Ehrlich II –2nd World Conference on Magic Bullets

Cefepime Neurotoxicity in Perspective BRAGATTI JA^1, SILVA D^1, CASTILHOS RM^1, AZAMBUJA M^1, GARCÉS EO^1, BARROS EG^2, THOMÉ FS^2, BIANCHIN MM^2 ^1Hospital de Clínicas de Porto Alegre, Brazil ^2Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil Background: Cefepime, a widely used antibiotic, is more effective than other cephalosporins against many Gram positive and negative microorganisms, and has a main role in the treatment of febrile neutropenia. Its safety profile was considered adequate, but increased mortality was recently found with its use. Most common neurological side effects are somnolence, disorientation, hallucinations, and epileptic seizures. Jallon et al. firstly reported on 19 patients who developed a confusional state that reverted after cefepime discontinuation. Cefepime?s neurotoxicity clearly results from its accumulation in the central nervous system (CNS). Excessive dosage and impaired renal clearance are important risk factors, as well as uremia, extreme ages, and meningitis. Our group employed three studies regarding clinical, EEG, and pharmacological issues of cefepime encephalopathy Methods: We previously published a series of seven patients with cefepime encephalopathy. Thereafter, we studyied the renal failure as a risk factor for this clinical entity, applying the Cockcroft-Gault formula to measure glomerular filtration rates (GFR). The third study, now ongoing, looks for detecting the incidence of cefepime encephalopathy in different grades of renal failure. When neurological manifestations during cefepime treatment are detected, we record an EEG and measure cefepime blood levels as soon as possible. Events are classifyied according to Naranjo?s algorhythm, and renal impairment as absent, slight, moderate, severe, and terminal. Results: In the first study, our patients used cefepime doses between 4 to 8 g/day. Somnolence, agitation, or myoclonic jerks developed after 2 to 9 days after starting treatment. Almost all patients had renal failure, and only one had cefepime dosage adjusted for renal function. The EEG pattern described by Jallon et al. were present in all patients. In the second study, we included 498 patients, 111 with renal failure. Five patients (1%) had encephalopathy, all in the renal impairment group. We also found that encephalopathy?s incidence increases with decreasing clearance rates. Indeed, in our present study, we hope to show that cefepime?s dosage is so important than renal function to the pathogenesis of cefepime encephalopathy.

Poor permeability of blood brain barrier for creatine: Autonomous brain synthesis of creatine, and consequences for creatine deficiency syndromes BRAISSANT O Inborn Errors of Metabolism, Clinical Chemistry Laboratory, Centre Hospitalier Universitaire Vaudois and University of Lausanne, 1011 - Lausanne, Switzerland. Abstract : In mammals, creatine (Cr) is taken up from the diet, or can be synthesized endogenously by a two-step mechanism involving L-arginine:glycine amidinotransferase (AGAT), which yields the intermediate guanidinoacetate (GAA), and guanidinoacetate methyltransferase (GAMT), which converts GAA to Cr. Cr is distributed through the blood and is taken up by cells with high energy demands through a specific Cr transporter, SLC6A8. It was thought for a long time that most, if not all, of the Cr needed by the brain comes from the periphery through blood brain barrier (BBB). However, our recent work has shown that AGAT and GAMT are expressed in CNS and that brain cells in vitro synthesize their own Cr. SLC6A8 is also expressed in CNS, but not in astrocytes, particularly in their feet sheathing microcapillaries at BBB. These data suggested that BBB has a limited permeability for Cr, and that CNS might depend more on its own autonomous Cr synthesis than on Cr supply from the blood. The brain is the main organ affected in patients suffering from Cr deficiency syndromes caused by either AGAT, GAMT or SLC6A8 deficiencies, which all three are characterized by an absence, or a severe decrease, of Cr in CNS, as measured by magnetic resonance spectroscopy. Because SLC6A8 is present in microcapillary endothelial cells, AGAT and GAMT deficient patients can be treated with oral Cr supplementation. However, due to the absence of SLC6A8 in the surrounding astrocytes, very high doses of Cr must be used, and the replenishment of cerebral Cr takes months and results only in the partial restoration of the cerebral Cr pool. Cr supplementation of SLC6A8 deficient patients is inefficient to restore cerebral Cr levels.

Low-dose steroids in critically ill patients: Who should be treated and when? BRIEGEL J Munich, Germany Ongoing and severe systemic inflammation affecting critically ill patients may cause adrenal insufficiency and steroid resistance. As this clinical entity is difficult to diagnose, many studies on low-dose corticosteroid therapy used inclusion criteria based on the underlying disease, clinical symptoms, time windows after onset of the disease, and biochemical testing. Septic shock is without doubt the best known disease with severe systemic inflammation and related adrenal insufficiency. In many studies, persisting septic shock after fluid and vasopressor resuscitation became the main inclusion criteria. Other target groups in low-dose corticosteroid trials were patients with ARDS, acute lung injury (ALI) due to pneumonia, cardiac surgery with cardiopulmonary bypass, acute pancreatitis, or trauma. Target group septic shock Septic shock and low-dose corticosteroid therapy have been extensively investigated during the last decade [1-6]. The main finding supported by all studies is that hydrocortisone 200 to 300 mg per day accelerates shock reversal, i.e. reduces the time on vasopressor therapy. Despite different inclusion criteria of septic shock (i.e. early vs. late septic shock, hypotension and poorly responsive to fluid and vasopressors resuscitation vs. the broader definition given by the ACCP and the SCCM), this finding is consistent and supported by the most recent trial, the large European CORTICUS study [1-6]. In the latter clinical trial, improvement in cardiovascular physiology did not translate in improved survival as it has been demonstrated earlier in the Annane study [1,2]. In addition, adjustment of the appropriate target group by means of corticotropin tests did not make a difference in the CORTICUS trial [1]. This is in marked contrast to the results of the Annane study, in which the benefit of hydrocortisone and fludrocortisone was found in the target group with a blunted response to corticotropin as defined by post-corticotropin cortisol increase of ¡Ü9 µg/dl. The reasons for the differences found in the two clinical trials remain to be discussed. First analyses revealed that patients in the Annane study were more severely ill (SAPS II: 59 vs. 49 pts.), had early septic shock (time window of inclusion: 8 vs. 72 hrs.), had more severe arterial hypotension and higher vasopressor doses at inclusion, did not respond to volume therapy for at least one hour, and had more pneumonia as underlying infection. The most important difference was the 28-day mortality of the placebo group which was 61% in Annane study and 31.5% in the CORTICUS trial. A post-hoc analysis of patients in CORTICUS who had a systolic blood pressure that persisted below 90 mm Hg at 1 day after fluid and vasopressor resuscitation (n=126) showed a rate of death of 56.1% in the placebo group and an absolute reduction in mortality of 11.2% in the hydrocortisone group. These results that are very similar to those reported by Annane [2]. CORTICUS patients who received the study drug within 12 hours after baseline did not show any significant differences in outcome when compared with patients who have received the study drug later. The results of the CORTICUS trial led to the new recommendation of the surviving sepsis campaign that only patients with septic shock poorly responsive to fluid resuscitation and high-dose vasopressor therapy should receive hydrocortisone therapy, but not all patients fulfilling the broader definition of septic shock given by the ACCP and the SCCM. The optimal time window for initiation of hydrocortisone in septic shock remains uncertain. Target group ALI-ARDS Ongoing systemic inflammation associated with excessive fibroproliferation in persistent ARDS has been proposed as another indication for corticosteroid therapy. Methylprednisolone treatment exceeding the dose of corticosteroid replacement therapy in the studies on septic shock by a factor of two improved pulmonary function and reduced multiple organ dysfunctions by profound immunomodulation of the persistent inflammatory process [7, 8]. The large-scale trial of the ARDS network, however, did not support this intervention [9]. Despite significant and substantial improvements in pulmonary physiology associated with a higher number of ventilator-free days and ICU-free days at day 28, the use of corticosteroid did not result in a lower mortality at day 60 after randomisation. An increased rate of return to assistant breathing associated with muscle weakness has been discussed as main reason why improved physiology did not translate in improved outcome. A post-hoc analysis revealed that patients with ARDS of 14 to 28 days duration (n=48) had even increased 60-day mortality when they were assigned to the corticosteroid group whereas a trend to improved survival was seen in the group recruited between day 7 and 13 after onset of ARDS [9]. It has been criticized that substantial imbalances at baseline in the latter post-hoc analysis group were present favouring the placebo group. [Regrettably not enough space on this page.]

The design of drugs which do not create resistance: folding inhibitors BROGLIA RA^1,2,3 AND TIANA G^1,2 ^1Department of Physics, University of Milano, Milano, Italy ^2INFN, sez. di Milano, Italy ^3The Niels Bohr Institute, University of Copenhagen. Copenhagen, Denmark Conventional protein inhibitors cap the active site of their targets, preventing the binding of the substrate. An alternative approach has been recently developed to block the activity of proteins and consists in preventing their folding to the native, biologically-active conformation. It is based on recent understandings on the overall folding mechanism of proteins, and in particular on the observation that some protein segments display residual native structure already in the denatured state of most proteins, and that the same segments often participate to the formation of the transition state, as shown by protein-engineering experiments. The theoretical picture which emerges is that folding is guided by Local Elementary Structures (LES) and stabilized by few, highly conserved (“hot”) amino acids. Docking of the LES give rise to the transition state and to the postcritical folding nucleus (FN), which inevitably grows into the native state. Intervening a folding reaction can, in principle, be achieved by interacting the polypeptide chain with peptides (called p-LES) whose sequence is identical to those of the LES that define the FN of the target protein. As the concentration of p-LES increases, the protein may nucleate by the assembly of the protein chain with peptide LES, leading to a nonproductive folding. This can be viewed as changing the folding from a unimolecular reaction to a bimolecular reaction. These are two important advantages of these non-conventional (folding) inhibitors with respect to conventional (active-site centered) ones. First, their molecular structure is suggested directly by the target protein. One needs not to design or optimize anything, just find the LES of the protein to be inhibited, because the design has been performed by evolution through a myriad of generations of the organism that expresses the protein. Moreover, the probability that the protein can develop resistance through mutations is much smaller than in the case of conventional drugs. In fact, a folding inhibitor binds to a LES, and a protein cannot mutate the amino acids of a LES under risk of denaturation. The above strategy has been applied to HIV-1 Protease, an enzyme which plays an essential role in the lifecycle of the virus. Consequently, its inhibition can control AIDS. The HIV-1 PR is an homodimer, each monomer containing 99 amino acids. It reaches the native conformation following a three-state mechanism in which each monomer folds independently of each other and afterwards they dimerize. We have identified the segments containing the residues 23-33 and 83-92 as those associated with the LES of the monomer, the segment 83-92 being the one that becomes more structured in the early folding events. Experiments in silico^1,2, in vitro^3 (enzyme) and in infected cells^4 (virus, also multi-drug resistant virus) have shown (see also 5) that peptides with identical sequence to the segment 83-92 (p-LES(83-92)) are able to inhibit enzymatic activity by inhibiting folding, as testified by circular dichroism experiments^3. Furthermore, “long-term” studies of pharmacological pressure conducted by passing in PBMC of a wild-type virus in the presence of p-LES(83-92) or Atazanavir (ATV) have shown the very high genetic barrier to mutations displayed by the LES peptide. In fact, genotipic sequenceing^6 showed that p-LES (83-92) did not select for any mutation leading to resistance, since the pattern of mutations at baseline and after 9 months of in-vitro pharmacological pressure were just alike. On the other hand, ATV selected for primary and/or secondary mutations. The transferibility of the folding-inhibition strategy to other proteins testify to the universality of the folding-inhibition scenario for the design of leads of drugs which are unlikely to generate resistance^7,8. 1. Broglia RA et al (2005) Protein Sci. 14:2668-2681 2. Broglia RA et al. (2007) La rivista del Nuovo Cimento 29:1-119 3. Broglia RA et al (2006) Proteins 62:928 4. Rusconi S et al (2007) Procs. Of the International School of Physics “E. Fermi”, Course CLXV on Protein Folding and Drug Design, IOS Press, Amsterdam, 293-299 5. Broglia et al (2008) Curr. Opin. Struct. Biol. 18:60-66 6. Ferramosca S et al., XVII International HIV drug resistance workshop, basic principles and clinical implications, June 10-14, 2008 Sitges, Spain (see http://merlino.mi.infn.it/publications/articles2/asbtract_2008.pdf) 7. Broglia RA et al. (2007) Proteins 67:469 8. Caldarini M et al (2008) Proteins (in press; see http://merlino.mi.infn.it/publications/articles2/lysozyme12fig.pdf)

Novel targets and magic bullets for treating cardiovascular disease BROOKS G University of Reading, Berkshire, UK Cardiovascular disease represents a major clinical problem affecting a significant proportion of the world’s population and it remains the major cause of death in the EU and the rest of the Western world. Furthermore, the burden on healthcare systems is increasingly high; the overall cost of cardiovascular disease to the EU economy is estimated to be in excess of 192 billion Euros per year. The majority of therapies currently available for the treatment of cardiovascular disease do not cure the problem but merely treat the symptoms. Furthermore, many cardioactive drugs have serious side effects and have narrow therapeutic windows that can limit their usefulness in the clinic. Thus, the development of more selective and highly effective therapeutic strategies that could cure specific cardiovascular diseases would be of enormous benefit both to the patient and to those countries where health care systems are responsible for an increasing number of patients. There is increasing evidence to suggest that targeting the cell cycle machinery in cardiovascular cells (e.g. cardiac myocytes, vascular smooth muscle cells (VSMCs), endothelial cells) provides a novel approach for the treatment of certain cardiovascular diseases, including post-infarct heart failure, restenosis, in-stent stenosis and bypass graft failure. It has been demonstrated that certain cell cycle molecules that are important for regulating terminal differentiation in cardiac myocytes (e.g. cyclins, cyclin-dependent kinases [CDKs], CDK inhibitors, E2F transcription factors) can be targeted to reinitiate cell division and repair in the myocardium post-infarction. Furthermore, cell cycle molecules that control excessive VSMC proliferation in disorders such as restenosis, in-stent stenosis and bypass graft failure have also been targeted effectively in recent laboratory and clinical studies. The results of these studies illustrate the exciting possibility of targeting components of the cell cycle machinery to develop magic bullets to improve cardiac function and prognosis for patients with heart failure and for patients with atherosclerosis.

A Therapeutic Conundrum: Low Molecular Weight Heparins in Patients with Kidney Dysfunction. BROPHY DF Virginia Commonwealth University, Richmond, VA, U.S.A. Background: Patients with chronic kidney disease (CKD) are frequently viewed as being at high risk for bleeding. In point-of-fact however, CKD patients not only maintain a bleeding tendency but also exhibit a high frequency of thromboembolic disease. Indeed, cardiovascular disease is the leading cause of death in CKD patients. Therefore, clinicians are frequently confronted with choosing the most appropriate antithrombotic therapy in CKD patients undergoing percutaneous coronary intervention. Clinical trials have shown that low molecular weight heparins (LMWH) have greater efficacy and less adverse effects compared to unfractionated heparin (UFH). However, CKD patients present a special circumstance such that LMWHs carry an increased risk of adverse bleeding in such patients compared to those with normal renal function. This presentation will provide perspective on the clinical use of LMWH in CKD patients. Methods: A Medline literature review was conducted to identify primary literature describing the efficacy and risk of adverse bleeding when UFH and LMWH drugs were used at therapeutic doses in CKD patients. Results: In major clinical trials, enoxaparin has been associated with increased bleeding rates. A recent meta-analysis carefully described the available data for all LMWH products in non-dialysis-dependent CKD patients with respect to bleeding. n the primary analysis, 4971 patients with a creatinine clearance < 30 mL/min had an increased risk of bleeding compared to those without renal insufficiency (5.0% versus 2.4%; odds ratio 2.3, [95% CI 1.2 to 4.3], p=0.01).In a secondary product-based analysis of these same data, enoxaparin use increased the rate of major bleeding to 6.0% in CKD patients compared to 2.4% in non-CKD patients (odds ratio 2.6, [95% CI 1.2 to 4.3]). The likelihood of a major bleeding event increased further when the enoxaparin dosing regimen was considered. Safety data for other LMWH is difficult to ascertain because of the relative few available data. Conclusions: These data suggest that non-adjusted enoxaparin dosing may lead to serious bleeding in CKD patients. The appropriate enoxaparin dose reduction has yet to be confirmed. The use of UFH is prudent in patients with advanced CKD.

Epinephrine Vasoconstrictor Drug-Drug Interactions Revisited BROWN RS Howard University College of Dentistry, Washington, DC, USA Drug-Drug interactions with regard to epinephrine vasoconstrictor local anesthetic formulations have been vastly overstated in the past. Such purported drug interactions include tricyclic antidepressants, non-specific beta blockers and cocaine. These supposed but mistaken interactions are widely published in many Dental Pharmacology Texts and have established problematic clinical considerations negatively influencing pharmacologic patient therapy. A portion of the rationale for such purported drug interactions include poorly designed studies and inapplicable case reports. The major misconceptions are due to misunderstandings of adrenergic pharmacology. Lack of understanding with regard to epinephrine’s beta two receptor’s influence upon blood pressure dynamics and the difference between alpha adrenergic local versus system effects are problematic. A misunderstanding of the positive attributes of local anesthetic vasoconstrictor action and limited knowledge of sympathetic activation and the actions of endogenous norepinephrine among dental clinicians has contributed to this problem and resulted in the misuse of pharmacotherapeutics. The lack of toxicity is further influenced due to epinephrine’s exceedingly short half-life. To add to the above, the mechanism of tricyclic antidepressant pharmacology is not common knowledge among dental clinicians and even such simple drug-drug interactions as the additive drug-drug interaction between cocaine and other local anesthetics is overlooked. Furthermore, many recent human studies which demonstrate the relative safety of epinephrine vasoconstriction will also be discussed. In conclusion, an evaluation of potential drug-drug epinephrine vasoconstrictor interactions is important with regard to clinical dental care and appears to limit fears regarding several noted but highly suspect previously accepted drug-drug interactions.

Magic bullets for the treatment of inflammatory bowel disease – yet to come? BRUNNER M Medical University of Vienna, Department of Clinical Pharmacology, Vienna, Austria Background: Inflammatory bowel disease (IBD), an immune-mediated chronic intestinal condition, encompasses two idiopathic inflammatory diseases of the intestinal tract: Crohn’s disease and ulcerative colitis. Currently, medical treatment for IBD aims at induction and maintenance of remission. Established therapies, such as 5-aminosalicylic acid compounds, corticosteroids, immunomodulators or calcineurin inhibitors, however, lack specificity or effectiveness and might cause significant long term side effects. Recent advances in the knowledge of pathogenesis and immunology of IBD has led to the development of novel therapies directly targeting specific aspects of the inflammatory process, such as cytokines and receptors involved in T-cell activation, selective adhesion molecule blockers, anti-inflamatory cytokines, modulators of the intestinal flora or monoclonal antibodies. The first monoclonal TNFα antibody, has been successfully introduced into clinical practice for the treatment of IBD approximately 10 years ago. Most other novel therapies are undergoing different stages of clinical evaluation. Conclusions: Although during the last years progress has been made in both defining the mechanisms underlying the development of inflammatory bowel disease and expanding the spectrum of effective therapies, it might be too early to know, whether the currently tested compounds will be routinely employed in IBD treatment. It seems rather unlikely, however, that one single drug will prove to be a magic bullet.

Transcriptional Changes Induced by Imatinib and Nilotinib in the Chronic Myelogenous Leukemia (CML) Cell Line K562 BRUENNERT D, KOCH A, GATTERMANN N, KRONENWETT R, HAAS R, NEUMANN F Heinrich-Heine-University, Duesseldorf, Germany Background: Nilotinib is a selective bcr-abl tyrosine kinase inhibitor that is 30-fold more potent than Imatinib in vitro. To examine the molecular and functional effects of Nilotinib and Imatinib we performed gene expression and functional analyses in K562 cells following in vitro treatment with the two tyrosine kinase inhibitors. Particular emphasis was put on 1539 genes which we found to be differentially expressed in primary CD34+ cells from patients with CML in chronic phase in comparison to CD34+ cells from normal bone marrow (Diaz-Blanco et al., Leukemia 2006). Methods: Affymetrix U133A 2.0 microarrays covering 21.722 probe sets were used to analyse the gene expression profile of 5x10^7 K562 cells after 24h in vitro treatment with Imatinib (0.5 µM) or Nilotinib (0.05 µM) (half maximal inhibitory concentration). FISH analysis confirmed the K562 cell line to be BCR-ABL positive. Gene expression data of the treated cells were compared with the data of untreated cells. In addition, proliferation (MTS Assay, Promega), apoptosis (Cell Death Detection ELISAPLUS, Roche) and cell cycle (FITC BrdU Flow Kit, BD Pharmingen) assays were performed. Results: Looking at those 1539 differentially expressed genes in K562 cells which distinguish patients with CML from healthy donors, we found that Imatinib led to a significant downregulation of 187 and upregulation of 45 genes. In general, the effect of Nilotinib with regard to the number of genes affected and degree of suppression was more pronounced resulting in the downregulation of 418 and upregulation of 41 genes. Of note, genes affected by Nilotinib included all genes altered by Imatinib such as those related to bcr-abl signalling. Downregulation of genes involved in cell cycle was only observed following Nilotinib exposure. The stronger effect of Nilotinib is in line with the results of cell cycle experiments showing that Nilotinib exposed cells had the lowest proportion of actively cycling cells. The proportion of apoptotic K562 cells was 5.5 fold greater following treatment with Nilotinib in comparison to Imatinib after 24 hours. Conclusion: Nilotinib is apparently more potent than Imatinib with regard to the number of genes downregulated and the degree of their suppression. Many of the suppressed genes are associated with bcr-abl signalling and cell cycle. Authors’ disclosure statement: Imatinib and Nilotinib were provided by Novartis. The work was supported by Leukaemieliga e. V.

according to registration: Bugelli Cainelli Gebara! Immunogenic and adjuvant activities of Bordetella pertussis proteins CAINELLI GEBARA VCB, Lopes APY, Wolf PS, Ferreira VRF, Quintilio W, Raw I Instituto Butantan, São Paulo, SP, Brasil Background: Adjuvants are essential components to enhance the immunogenicity of vaccine antigens. Extracts of bacterial origin are well known and widely used immunomodulatory substances. B. pertussis is a promising candidate since it produces several components acting on the immune system of the host. Methods: The proteins were purified by anion exchange chromatography (Hitrap Q Sepharose HP) followed by extraction from a preparative gel (SDS-PAGE), and were further analysed by mass spectrometry and tested in mice. Female BALB/c mice, 4-6 weeks old were subcutaneously injected 2 times at weekly intervals with the proteins (1µg/0.1ml/mouse) alone or mixed with DPT (Diphtheria-Pertussis-Tetanus) vaccine formulated without aluminum hydroxide (NA-DPT), (2µl/mouse). DPT vaccine containing aluminum hydroxide (DPTBut) was used as control (2µl/mouse). Non-immunized mice (saline solution injected) were used as control. Blood was collected 1 day before and 20 days after the first immunization, and mice were challenged by intracerebral route with live B. pertussis. Sera were assayed for antibodies and isotypes. ELISA was used for all evaluations. Statistical analyses included one-way ANOVA followed by Dunnet’s multiple comparison tests considering p<0.05, and ^2 test, with Yates correction. Results: We have purified two B. pertussis proteins, and identified by mass spectrometry as the 73 kDa N-terminal -domain of BrkA autotransporter protein and the Cpn60/60 kDa chaperonin. Immunizations with 73k+60kDa+NA-DPT elicited a significant increase (p<0.01) of the antibody response against pertussis, and of pertussis specific IgG1 and IgG2a, when compared to DPTbut immunization. The former group have also showed a better protection rate (42%) against the challenge than the latter (27%). Conclusions: Our results suggest that mouse immunization with both proteins mixed with NA-DPT could stimulate Th1 and Th2 cells, induce significant higher levels of IgG1 and IgG2a antibodies, and mediate a partial protection against the challenge. Therefore, these proteins are good candidates to be exploited as adjuvants for inclusion in vaccination protocols. Financial Support: FAPESP, Fundação Butantan, CNPq

Online method for measuring of the activities of antibiotic efflux pumps in Escherichia coli and Pseudomonas aeruginosa BUIVYDAS A^1,2, SENČILO A^1,2, STUKAITĖ S^1, BAMFORD D^2,DAUGELAVIČIUS R^1 ^1Čiurlionio 21/27, Department of Biochemistry and Biophysics, Vilnius University, Vilnius, Lithuania LT-03101; ^2Viikinkaari 5 (PO Box 56), Institute of Biotechnology, University of Helsinki, Helsinki, Finland. Background: Multidrug resistance (MDR) pumps, decreasing intracellular concentrations of antibiotics are major causes of resistance in opportunistic pathogens such as Pseudomonas aeruginosa. This phenomenon ensures the viability of bacteria under high concentrations of antibiotics and complicates the treatment of infected patients. Fast methods are needed to determine the efficiency of drugs inactivating MDR pumps for the successful treatment of patients. Methods: Aerated suspensions of Escherichia coli and P. aeruginosa cells were studied in thermostated reaction vessels, and changes in the extracellular concentration of indicator lipophilic cation tetraphenylphosphonium (TPP^+) was monitored using selective electrodes. Tetracycline was used as the model antibiotic, and, phenylalanyl arginyl β-naphthylamide (PAβN), reserpine and chlorpromazine were used as inhibitors of the different MDR pumps. Results: Depending on the outer membrane permeability, membrane voltage and activity of the MDR pumps, wt cells and the main MDR pump mutants of P. aeruginosa and E. coli accumulated different amounts of TPP^+. Addition of tetracycline and the pump inhibitors caused detectable alterations of the amount of TPP^+ accumulating. Using the outer membrane-permeabilizing and the plasma membrane depolarizing compounds, activity of the pumps, affinity of the substrates and efficiency of the inhibitors were evaluated. Conclusion: Online monitoring of TPP^+fluxes across the bacterial envelope can be applied for studies of the activity of MDR pumps in E. coli and P. aeruginosa, for determination of the pump selectivity to substrates and for evaluation of the efficiency of pump inhibitors.

Red Blood Cell Membrane Fatty Acid Analysis in Never-Medicated First-Episode and Chronic Medicated-Schizophrenic Patients BURETIĆ-TOMLJANOVIĆ A^1, GIACOMETTI J^1, NADALIN S^1, RUBEŠA G^2, VULIN M^3, TOMLJANOVIĆ D^4 ^1School of Medicine, University of Rijeka, Rijeka, Croatia; ^2Clinical Medical Centre, Rijeka, Croatia; ^3Psychiatric Hospital Lopača, Rijeka, Croatia; ^5Private psychiatric practice, Rijeka, Croatia Background: Reduced n-3 and n-6 polyunsaturated fatty acids (PUFAs) content in red blood cell (RBC) membranes and abnormal membrane phospholipid metabolism were repeatedly implicated in the etiology of schizophrenia. Methods: Gas-chromatography analysis of fatty acids content in RBC was performed in the group of never-medicated first-episode schizophrenic patients, chronic medicated patients and healthy controls. Differences between group means were investigated using ANCOVA. Group, sex and smoking status were used as predictors, while covariates were body mass index (BMI) and age. The level of statistical significance was set to 0.01. Results: Three groups of subjects significantly differed in total n-3 fatty acid, 22:6n-3, 22:5n-3 and 22:0 RBC content, delta-9-desaturation (D9C18) index, peroxidizability index (PI), double bond index (DBI), and DBI/PI ratio. BMI was significantly correlated to average chain length (ACL), D9C18 index and PUFA/monounsaturated fatty acid ratio. First-episode patients had significantly higher total saturated fatty acid (SFA) content, particularly 18:0, lower total PUFAs content, particularly 18:2n-6 and 22:5n-3, lower D9C18 index, PUFA/SFA and DBI/PI ratios compared to controls. The differences between chronic medicated-patients and controls showed similar pattern. Only minor but highly significant differences were found between two groups of schizophrenic patients (22:0, 22:5n-3). Conclusions: 1) The results confirmed a disturbance of lipid homeostasis in RBC membranes as intrinsic feature of schizophrenia. 2) Reduced total n-3 FAs and total PUFAs content, reduced PI, DBI and D9C18 index, and higher total SFA and 18:0 content were found regardless of illness duration and antipsychotic treatment. 3) Nicotine usage was not a significant predictor of the FAs composition in RBC membranes of investigated subjects. 4) Drugs affecting phospholipid metabolism and providing recovery of lipid homeostasis in cellular membranes might have beneficial effects in schizophrenia.

Conceptual basis for cancer treatment: from single drugs to kits with serial programmed actions of multiple substances-drugs BURLAKA D Department of Biotechnical Problems of Diagnostics, Institute of Cryobiology and Cryomedicine National Academy of Sciences of Ukraine, Kyiv, Ukraine. Background: Well-known paradoxical effect that both high-dose estrogens and anti-estrogens cause tumor regressions. In this case important is an understanding of the properties of a tumor. Aims: 1) To study of the influence of the same hormonal environment on tumor growth and character of change of the receptors level in tumors with different hormone sensitivity. Methods: Concentration femtomoles/mg protein (fM/mg) of estrogen receptor (ER) was determined in transplanted mice mammary tumors with intensive reproduction mode (2-6 parturition without lactating) (n=20) and virgin (n=20). The levels of ER were determined by means of the dextran-coated charcoal technique. Mice of line С3H/Sn (n=69) contained in cages on 5 female +2 male (normal reproduction) in a room with daylight. Virgin female mice (n=47). Results: The data provides both estrogen-positive and –negative tumors that actually are evoked from the same tissue, so that one can determine at what level(s) the hormone sensitivity or dependency exists. The widely accepted hypothesis that the interaction of estrogen with its cellular receptors determines the hormone dependency of mammary tumors should now be challenged on the basis of the following observations: a) tumors occuring in experimental animals with permanent high estrogen levels are receptor-positive(~20-100fM/mg) and with low estrogen levels (<2fM/mg) are receptor-negative; b) tumors regrowing after complete or partial regression as a result of endocrine ablation or hormone administration are no considered to be autonomous or hormone dependence but environment dependence; c) growth of a tumor at cyclically changing hormonal level leads to heterogeneity of mammary tumors that complicates hormone therapy. Conclusions: 1) Series of parameters of a tumor such as the invasiveness, heterogeneity and others are consequence of adaptic properties of tumoral cells. 2) Ability of tumoral cells to adapt for change of a surrounding microenvironment answers on a question on a paradoxicality of a hormone therapy. 3) High sensitivity of tumors to change of steroids concentration (10^-9M) discovers an epigenetic path of redifferentiation a tumor oncogenome i.e. multiple-stage therapy.

Pro-Atherogenic and Pro-Inflammatory Alterations in Mononuclear Cell Populations Induced by Oxidized Low Density Lipoprotein (oxLDL) and High Glucose Levels in Type II Diabetic Patients. Anti-Inflammatory Drugs, an Alternative? BUSTAMANTE, M^1, DÍAZ, F^1, MUÑOZ, M^1, GUZMÁN, C^1, LLANCAQUEO, A^1, NÚÑEZ, L^1, CAMPOS, L^1, RIVAS, CI^2, VERA, JC^2, GROSS, H-C^3& BACHEM, M^3. ^1Laboratory of Molecular Diagnosis, Catholic University of Concepción, Concepción, Chile; ^2Department of Phatophysiology, University of Concepción, Concepción, Chile; ^3Einrichtung Klinische Chemie, Uni-Klinikum, Ulm, Germany Background: Type II diabetes mellitus is a risk factor in the development of atherosclerosis. Two factors are central to the effect of diabetes, oxidative modification of LDL and high glucose concentrations. We hypothesized that mononuclear cells from diabetic patients in the presence of oxLDL and high glucose levels undergo pro-inflammatory alterations, which would enhance the development of atherosclerotic plaques. Therefore, our aims were: 1) To measure the induction of necrosis and apoptosis in mononuclear cells from diabetic patients, 2) to measure the release of pro-inflammatory interleukins in stimulated cells, and 3) to analyze the possible use of meloxicam as an alternative treatment. Methods: This study included cells from 140 diabetic patients and 105 controls. The individuals were grouped by age, sex and obesity for the measurement of necrosis and apoptosis; 15 diabetic and 15 controls for quantification of interleukins; and 20 patients and 20 controls to study the effect of meloxicam (7,5 mg/day/30days). The results are presented as the mean ± SD of at least three independent experiments (a value of p < 0,05 was considered as significant, t-test). Results: Cell necrosis and apoptosis was increased in mononuclear cells from obese, diabetic patients older than 50, without significant sex-related differences. Moreover, the release of pro-inflammatory interleukins, in particular those related with the development of atherosclerosis, was also increased in diabetic cells. Interestingly, meloxicam treatment decreased necrosis, apoptosis and release of pro-inflammatory interleukins from diabetic mononuclear cells. Conclusions: Some of the pro-inflammatory effects observed when diabetic cells are subjected to the synergistic action of oxLDL and high glucose concentrations, such as necrosis, apoptosis and interleukin release, are reduced by treatment with meloxicam. Acknowlegment: FONDECYT 1040977, DIP-UCSC, Prof. Max Bachem.

Tacrolimus: a highly effective new therapy for chronic glomerular diseases? BUTANI L Professor and Chief, Pediatric Nephrology, Co-Instructor of Record, 3rd year Pediatric Clerkship, UC Davis Medical Center, 2516 Stockton Boulevard, Sacramento, CA 95817, USA Abstract: Tacrolimus is a potent immunosuppressive agent that has been demonstrated to be superior to cyclosporine after renal transplantation as an effective and safe anti-rejection drug. Based on the excellent outcomes in the transplant population, the use of tacrolimus has been extended to patients with a variety of different chronic glomerular diseases that are immune-mediated, such as steroid resistant nephroses and lupus nephritis, to name a few. The aim of this presentation is to critically appraise data related to the safety and efficacy of tacrolimus in adults and children with immune mediated renal diseases, outside the transplant setting. New evidence discussing the potential benefits and side-effects of long-term tacrolimus use (such as subclinical nephrotoxicity) will be discussed, so as to allow the audience to make an evidence-based assessment on whether tacrolimus is a reasonable strategy to treat patients with chronic glomerulonephritis.