. The rats were administered graded doses (0.75, 1.5, 3 and 6 g/kg p.o) of the extract daily for 6 weeks and the effects on clinical signs, body weight, food and water consumption, organ weight, haematology, histology as well as serum, hepatic and renal biochemical parameters were measured. Results: The best MIC and MBC values for the microorganisms sensitive to the extract were 0.00078 and 0.00315 mg/ml respectively. The greater and remarkable antimicrobial activity of the (EA) extract of CG was recorded with Staphylococcus aureus, Proteus vulgaris and Bacillus cereus T. Like loperamide (3 mg/kg body weight) a single oral dose of this extract (375, 750 mg/kg body weight) produced a significant decrease in the severity of diarrhoea. The extract produced a decrease in intestinal transit (10.26 - 30.75 %,), and unlike atropine, it significant inhibited castor oil-induced enteropooling. However, it did not alter the electrolyte concentration in intestinal fluid as compared to castor oil-treated rats. Body weight of dosed and control rats increase throughout the duration of treatment but food and water consumption were not significantly affected. The relative weights of the liver, lungs, heart and kidneys remained normal whereas a significant change was observed in that of the spleen. The hematocrit level was increased in treated animal. Our data demonstrates a significant increase in serum concentrations of aspartate amino-transferase, alanine amino-transferase, total cholesterol and glucose with high-dose of CG treatment tested (3 g/kg). CG also caused a significant reduction in hepatic malondialdehyde concentration. Renal urea and creatinine levels were reduced significantly in test groups. Histological findings reveal a characteristic progression treatment-related effect on liver, kidneys and lungs. The acute toxicity LD50 was estimated at 14.5 and 11 g/kg body weight for male and female respectively, but dose-related mortality of 30 and 50 % was observed during the sub-acute toxicity. Conclusion: 1- Our observations confirm that (EA) extract of the stem bark of (CG) possesses strong anti-microbial activity in vitro against some pathogenic microbial strains involved in diarrhoeal infection.2- It also possesses a significant anti-diarrhoea activity due to its inhibitory effect both on gastrointestinal propulsion and fluid secretion. 3 - The findings on the toxicity study have once more highlighted the limitations of the acute toxicity LD50 testing and suggest that (CG) may exert varied toxicological effects when administered orally either acutely (up to 4 g/kg p.o.) or sub-chronically (up to 0.75 g/kg p.o.) in rats.	Secretin and Autism KÖVES K^1, HEINZLMAN A^1, SZABÓ G^2 ^1Semmelweis University, Budapestand ^2Albert Szent-Györgyi Medical University, Szeged, Hungary. Background: Leading morphological disorders in autism are found in the cerebellum and in the frontal and parietal cortices. In autistic postmortem cerebellum the level of AMPA glutamate receptors, GAD and reelin level decreased compared to control values. Increased homovanillic acid level in the cerebrospinal fluid, hyperserotoninemia and endorphinemia, changed serotonin metabolism in the brain are the most consistent disorders in autism. The researchers look for compounds to treat autism. At this moment there are no therapeutic agents which are able to generally improve the autistic phenomena. Inspite of this fact there are many compounds that are used for treating autistic patients and at least in part of the cases they seem to be effective. One of these compounds is secretin. Its significance in autism was first suggested by Horvath and his co-workers (1998, 1999). Their observation facilitated us to explore the occurrence of secretin in the nervous system. Methods: In our laboratory with the use of immunohistochemistry secretin immunorectivity was looked for in colchicine treated rats, intact cat and human samples. We have also investigated the role of secretin given intracerebroventricularly (icv) on the behaviour of mice with genetic cerebellar atrophy testing open field activity, novel subject approaches and rearing. Results: Secretin was found in the pyramidal cells of the motor cortex, Purkinje cells, a subpopulation of central cerebellar nuclei, the mesencephalic nucleus of the trigeminal nerve, the superior olivary nucleus, the cells of trapezoid body, a subpopulation of the cells of spinal and trigeminal ganglia. Our data show, that besides a well established synthesis of secretin in the gastrointestinal tract, several nerve structures can produce secretin. Icv administration of secretin ameliorated the hypermotility of mice which is characteristic for this genetically modified strain. Conclusion: Our data and those available in the literature indicate that secretin, besides its gastrointestinal role, is a neuropeptide as well. It was found by Gershon and D’Antreaux (2003) that the number of secretin producing S cells in autistic patients is half of the normal. Because secretin is present in Purkinje cells, its level has to be dramatically decreased in the case of cerebellar atrophy. Tthe low level of secretin may be responsible for some autistic symptomes.
Vitamin A and D Derivatives; Potential MAGIC BULLETS with Antithrombotic and Antineoplastic Applications KOYAMA T, CHUNG J Tokyo Medical & Dental University, Tokyo, Japan. Background: Tissue factor (TF) is a membrane-bound glycoprotein that is essential for activation of the coagulation pathway. Although synthesis of TF is tightly regulated, its expression can be induced by a variety of agonists, such as tumor necrosis factor (TNF) and oxidized low-density protein (LDL). Induction of TF expression in endothelial cells, monocytes, or malignant cells is associated with fatal thrombotic disorders, such as disseminated intravascular coagulation (DIC) and atherosclerotic thrombosis. Downregulation of the pathological expression of TF appears to be a critical strategy to prevent and treat various thrombotic disorders. A vitamin A derivative, all-trans retinoic acid (ATRA) and the active form of vitamin D3, 1,25-dihydroxyvitamin D3 (D3), and their synthetic analogs can induce myeloid leukemia cells to differentiate and inhibit the clonal growth of those cells. Methods & Results: Because of dramatic clinical experiences of rapid amelioration of DIC in acute promyelocytic leukemia patients treated with ATRA, we found the anticoagulant effects of RA derivatives. They evoke an anticoagulant effect by upregulating the expression of an anticoagulant glycoprotein thrombomodulin (TM) and downregulating the expression of TF in leukemia cells or cytokine-stimulated vascular endothelial cells and monocytes. Since RAs and D3 are similar in their mechanisms of action, we also investigated the anticoagulant effects of D3. D3 and its analogs downregulated TF and upregulated TM expression in monocytic cells, counteracting the effects of TNF and oxidized LDL. Pathogenic expression of TF mRNA in monocytic cells was markedly downregulated by D3 and its synthetic analogs. We have recently found that D3 suppresses basal and TNF-induced TF expression in monocytic cells by inhibition of AP-1 and NF-B activation pathways. D3 analogs also effectively downregulate TF in several cancer cells. The more potent D3 analogs, which have far stronger binding affinity to D3 receptor (VDR) have been synthesized. Conclusions: Several studies report that the D3/VDR system has a physiological role in vivo in the maintenance of antithrombotic homeostasis. We propose that synthetic retinoids and D3 derivatives could be developed as a new type of antithrombotic agent, which will ameliorate the procoagulant character of abnormal cells and act as antineoplastic agents.	Effects of Intracarotid Injection of Methylprednisolone on Cellular Oedema after Osmotic Opening of the Blood-Brain Barrier in Rats KOZLER P^1, POKORNÝ J^2 ^1Department of Neurosurgery, Central Military Hospital, 1^stFaculty of Medicine, Charles University, Prague, CZ, Czech Republic ^2Institute of Physiology, 1^st Faculty of Medicine, Charles University, Prague, CZ, Czech Republic Background: In our work we studied methylprednisolone (MP) for its effects on the permeability of cytoplasmatic membranes of neuronal populations in the rat. Methods: We used a standard model of cellular oedema induced by water intoxication, applying MP selectively into the internal carotid artery(ICA) after opening the blood-brain barrier (BBB) with mannitol. The results were assessed under fluorescence microscopy in keeping with the Intracellular Distribution Index of Evans Blue (IDI EB) in the neocortical field (Cortex) and in hippocampal areas CA1, CA3 and GD (gyrus dentatus). [The IDI EB values range from 0 to 2. IDI EB=1 matches equal extra/intra cellular EB distribution, IDI EB>1 means dominant intracellular EB distribution, IDI EB<1 means dominant extracellular EB distribution]. Evans blue (EB) was applied similarly as MP. Three different experiments were carried out. In experiment 1 - EB alone and no MP was applied. In experiment 2 - 5.4 mg/kg MP and EB were applied. In experiment 3 - 54 mg/kg MP and EB were applied. Results: In experiment 1 the IDI values were high (>1), indicating the presence of large quantities of EB in the cells. In experiments 2 and 3 the IDI values were low (<1), indicating more EB outside than inside cells. IDI differences between experiments 2 and 1 and experiments 3 and 1 were statistically significant (p<0.05). Conclusions: In our view, this amounts to morphological evidence of cell membrane integrity restored under the effect of MP.
Chlorhexidine Gluconate - Local Antimicrobial Agent in Aid of Prevention and Treatment of Periodontal and Peri-implant Diseases KOZLOVSKY A, ISRAELI-TOBIAS CH, BAR-NESS GREENSTEIN R, HIRSHBERGA The Maurice and Gabriela Goldschleger School of Dental Medicine, Tel Aviv University, Tel Aviv, Israel. Background: Chlorhexidine gluconate (CHX) is the most effective oral antimicrobial and anti-plaque agent and it is the first drug of choice in aid of prevention and treatment of periodontal and peri-implant pathology in clinical situations, mostly when mechanical oral hygiene is compromised or impossible. Therefore, the aims of our two separated studies were: 1. To evaluate the influence of CHX on the process of wound healing 2. To assess the substantivity of CHX to titanium surfaces. Methods: 1. Excisional wounds ( 5 mm in diameter) were made in the center of the palate of 125 Wistar male rats. CHX, phenolic compounds, amine/stannous fluoride solutions and saline as a control, were applied daily for 1 minute. Six animals were sacrificed from each group at 3, 7, 14 and 21 days post-operatively. Wound areas were measured photographically. The epithelialization rate was determined histologically. 2. Saliva coated machined (M), and sandblasted and acid-etched (SLA), titanium discs were soaked in CHX solution. The levels of adsorbed and desorbed CHX were measured spectrophotometrically and bacterial inhibition on S. mutans bacterial lawns were calculated. Results: 1.The mean area of wounds decreased significantly with time (p<0.001) in all experimental and control groups with CHX and phenolic compounds solution presenting the best rate of epithelialization (-10.56 SE 11.05 and -10.06 SE 6.80 respectively). 2. Of the available CHX, 3-8% was adsorbed, SLA surface adsorbed significantly more CHX than M surface (p<0.001) and discs immersed in 0.2% CHX adsorbed significantly higher levels of CHX as compared to 0.1% solutions (p<0.001). A part of the CHX adsorbed to SLA and M discs (0.6 % and 1.1% accordingly) was desorbed following 24h.The levels of desorbed CHX were significantly affected by CHX concentration (p=0.013).The antimicrobial assay revealed that 0.2% CHX solution and SLA discs presented a significantly higher inhibition level than 0.1% CHX and M discs. Conclusions: 1. Use of CHX as local antimicrobial agent do not impair wound epithelialization and may be used for post operative infection control. 2. The demonstrated evidence of substantivity of CHX to saliva-coated titanium surfaces supports its use onto implant surfaces.	Nifedipine, the most citated, studied, experienced and debated ca-antagonist: where has it gone? KRAGTEN JA, Dunselman PHJM Atrium medical centre Parkstad, Heerlen, The Netherlands. Of all dihydropyridine Ca-antagonists, that have been introduced since the 1960s, nifedipine was not only the first, but probably since that time, the most prescribed, debated and studied Calcium antagonist. Introduced as the third possibility to treat patients with anginal discomfort, after nitrates and betablockers, it was very much appreciated at that time. Not having the possibility to treat patients with PCI and or routine CABG, adding medication to triple treatment on the coronary care dept. was a standard treatment in unstable or worsening angina, however often proved to work insufficiently for those patients. The first formulations of Nifedipine - short acting – had to be administered 4 times a day, causing massive fluctuations in plasma levels during the day. These short-acting formulations were far from ideal and in 1996 several studies, involving various dosing regiments (up to 120mg/day!) reported possible dangerous effects in secondary prevention. The reports given by Furberg (1995) caused an intensive debate on the safety of these drugs both in the treatment of angina pectoris as in the treatment of hypertension. Following this debate and with the knowledge of the farmacokinetic profile, long-acting formulations of the drug were developed and studied in large scale randomised controlled trials, both in hypertension and angina pectoris. The results of these trials, presented in the period 2000 – 2004 demonstrated both the safety and the positive effect of long acting formulations of Nifedipine, especially the GITS formulation. As a consequence of these results, guidelines for both hypertension and angina pectoris have recently been reconsidered, and have put the modern, long-acting formulations of the ca-antagonists in a pole position. Within this group of therapeutics, Nifedipine GITS has a unique position and cannot be replaced by just an other Nifedipine formulation, which has not been proven effective and safe in large clinical trials in order to prevent the history to repeat itself.
Fusion Proteins For Flexible Vaccine Antigen Targeting To Cell Surface Receptors KRATZER R^1,2, MAUVAIS FX^1,2, VAN ENDERT P^1,2 ^1INSERM U580, Paris, France; ^2Université Paris Descartes, Paris, France. Background: The initiation of an immune response requires that relevant antigens gain access to the appropriate intracellular compartments to be broken down to peptides, which can then be loaded on major histocompatibility complex (MHC) class I and/or class II molecules. Peptide presentation by cells providing efficient costimulation is important for priming and boosting T cell responses, a requirement met best by dendritic cells (DCs). Targeting of protein vaccines to antigen presenting cells is therefore an attractive strategy for eliciting cellular immune responses. However, because of the difficulty of coupling frequently hydrophobic proteins to targeting devices such as antibodies, its potential has been little exploited. This problem could be bypassed by using fusion proteins. Methods: We have produced several antigens, including hydrophobic viral proteins with an interest as vaccines, as fusion proteins. The soluble proteins also comprise protein G domains for binding to immunoglobulins, and ubiquitin for proteasome targeting in cross-presentation pathways. The behavior of the fusion proteins has been analyzed in situ by fluorescence microscopy. The humoral and cellular immune response of immunized mice has been evaluated by Enzyme-linked immuno-sorbent assay and flow cytometric analyses. Results: Complexes between fusion proteins and suitable antibodies bind specifically to DCs and are internalized into endolysosomal compartments. The fusion proteins are rapidly transported in vivo to CD169+ and later to CD35+ cells in the draining lymph nodes after subcutaneous immunization. Using the model antigen ovalbumin, we show that the fusion proteins, when coupled to various antibodies, elicit a humoral immune response and both MHC class I and class II restricted T cell responses in vitro and in vivo with an at least 100-fold greater efficiency than antigen alone. Multiple-cytokine-producing CD8+ effector T cells are generated. Conclusions: 1) The results demonstrate the potential of the strategy for vaccination by initiating a potent immune response. 2) This new tool can be used to stimulate the immune system in a suitable way by targeting the antigen to specific cell surface receptors. 3) A major advantage of this strategy is the possibility to easily compare immune responses by simply exchanging the targeting antibody.	The impact of interferon-β treatment on the blood-brain barrier KRAUS J Paracelsus Private edical University and Salzburger Landesklinken, Christian-Doppler-Klinik, University Hospital of Neurology, Ignaz-Harrer-Straße 79, 5020 Salzburg, Austria., Phone: [phone], Fax: [phone], email: [e-mail] Molecular alterations at the blood-brain barrier (BBB) are essential in the pathogenesis of multiple sclerosis (MS). The implementation of the immunomodulators as the first evidence-based treatment option in the mid 1990s opened a new era in multiple sclerosis therapy. Interferon β (IFN β) is commonly suggested to act as an immunomodulator of the cytokine network. However, there is increasing evidence that IFN-β leads to a stabilization of BBB integrity in MS patients. This paper will show recent advances in MS with focus on the BBB and will present the author’s contribution on revealing direct and indirect effects of IFN—β on the BBB. It will summarize recent work both with MS patients as well as experimental in vivo and in vitro data. The understanding of IFN β-derived stabilization of the BBB will not only provide new insights in the pathogenesis of MS but also might be helpful in the development of new, more specifically designed drugs in the treatment of MS.
Effects of Small-volume Resuscitation Using Hyperosmolar Saline Colloid Solution on Regional Blood Flow and Ischemic Tissue Injury KREIMEIER U^1, MESSMER K^2 ^1Dept. Anaesthesiology, ^2Dept. Surgical Research; Univ. Hospital Munich, Germany Background: The concept of Small-volume Resuscitation (SVR) – the rapid infusion of a small dose (4 ml per kg B.W.) of 7.2 – 7.5% NaCl/colloid solution – has been advocated for initial therapy of severe hypovolemia and shock. The solution seems to encompass specific pharmacologic effects, highly relevant for prevention of multiple organ dysfunction syndrome (MODS) induced by trauma and shock. Methods and Results: - Hemorrhagic shock Using the radioactive microspheres method we demonstrated the positive circulatory effect of 7.2% saline given in an amount as small as 1/10 of actual blood loss. In a standardized animal model of severe hemorrhagic hypotension in dogs macrohemodynamic parameters and blood volume were normalized and nutritional blood flow (RBF) recovered within 5 min. RBF was completely restored when combining 7.2% saline with 10% dextran 60 (HHS; hyperosmolar-hyperoncotic solution). - Endotoxin shock In a pig model of endotoxin shock elicited by continuous i.v. infusion of S. abortus equi leading to a hyperdynamic state with severe circulatory and pulmonary deterioration, 4 ml/kg B.W. of HHS led to significant enhancement of RBF particularly in the small intestine and kidneys. - Reperfusion injury Using the hamster dorsal skin-flap for analysis of leukocyte-endothelial interaction HHS proved to ameliorate the activation of polymorphonuclear neutrophils (PMNL). Functional capillary density was augmented and extravasation of macromolecules diminuished. - Up-regulation of beta2-integrins In in-vitro experiments, hyperosmolar saline attenuated N-fomyl-methionyl-leucyl-phenylalanine (fMLP) stimulated expression of adhesion molecules on PMNLs. fMLP-stimulated up-regulation of beta2-integrins was diminished. - Subarachnoid hemorrhage In a standardized model of subarachnoid hemorrhage (SAH) in rats treatment with 7.5% NaCl plus 6% dextran 70 resulted in lowered intracranial pressure, improved neurological recorery and less morphological damage. Conclusions: 1) Small-volume hyperosmolar colloid resuscitation from severe hemorrhage and shock rapidly mobilizes endogenous water. 2) Nutritional blood flow is enhanced and reperfusion injury diminuished. 3) Following subarachnoid hemorrhage hyperosmolar saline dextran solution improves neurological outcome.	Anti-Idiotypic Vaccines Against Autologous Antigens: Designed Ankyrin Repeat Proteins as Scaffolds to Break Self Tolerance? KRICEK F^C, VOGEL M^A, KELLER-GAUTSCHI E^A, BAUMANN M^A, RUF C^C, EFFENBERGER F^C, AMSTUTZ P^B, STADLER BM^A. ^aInstitute of Immunology, Inselspital, CH-3010 Bern, Switzerland ^bMolecular Partners, University Zürich, Winterthurerstr. 190, CH-8057 Zürich, Switzerland ^cNovartis Institute for Bio Medical Research GmbH 8 Co KG, Brunner Strasse 59, 1235 Wien Austria According to the network hypothesis antibodies may act as antigens to induce an anti-idiotypic immune response. The binding site of an anti-idiotypic antibody thus represents the internal image of an epitope present on the original antigen. When formulated as a vaccine, it has been recently demonstrated that anti-idiotypic antibodies or antibody fragments may be used to regulate the immune responses associated with autoimmunity, cancer or allergy. In allergy, binding of IgE to its high affinity receptor (IgERI) is a key pathogenic event. A humanized monoclonal anti-IgE antibody (Omalizumab, Xolair^®) which inhibits the IgE / IgERI interaction has been developed for the treatment of severe allergic asthma. Recently we described the isolation of anti-idiotypic antibody fragments specific for the omalizumab-like anti-IgE antibody BSW17 from a non-immune human Fab phage display library. Immunization of rabbits with these Fabs induced a neutralizing antibody response against human IgE. Novel binding molecules based on designed ankyrin repeat proteins (DARPins) may serve as an alternative to antibodies with improved potency to break self tolerance. Using a consensus DARPin library design strategy employing varying repeat numbers and randomized surface residues, anti-idiotypic binders against mAb BSW17 were identified which are now being further evaluated as putative anti-idiotypic vaccine candidates.
Molecular Changes Induced by Stress Factors in Cerebral Endothelial Cells KRIZBAI IA, FARKAS AE, NAGYOSZI P, FAZAKAS C, WILHELM I Institute of Biophysics, Biological Research Centre, Szeged, Hungary Being located at the interface of blood and brain, cerebral endothelial cells (CECs) are primary targets of different environmental stimuli. The aim of our study was to reveal molecular mechanisms activated by oxidative, hyperosmotic, and calcium depletion induced stress in CECs using an in vitro model of the blood-brain barrier (BBB). There is increasing evidence that the cerebral endothelium and the BBB play an important role in the hypoxia induced brain damage. We have shown that oxidative stress induces a downregulation of the tight junction protein occludin which is more pronounced in the absence of glucose. Furthermore, oxidative stress leads to disruption of the cadherin - catenin complex and an activation of ERK1/2, which is more intense in the absence of glucose. These results indicate that one of the causes of the BBB breakdown is the structural alteration of the junctional complex caused by oxidative stress, a process in which ERK1/2 may play an important role. Hyperosmotic stress elicited by mannitol has been successfully used to reversibily open the BBB. We have shown that hyperosmotic conditions induce protein phosphorylation on both Ser/Thr and Tyr residues. Among the targets of protein tyrosine phosphorylation is the adherens junction protein beta-catenin. Phosphorylation of beta-catenin on tyrosine residues caused its subcellular redistribution and its dissociation from cadherin and alpha-catenin. All these effects were Src kinase dependent. Osmotic stress is able to induce tyrosine phosphorylation of Axl followed by activation of Akt as well. Moreover, Axl was also cleaved in response to osmotic stress resulting in a 50-55 kDa double degradation product. This process was mediated by a metalloproteinase-dependent cleavage, followed by a proteasomal cleavage. We have shown that besides changes in junctional protein expression and localization calcium removal induces significant changes in the morphological parameters of CECs as well as revealed by atomic force microscopy. These changes could be partially inhibited by the Rho-dependent kinase inhibitor Y27632 suggesting a role for ROCK in mediating the effect of low calcium concentration in CECs. Our results show that multiple signaling pathways are activated by different stress factors in CECs.	Pharmacokinetic and Pharmacodynamic Modeling of Recombinant Human Erythropoietin. KRZYZANSKI W Department of Pharmaceutical Sciences, University at Buffalo, USA Erythropoietin is a hematopoietic growth factor stimulating the production of RBC. Recombinant human erythropoietin (rHuEPO) has been indicated for treatment of anemias associated with renal failure, cancer chemotherapy, and HIV antiviral therapy. rHuEPO is an alternative treatment for blood transfusions and its clinical endpoint is to elevate hemoglobin levels. The major clearance mechanism for rHuEPO is binding to its receptor (EPOR) followed by internalization and degradation. EPOR are mostly expressed on bone marrow erythroid progenitor cells. Upon binding to its receptor rHuEPO initiates several intracellular signaling pathways leading to inhibition of cell death, increase in proliferation, and acceleration of the differentiation processes. All of which causes an increase in number of progenitor cells in bone marrow, and reticulocytes and RBC in blood. There will be presented several pharmacokinetic (PK) and pharmacodynamic (PD) models that have been developed to describe serum rHuEPO concentrations and reticulocyte, RBC, and hemoglobin responses obtained from clinical trials and studies in animals. The focus will be on applications of concepts target-mediated disposition and lifespan based indirect response in PK/PD modeling of rHuEPO. Topics will encompass the nonlinear structure of presented models and stress usefulness of mathematical models in assessment of rHuEPO efficacy and potency. Conclusions will include possible modifications of the PK/PD models to account for neglected so far processes and applications of existing models in development of new protein drugs exhibiting a similar to rHuEPO mechanism of action.
Thiopurine S-methyltransferase and inosine triphosphate pyrophosphohydrolase genes in Japanese patients with inflammatory bowel disease in whom adverse drug reactions were induced by azathioprine/6-mercaptopurine treatment KUBOTA Takahiro^1 and UCHIYAMA Kan^2 ^1Chiba Institute of Science, Chiba, Japan; ^2The Jikei University School of Medicine (Kashiwa Hospital), Chiba, Japan Background: The thiopurine S-methyltransferase (TPMT) and/or inosine triphosphate pyrophosphohydrolase (ITPA) gene mutations are suggested to be closely related to adverse reactions induced by azathioprine (AZA)/6-mercaptopurine (6MP) in Japanese. Screening for mutant alleles may be useful for predicting development of the most serious adverse reactions, agranulocytosis and acute bone marrow suppression. Methods: Gene mutations for TPMT and ITPA, major AZA/6-MP-metabolizing enzymes, were investigated in 103 healthy Japanese (45 males, 58 females; mean 32.1±8.35 years old) and 16 Japanese patients (9 males, 7 females; 39.1±15.4 years) with inflammatory bowel disease, in whom AZA/6MP treatment induced adverse reactions. TPMT*2 (238g>c) and *3C (719a>g) were analyzed using an allele-specific PCR method, and the TPMT*3A (460g>a, 719a>g), *3B (460g>a) and ITPA 94c>a mutations by PCR-RFLP. All experiments were approved by the Ethics Committee of Jikei University School of Medicine. Results: Analysis of ITPA genes in the 103 healthy subjects showed that 75 (72.8%) were wild-type, while 25 (24.3%) were heterozygous mutations, and 3 (2.9%) homozygous mutations. The frequency of the 94c>a mutant allele was 15% (0.150, 95% confidence interval: 0.108-0.206). The TPMT gene was the wild-type in all 16 patients, whereas the overall ITPA gene 94c>a mutation detection rate was 50%, with rates of 83.3% in patients with acute bone marrow suppression and 75% in those with agranulocytosis. The 94c>a allele frequency was 31% (0.313, 95% confidence interval: 0.180-0.486), which was significantly higher than the Japanese standard (chi-square test, p<0.05), while adverse reactions developed earlier in patients with that mutation. In half of the patients, no gene polymorphism was noted, suggesting involvement of drug interactions and reduction of TPMT activity. Conclusions: In predicting thiopurine adverse reactions in Japanese, it may be favorable to perform screening for the 94c>a mutation, followed by determination of in vitro TPMT activity when the gene is the wild-type to investigate drug interactions.	Memorabilia of Paul Ehrlich in modern Strzelin (Strehlen) and Wroclaw (Breslau). KUCHAR E, KUCHAR I, SZENBORN L  Department of Pediatrics and Infectious Disease, Medical University in Wroclaw (Breslau), Poland. Background: Paul Ehrlich (1854-1915), the best known for discovery the idea of chemotherapeutics, as he expressed it “magic bullets”, chemical substances that have a specific affinity to pathogenic microorganisms, and can destroy them, was given a Nobel prize in 1908. We wish to commemorate Paul Ehrlich on the centennial of his Nobel Prize in Physiology or Medicine in 1908 as he is the most famous person born in Strehlen, now Strzelin, little city in downer Silesia, region where our Medical University is located. Methods. Descriptive study; we visited Strzelin and Wroclaw tracing memorabilia of Paul Ehrlich. Photographic and written documentation was made. Results. Strzelin (to 1945 Strehlen) is now a little calm town located at Olawa river with 13.300 inhabitants. Ehrlich’s family house situated at the market square was destroyed during second world war in 1945 and a pharmacy building occupies this place now. There is an monument commemorating Paul Ehrlich at the crossing of present Dzierżoniowska and Bolka I Świdnickiego streets founded in 2004. Wroclaw (to 1945 Breslau) with about 630 000 inhabitants is rapidly growing modern city, the fourth largest in Poland. It is the capitol of downer Silesia Province. Paul Ehrlich finished St. Magdalene Gimnasium located on present Szewska street (former Schubrücke) near St Magdalene Church in 1872. The gymnasium founded in 1643 was completely destroyed during second world war in 1945. Then Paul Ehrlich studied medicine at the Breslau University. The University (Schlesische Friedrich-Wilhelm-Universität zu Breslau) founded on 3 August 1811 by merging of Leopoldinum Academy dated from 1702 with Viadrinia University established in 1506 in Frankfurt upon Oder and included 5 faculties: catholic theology, evangelic theology, law, medicine and philosophy at his time. At present Wroclaw University (Uniwersytet Wrocławski), its direct successor educates over 38.000 students and consists of 10 faculties.
Tolerability of pirymethamine/sulpha chemotherapy in children with congenital toxoplasmosis treated in Department of Pediatrics and Infectious Disease, Medical University in Wroclaw, Poland. KUCHAR E, KUCHAR I, SZENBORN L, JASONEK J, ZYZAK K. Department of Pediatrics and Infectious Disease, Medical University in Wroclaw (Breslau), Poland. Background: Toxoplasmosis is a wide-spread protozoan diseases occurring in congenital and acquired form. Congenital infection with rate reaching 1 per 1000 of newborns may involve the retina and brain with long-life sequellae. Despite the discovery of new antibiotics combined therapy with pirymethamine/sulpha has been the first line treatment for congenital toxoplasmosis for more than 20 years. The aim of the study was to sum up single centre experience with tolerability of the treatment in last 8 years. Methods. Descriptive retrospective study; 12 children with congenital toxoplasmosis treated with standard doses of pirymethamine/sulpha and folinic acid supplementation in the Department of Pediatrics and Infectious Diseases in Wroclaw, Poland between 2000 and 2007 were enrolled. Disease was diagnosed with serologic tests in all patients but one, in whom the diagnosis was made by PCR. Results. 12 children aged from 5 days to 3 years (8 girls and 4 boys) were treated for 3 weeks to 13 months (median 9 months). We observed thrombocytopenia in 3/12, anemia in 3/12, leucopenia in 2/12, cholestasis in 2/12 and elevated aminotransferases in 1/12 treated children respectively. The therapy had to be suspended for 4 to 6 weeks in 3/12 children due to thrombocytopenia mainly. Therapy was effective in all treated children with 100% survival in a follow up ranging from 1 to 8 years. Conclusions. Chemotherapy of congenital toxoplasmosis with pirymethamine/ sulpha and folinic acid supplementation in infants is effective and generally well tolerated. The most common undesirable effects include brainstem toxicity and need regular complete blood count monitoring.	Benzimidazoles and surgery in cardiac hydatidosis: efficacy in prevention of disease relapse KUDAIBERDIEV T National Center of Cardiac Surgery and Organ Transplantation, Bishkek, Kyrgyz Republic Background: Echinococcosis is a pathology encountered in animal husbandry countries. Cardiac echinococcosis (CE) is a rare entity that might cause serious life-threatening complications. Management strategies include surgical removal (cystectomy) of hydatid cysts and adjunctive benzimidazole derivatives therapy (BDT). However, there are no randomized trials on the recurrence rate of CE in patients treated with BDT therapy as adjunct to surgery. We aimed to analyze case series on surgical correction of CE with adjunctive BDT reported in literature and own experience to define the recurrence rate in such combinative treatment and analyze cases with recurrence of disease. Methods: We made search in MEDLINE using MESH terms – “cardiac echinococcosis” and “heart diseases” for a period between 1998 and 2008 years. Overall, there were 166 articles retrieved, among them 14 were reporting series (2 to 62) of patients (pts) treated with surgical correction and BDT. Results: Fourteen studies reported included overall 164 pts with CE (age range 9-75 years, and 88 pts were female). The cyst localization was – left ventricular (LV, including interventricular septum (IVST) aspect) – 37.1% (61 pts, including IVST aspect), right ventricular (RV) – 18.9% (31 pts), IVST –17.6% (29 pts), right atrial – 9.1% (15 pts), left atrial -1.8% (3 pts), pericardium – 9.1% (15 pts), sinus of Valsalva – 0.6% (1 pt); 5 pts (3%) had cysts in LV and pericardium, 4 pts (2.4%) – RV and pericardium. Of 164 pts 40 (24.3%) pts had associated lesions in liver (17 pts), lungs (12 pts), spleen (2 pts), and aorta (1 pt). Multiple lesions were found in 8 pts: brain, spine and spleen – 1 pt, brain and kidney – 1 pt, liver and lung - 4 pts, liver and spleen – 1 pt, liver, lung and brain – 1 pt. Surgical removal of hydatid cysts was performed using off-pump cardiac surgery in 47 pts (28.7%) and using cardiopulmonary bypass – 117 pts (71.3%). Therapy with albendazole was started in 81 pts, with mebendazole – in 29 pts. The follow-up period varied between 3 months to 12 years. The CE recurrence rate for albendazole was 3.7% (3 of 81 cases), and 3.4% for mebendazole (1 of 29 pts). The CE relapse occurred in pts taking albendazole with multiorgan, multiple and complicated lesions (ruptured cysts). Few pts were taking albendazole prior to operation (2 weeks-12 weeks) due to previous lesions in other organs. Cysts characteristics for these pts were distinctive by their solid, unviable and inactive nature, posing less complicated removal during cardiac surgery as compared with cysts at active stage, which might have risk of rupture and dissemination of daughter cysts during surgery. Conclusion: 1. BDT as adjunct to surgery for cardiac echinococcosis is accompanied by recurrence rate of 3.7%. 2. The CE relapses are related to multiple damage, rupture of cysts and complicated course of disease. 3. Albendazole taken prior to surgery might facilitate the successful surgical removal of cardiac cysts.
Towards a Magic Bullet for the Metabolic Syndrome – Use of the Ginkgo Biloba Extract KUDOLO GB University of Texas Health Science Center, San Antonio, TX, USA. Background: The metabolic syndrome represents a collection of inter-related metabolic defects commonly associated with diabetes (e.g. dyslipidemia, obesity and hypertension) that increase the risk of cardiovascular disease. Ingestion of the dietary supplement Ginkgo biloba Extract might ameliorate the metabolic syndrome by reduction of oxidative stress in blood plateletes and the normalizationation of pancreatic beta-cell function in especially T2DM patients with pancreatic exhaustion. Since hyperinsulinemia is a hallmark of T2DM, it is important to verify that increased insulin production is not due to increased insulin resistance. The primary aim is to measure the effect of the ingestion of Ginkgo biloba extract on whole body insulin sensitivity in subjects with normal glucose tolerance (NGT), impaired glucose tolerance (IGT, pre-diabetes) and full-blown T2DM. Methods: Subjects with NGT (n = 10; age, 44.2  13.9 years old), impaired glucose tolerance (IGT) (n = 8; age 51.3  6.6 years old) and T2DM (n = 8, 51.6  15.2 years old) completed a randomized, double-blind, placebo-controlled crossover study, with each arm lasting 3 months. After ingestion of either Ginkgo biloba extract (120 mg/day as a single dose) or placebo for each arm, a 2-step (each lasting 2 hours) euglycemic insulin clamp was performed, using [^3H]glucose intravenous influsion and whole body glucose metabolic rate (M-value) was calculated during the last 30 min of each step. Results: At the low insulin infusion rate (10 mU/m^2/min) the glucose metabolic rates (M values) were 3.5  1.5 vs. 3.0  0.5 mg/kg (P = 0.16), 3.0  0.4 vs. 2.8  0.8 mg/kg (P = 0.19) and 2.6  0.7 vs. 2.4  0.5 mg/kg (P = 0.09) for the placebo and Ginkgo biloba cycles, in the NGT, IGT and T2DM subjects, respectively. At the high insulin infusion rate (40 mU/m^2/min) the M values were 7.3  2.3 vs. 8.1  2.5mg/kg (P = 0.07), 6.2  1.6 vs. 6.5  2.1 mg/kg (P = 0.32) and 3.6  1.6 vs. 3.5  1.0 mg/kg (P = 0.34) for placebo vs. Ginkgo biloba cycles, in the NGT, IGT and T2DM subjects, respectively. Conclusions: The ingestion of 120 mg of Ginkgo biloba extract as a single for 3 months had no significant effect on insulin resistance in non-diabetic subjects or those with pre-diabetes (IGT) nor did it exacerbate the disease in those with full-blown T2DM. Considering the other benefits of ingesting Ginkgo biloba, this is as close to a magic bullet as there is fort he metabolic syndrome. Acknowledgement: This study was supported by funds from NIH grants M01-RR-01346 and R01-AT-00832 (National Center for Complementary and Alternative Medicine, NCCAM).	In silico Approaching to Cisplatin Toxicity KUDUK-JAWORSKA J^1,JAROSZEWICZ^1 I, JAŃSKI^1 J, CHOJNACKI^2 H ^1Faculty of Chemistry Wrocław University, Wrocław, Poland ^2Institute of Physical and Theoretical Chemistry, Wrocław University of Technolgy, Wrocław, Poland Background: A new approach to evaluation of the mode of action in platinum anticancer drugs arena are quantum chemical in silico studies which have, so far, focused on cisplatin hydrolysis and then binding to purine bases of DNA. These reactions are believed to be the basis of anticancer activity of cisplatin and its analogues. Contrary this, we concentrated on the toxicity of cisplatin, particularly in comparison with non-toxicity of its trans isomer. Thus the impact of platinum(II) on sulphur-containing molecules, present in serum, was the object of this study. In serum, because of the high chloride concentration, cisplatin and transplatin are found in chloro- form. Aim: theoretical studies on reactions of non-hydrolyzed (NH3)2PtCl2 with thiols. Methods: The following systems were investigated: (1) cisplatin (transplatin) with CH3SH; (2) cisplatin (transplatin) with L-cysteine. The electronic structure for molecular systems was studied at non-empirical all-electron level by using density functional (DFT) or Moeller-Plesset (MP2) methods within the correlation consistent cc-pVTZ basis set. In the case of platinum the widest Huzinaga basis set with polarization functions was used. At the first stage, the optimization was performed at the all-valence MOPAC-PM6 method following the B3LYP density functional or MP2 formalism in the next step. The B3LYP density functional was applied using GAUSSIAN-03 program package. The numerical calculations have been performed in part at Wroclaw Networking and Supercomputing Center. Results: The order of reactivity in the impact of cysteine on Pt(II) was: transplatin (PCM)> cisplatin (PCM) > transplatin (gas phase) > cisplatin (gas phase); The order of reactivity in the impact of CH3SH on Pt(II) was: transplatin (gas phase) > transplatin (PCM) . > cisplatin (PCM) > cisplatin (gas phase) Conclusions: (1) Quantum chemical in silico methodology was applied to new evaluation of energy interaction in P(II) – thiol systems. (2)The established rows of reactivity showed good agreement with results of earlier biochemical and kinetic studies.
Experimental Evidence for Killing the Resistant Cells and Raising the Efficacy of Cytostatics by a New Anticancer Drug Candidate (Culevit Infusion) Developed on the Basis of the Passive Antitumor Defence System Kulcsar G^1, Gaal D^2, Toth Z^1, Kulcsar PI^1, osz E^1, Berente Z^1, Halasz H^1 ^1Univ. Pecs, Pecs, Hungary ^2National Institute of Oncology, Budapest, Hungary Background: While other research groups, realizing the inefficiency of the immune system against cancer, studied immune escape or response modifiers we thought that the failure of tumours to develop in the majority of the population during their lifetime despite ineffective immune surveillance indicates the exis­tence of other defence system(s) whose agents should lie in the circulatory system (CS). It is well known and used for tumour detection (as in PET) that the uptake of most substances (amino acids, monosacharides, nucleobases, etc.) occurring in CS is strictly regulated by normal cells, whereas it is unregulated and elevated by tumour cells. Considering that many molecules in the living system have more than one role we supposed that some of the accumulated substances might be the agents of a defence system capable of killing emergent cancer cells. We substantiated this hypothesis by experimentally selecting 16 substances of CS (of 89 investigated) whose mixture had toxic effects in vitro and in vivo on all tumour cell lines investigated, but not on normal cells and animals. Knowing the 16 agents protected by patents in many countries, the development of a medicine in an infusion form is at the preclinical phase. Methods: The infusion was administered i.p. as injection 8 times a day for 10 days. Results: Significant inhibitory effects of the infusion have been observed in the case of all tumours (P-388, S-180, B-16, MXT, Colon-26, He/De, Ne/De, HL-60) investigated. The infusion compared to cytostatics (5-FU, Cisplatin) had slightly better inhibitory effect on Colon-26. It is important to note that the infusion significantly increased the effects of both cytostatics when they were used simultaneously. It appeared that the components of the infusion could also kill multidrug resistant cells (AT3B-1, MCF7/ADR) and the infusion abrogated the resistance towards 5-FU of B16 melanoma. No lethality, toxic effects, adverse clinical symptoms were observed in toxicity studies. Conclusion: The Culevit infusion, besides having significant inhibitory effect on tumours, can improve the efficacy and reduce the side effects of chemotherapy and radiation and can decrease the possibility of relapse.	New Animal Models for Psychotropic Drug-Drug Interactions Kulikov AV, Bazovkina DV, Osipova DV Institute of Cytology and Genetics, Russian Academy of Sciences, 630090 Novosibirsk, Russia. Background: The involvement of brain neurotransmitters and trophic factors in the mechanisms of psychotropic drug action is commonly accepted. The existent animal models are based on the effects psychotropic drugs on the behavior induced with chronic stress or knockout of the genes involved in the regulation of brain neurotransmission. Here new models for study the role of serotonin and cytokines in psychotropic drug-drug interaction were presented. Methods: The three new genetic models were created: 1) the ASC/Icg (Antidepressant Sensitive Catalepsy) mouse line selectively bred from a backcross population between CBA/Lac and AKR/J strains for high predisposition to catalepsy; 2) the AKR.CBA-D13Mit76 congenic mouse line with the 61-70 cM CBA-derived fragment of chromosome 13 transferred to the AKR genome (Kulikov et al., Genes, Brain Behav., 2008, 7:506-512); and 3) the B6-1473G congenic mouse line with the 1473G allele of the tph2 gene decreasing activity of the rate-limiting enzyme of serotonin synthesis in the brain, tryptophan hydroxylase-2, transferred to the C57BL/6J genome. Results: The major gene defining predisposition to catalepsy was mapped on the 61-70 fragment of mouse chromosome 13 and linked to the Il6st gene coding the gp130 protein associated with cytokine receptors. The ASC mice showed numerous depressive-like traits and altered serotonin neurotransmission compared with the parental CBA and AKR strains. Chronic antidepressant treatment decreased catalepsy in ASC, but did not affect the trait in CBA mice. The transfer of the 1473G allele of the tph2 gene to the C57BL/6 genome significantly affected the intermale aggression and depressive-like immobility in the forced swim test. Conclusion: 1. The ASC mouse line meets face, predictive and construct validity criteria of animal model of depression and antidepressant drugs screening. 2. The AKR.CBA-D13Mit76 congenic mouse line with altered gp130 protein is a promising model to study the interaction between cytokines and psychotropic drugs. 3. The B6-1473G congenic mouse line with altered tph2 gene is a valuable model of the interaction between serotonin and psychotropic drugs. Authors’ disclosure statement The study was supported with Russian Foundation for Basic Research (grant #07-04-00209), Integration Program of the Siberian Division of Russian Academy of Sciences (grant #5) and Russian Program “Molecular and Cell Biology” (grant #10.11).