Ehrlich II –2nd World Conference on Magic Bullets

Results: High grade protection against virulent virus challenge was found after immunization with the pcDNA3.1-gD plasmid and with the gE-del virus. A medium grade, but still satisfactory protection was noted following immunization with the FpgD1/313 recombinant glycoprotein. Considerable response of peripheral blood leukocytes (PBL) in lymphocyte transformation test (LTT) was found in the mice immunized with FpgD1/313, with pcDNA3.1-gD plasmid as well as with live ANGpathE-3-3 virus. In vitro, secretion of Th 1 (TNF, IFN- and IL-2) and Th 2 (IL-4 and IL-6) cytokines was followed in purified PBL as well as in splenocyte cultures coming from immunized and control animals. The leukocytes from FpgD1/313 immunized mice showed increased secretion of both, Th 1 and Th 2 cytokines. The secretion of IL-4 and TNF was high by PBL of FpgD1/313 immunized mice (as compared to mock-immunized animals); the splenocytes from FpgD1/313 immunized mice showed extensive IL-4 production and a slightly elevated IL-6 synthesis.

Conclusions: The classical purified subunit vaccine (especially in combination with a novel adjuvant), might be a more powerful immunogen than a single recombinant glycoprotein.

Authors’ disclosure statement. Introduction of new adjuvants, which shift the cytokine production towards the stimulation of helper T-cells indicates a promising development. Even when the immunotherapeutic use of HSV vaccine is effective only partially, it still might represent an alternative to chronic chemotherapy of recurrent labial and/or genital herpes.

Prevalence And Characteristics Of Verotoxigenic Producing Escherichia Coli O157:H7 Isolated From Goats And Cattle Carcasses In Tanzania RAJI MA Dept of Veterinary Pathology and Microbiology, Ahmadu Bello University Zaria, Zaria, Nigeria The prevalence of Verotoxigenic producing Escherichia coli (VTEC) in cattle and goats carcasses were investigated between September 2002 and December to June 2003 by cultural and immunomagnetic separation technique. A total of 167 Escherichia coli colonies from carcasses of cattle (300), and goat (263), from Morogoro and Dar-es-salaam were isolated in this study. TEC O157 strains were recovered from 17 (5.67%) cattle carcasses and none from goats. Of 167 E.coli strains, 17 were grouped into sorbitol non-fermenting and glucuronide negative and 29 strains were sorbitol positive and glucuronide positive. The remaining 38 were sorbitol negative and glucuronide positive.V Using Reversed passive latex agglutination kit from Denka Japan indicated that all isolates produced verotoxin. Further characterization of the VTEC isolates showed that 1(4%) of the bovine VTEC strains was positive only for stx1. Stx2 gene alone was detected in 4(20%) of bovine isolate. Both stx1 and stx2 gene were present in one (4%) of bovine isolates. Eae A was detected in 4 (20) of bovine isolates. Stx 1, stx2 eae A and Ehly A were present in one (4%) bovine isolates. Other bacterial agents such as Pseudomonas spp, Proteus spp and coliforms were also isolated. The VTEC O157 isolates were resistance to gentamicine, chloramphenicol, streptomycin, and amoxsylav. This study is the first attempt to investigate the prevalence of VTEC O157 in goats and cattle carcarsses in Tanzania. Cattle carcasses are contaminated with verotoxigenic Escherichia coli O157:H7 in this region.
Effectiveness Of Antibiotics In The Management Of Acute Exacerbations Of Chronic Obstructive Pulmonary Disease. Do Antibiotics Improve Patient Outcomes – Evidence To Date RAM FSF^1, Rodriguez-Roisin R^2, Granados-Navarrette A^2, Garcia-Aymerich J^2, Barnes NC^3 ^1Massey University, Auckland, New Zealand; ^2University of Barcelona, Barcelona, Spain; ^3London Chest Hospital, London, United Kingdom Background: True efficacy of antibiotic use in the treatment of acute exacerbations of chronic obstructive pulmonary disease (AECOPD) remains controversial. Most patients are treated with a broad-spectrum antibiotic during an exacerbation, and a number of controlled trials have shown their beneficial effects. However, some trials have shown a lack of positive effect with antibiotic therapy. Despite the many clinical trials and guidelines the role of antibiotics in the management of AECOPD remains a matter of debate. Methods: We undertook a systematic review with meta-analysis of all available high quality randomised controlled trials. Trials were included in the review if an antibiotic was compared to placebo in patients with AECOPD. Trials were searched for on the Cochrane database, MEDLINE, EMBASE, Web of Science, key respiratory web sites, journals and also hand searching of journals. Results: Eleven trials with 917 patients with moderate-to-severe COPD exacerbations were included in the review. Antibiotic therapy reduced relative risk (RR) of mortality by 77% (RR 0.23; 95%CI 0.10, 0.52), treatment failure by 53% (RR 0.47; 95%CI 0.36, 0.62) and sputum purulence by 44% (RR 0.56; 95%CI 0.41, 0.77). The number of patients required to be treated (NNT) with antibiotics to save one life was 8 (95%CI 6, 17) to avoid one treatment failure 3 (95%CI 2, 4) and to avoid one patient having purulent sputum was 8 (95%CI 5, 14). However, and as expected, antibiotic use increased the risk of diarrhoea (RR 2.86; 95%CI 1.06, 7.76). Conclusions: Our results clearly show that in moderate-to-severe exacerbations of COPD associated with increased cough and sputum purulence, antibiotic therapy regardless of antibiotic choice, is efficacious in decreasing risk of mortality, treatment failure and sputum purulence. This review supports the use of antibiotics for patients with AECOPD who are moderately or severely ill. There should now be no doubt that antibiotic chemotherapy plays an important role in the management of patients with AECOPD.	Ciprofloxacin Resistance Profile In Klebsiella Pneumoniae Isolates During 2002-2007 In Paediatric Septicaemic Cases Of A Tertiary Care Hospital In India RANDHAWA VS, GUPTA LK*, MEHTA G Deptt. Of Microbiology and Pharmacology*, Lady Hardinge Medical College and associated Kalawati Saran Children’s Hospital New Delhi, India Ciprofloxacin was introduced in the late 1980s as an efficacious and safe drug. It’s usage in India increased phenomenally in the period after the local outbreak of plasmid mediated chloramophenicol resistant S. Typhi in 1993, which resulted in the change of the drug of choice of typhoid fever from chloramphenicol to ciprofloxacin. The effectiveness of ciprofloxacin in numerous microbes and significant reduction in its cost has led to its rampant empiric use in many clinical conditions. The current study is to assess the trends of ciprofloxacin resistance in Klebsiella pneumoniae. More than five hundred and fifty isolates of Klebsiella Pneumoniae were isolated from septicaemic cases admitted in Kalawati children hospital during 2002-2007. The age of the patients varied between newborn to 14 years. The isolates were identified by standard microbiological (including biochemical) techniques. The resistance profile of the isolates was analyzed by antibiotic disc diffusion technique. The percentage of strains resistant annually to ciprofloxacin were calculated. The ciprofloxacin resistant isolates rose from 26.9% in 2002 to 63.8% in 2007. This resistance to ciprofloxacin is due to multiple chromosomal mutations and is associated with drug resistance to other antimicrobials. Thus, a rational and judicious use of ciprofloxacin is necessary, besides efforts to prevent spread of ciprofloxacin resistant K. pneumoniae strains in the hospital.
Polymyxin B Sulphate: A Brief Overview RANGASWAMY V, HIREMATH A, GUDURI B Reliance Life Sciences, Industrial Biotechnology Group, DALC, Navi Mumbai, India Background: Polymyxin B sulphate, an antibiotic discovered in 1947, is used in the treatment of infections caused by gram-negative bacteria, particularly the Pseudomonas aeruginosa and Escherichia coli. They are decapeptides with antimicrobial spectrum that includes gram-negative bacteria, prominently Pseudomonas aeruginosa. Many questions remain unanswered about polymyxins since most of the research was carried out before the 1980s and the methods for evaluation of the antibiotic were not as advanced as today. Therefore, a detailed investigation on the polymyxin production by Bacillus polymyxa is warranted. Methods: Optimization of nutritional and physiological parameters for high yield of polymyxin B sulphate production was done at shake flask level. The process was scaled up to 10 L in a bioreactor. A process for purification of the antibiotic was developed. Results: By optimization, a yield of 2 g/L of polymyxin B sulphate could be obtained. The process was scaled up and validation at 10 L level in a bioreactor. Downstream processing for isolation of the antibiotic involved steps including charcoal adsorption, chromatographic separation on Amberlite resin and solvent precipitation. A recovery of 33 % could be obtained. Conclusions: A detailed study for optimized production of polymyxin B sulphate was done. The downstream process to isolate the antibiotic from the broth was developed. Authors’ disclosure statement: Some of the data being in the patenting stage cannot be presented	The Vienna Vaccine Safety Initiative (Vivi) – An International Scientific Forum Aiming To Promote Evidence-Based Vaccine Safety Research And Communication RATH B, LAIMER T, VYHNANEK P, ROTH-MANGEL S, MAURER W, POLLAK A Department of Pediatrics and Adolescent Medicine, Medical University of Vienna, Austria Background: Recent trials have confirmed the safety of recommended vaccines, but rumours surrounding the safety of human vaccines spread quickly via the international media causing confusion and anxiety among patients, parents and physicians alike. Methods: In view of these concerns, The Vienna Vaccine Safety Initiative (ViVI; http://www.meduniwien.ac.at/kikli/vivi/) has been founded in 2008 as an international scientific forum aiming to promote evidence-based vaccine safety research and communication. Results: Initial projects are dedicated to the role of the media in vaccine risk communication, and to perceptions of vaccine safety among vaccine recipients as well as providers. The goal is to generate feedback for the scientific community. Interim Results of a study assessing vaccine safety perceptions among parents in Vienna will be presented along with a survey of vaccine safety training and awareness among pediatricians in collaboration with the International Pediatric Association (IPA). Conclusion: The Vienna Vaccine Safety Initiative has been formed to address the urgent need to promote vaccine safety training and research. Additional projects will serve to generate awareness of international vaccine safety standards in the public as well as pilot projects in collaboration with international vaccine safety experts from the developed and developing world. It is hoped that the activities of this new initiative will be able to generate concepts for evidence-based action towards accurate AEFI reporting while strengthening trust in vaccines.
Interfacing Cell’s Membrane And Drugs’ Mechanical Properties To Control Bioavailability: Application To Lipinski’s 2^nd Rule And Resistance To Drugs RAUCH C School of Veterinary Medicine and Science, Nottingham University, Sutton Bonington, Leicestershire, United Kingdom Background: An important bottleneck in drug discovery is determining the appropriate properties of a drug that facilitate its delivery. Lipinski has produced four rules that identify statistically the properties required for oral compounds to achieve maximum bioavailability (1). The second rule stipulates that drugs must have a molecular weight (MW) inferior to 500. As for drugs small enough their MW is proportional to their volume, Lipinski’s 2^nd rule suggests that drugs’ volume is a limiting parameter when they cross membranes. Furthermore, as to be bioavailable, drugs must traverse lipid membranes; this rule suggests that a specific interaction occurs between cells’ membrane and drugs’ volume, affecting their bioavailability. Methods: Based on this premise and using concepts associated with membrane biology and complex systems physics it is possible to demonstrate that, indeed, a universal MW cut off exists identical to Lipinski’s 2^nd rule. Results: The universal MW cut off which defines Lipinski’s 2^nd “Law” is given by: MWc^2/3=(4/3^1/2)^2/3(hRkBT/8kc), where MWc, h, R, kB, T, kc are, respectively, the critical MW, membrane thickness, endocytic vesicle radius, Boltzmann’s constant, temperature (in Kelvin) and membrane bending modulus. The theory provides a numerical value MWc=456 very close to 500. Finally, this universal law regarding a MW cut off can also predict multi drug resistance levels. For example, relative resistance levels (selected using actinomycin D) to control in lung-derived cell line plotted as a function of drugs’ MW (Fig.1A) agree well with the power law given by Lipinski’s 2^nd “Law” (Fig.1B; R^2=0.83). Figure 1:   Conclusions: Given the formulation of Lipinski’s 2^nd “Law”, it is now possible to modulate and predict how drugs cross the membrane of cells of either sensitive or resistant cells (not shown). References: (1) Lipinski CA, Lombardo F, Dominy BW, and Feeney PJ. Advanced drug delivery reviews 46: 3-26, 2001.	Computational Investigation Of Infectious Disease Mechanisms: From HIV-1 Virus Inhibition To Bacterial Resistance To Antibiotics RAUGEI S International School for Advanced Studies, Trieste, Italy Computer simulations have become an invaluable tool to study at atomistic level the dynamical behavior of biomolecules inside cells and to predict how they might react to the presence of prospective drugs. In this seminar, recent applications of molecular modeling and molecular dynamics simulations to the understanding of the functional activity of proteins involved in relevant infectious diseases are presented. Specifically several aspects of HIV-1 protease and HIV-1 integrase, two proteins encoded HIV-1 virus, will be discussed. In addition, new insights on the hydrolysis mechanism of penicillin-like antibiotic, such as cefotaxime, by beta-lactamases and the onset of resistance to beta-lactamase inhibitors, like clavulanic acid and tazobactam, will be reported.
Acetaminophen: The Global Pain Killing Magic Bullet Of The New Millennium RAY SD, BULKU E, ZINKOVSKY D, LAHOTI T Mol. Tox. Labs, A&M Schwartz Coll. of Pharmacy & HScs, Long Island University, Brooklyn, NY, USA Background: For decades now, acetaminophen (APAP) has enjoyed untainted popularity as the most effective and possibly the safest over-the-counter analgesic of the world. It has maintained its prestige by itself and in combination with several other drugs such as morphine, codeine, aspirin etc., which has mesmerized the field of medicine as the “Magic Bullet for Pain Management’. APAP uses COX1?/?COX2?/COX-3 as instruments to subdue pain, but unfortunately its safety and efficacy overshadows its potential to cause hepatotoxicity. Our laboratory has used APAP as a model toxin for decades to understand mechanisms of cell injury and cell death, and we were the first to report its potential to induce genomic injury and apoptotic cell death in the liver. APAP is unique because of its ability to communicate with various macromolecules and all the cellular components (membrane, ER, mitochondria, nucleus). CYP450-mediated biotransformation of APAP produces a highly reactive intermediate, NAPQI, and oxyradicals such as ROS, RNS & ROS-RNS hybrids. Concerted actions of all these dramatically deplete intracellular antioxidants, induce massive oxidative stress and provide a platform to initiate molecular changes that orchestrate various forms of cell death in the liver. Methods: This study (♂ICR mice) was designed to explore some of the molecular mechanisms after exposure to toxic doses of APAP (500 mg/Kg, ip) for 0 - 24 hours. Livers were analyzed for tissue biochemistry, ultrastructural changes & differential expression of pro- and antiapoptotic genes. Results: APAP induced massive liver injury (ALT) coupled with massive oxidative stress (lipid peroxidation) and genomic injury (DNA fragmentation). Gene expression studies revealed considerably reduced anti-apoptotic genes (bcl-2, bcl-XL) and elevated levels of pro-apoptotic genes (bcl-Xs, p53). Ultrastructural studies revealed megamitochondria formation and evidence of cytochrome c leakage a unique hallmark of mitochondrial route of apoptosis. Conclusion: In addition to the classic apoptotic markers, this in vivo study clearly suggests APAP’s ability to use the mitochondrial cytochrome c-release signaling system as a unique mechanism to propel this cell suicidal process. Since this pathway is maneuverable, it may have tremendous therapeutic implications in liver injury management.	Lapatinib: New Expectations RAZIS E Hygeia Hospital, Athens, Greece In the recent years there has been substantial improvement in the management of Her2Neu overexpressing breast cancer. The addition of trastuzumab, a humanized anti Her2 monoclonal antibody to the treatment of both metastatic and early disease has resulted in impressive amelioration of Disease Free survival and overall survival for these patients. However a significant number of Her2Neu overexpressing tumors have either primary resistance or develop it after response to trastuzumab. The mechanisms of resistance are varied and are the topic of extensive research, but it is clear that new molecules are needed for the management of refractory cases. Lapatinib is a small, orally administered, reversible dual Erb 1 and Her2 inhibitor. It is a tyrosine kinase, which inhibits Erb phosphorylation and survivin thus inducing apoptosis. Lapatinib inhibits downstream signaling from the hetero-and homo-dimerization of EGFR, Erb2 and possibly other Erb family members, thus preventing or delaying the development of resistance. Original phase I studies showed that lapatinib is active in nearly pretreated Her2 positive metastatic breast cancer with a median response duration of 5.5 months. Most frequent drug related event included diarrhea (42%) rash (31%) nausea (13%) pruritus and fatigue (10%). The development of rash did not seem to correlate with response. The optimally tolerated dose was 1500mg/day. Subsequent phase II trials at that dose level yielded a response rate of 4-8% in heavily pretreated patients but in the first line therapy of FISH positive patients the ORR was 28%. The next study was a randomized phase III trial in Her2 positive pretreated patients who received either capecitabine 2500mg/m^2/day or capecitabine (same dose) plus lapatinib 1250mg/day. In this study median time to progression and progression free survival were statistically significantly longer in the combination group. Furthermore some responses were seem in CNS disease. Additional studies in this area reveal modest responses in CNS metastasis. Further ongoing studies of lapatinib efficacy include randomized trials in combination with other chemotherapy agents (such as the taxanes) combination with trastuzumab for complete Her2 signaling blockade and combinations with hormonal therapy (letrozole). Also ongoing are trials in the neoadjuvant adjuvant setting and in CNS disease. Extensive investigation into the mechanism of resistance to Her2 blockade is also in progress specifically into molecular determinants of resistance such as PTEN, IGF-1R and akt and into the best method for identification of responders. Last but not least a clear grasp of the cardiac toxicity associated with lapatinib is necessary. Because erb2 signaling is important for cardiac function there were concerns that lapatinib had the potential of cardiac toxicity. The incidence of symptomatic and asymptomatic decrease in LVEF among the 1674 patients treated with lapatinib in the phase III trial was only 1.3% and the drop was reversible but ongoing studies are in progress. Finally, ongoing studies in other Her2 overexpressing solid tumors are also in progress.
Antimicrobial Peptides From Bacteria: Towards Novel Magic Bullet Strategies? REBUFFAT S, PEDUZZI J, ZIRAH S Muséum National d’Histoire Naturelle-CNRS, Chimie et biochimie des substances naturelles, Paris, France Background: Probably the oldest and most widespread antimicrobial strategy in living organisms is the use of antimicrobial peptides. Bacteria secrete gene-encoded antimicrobial peptides they use for microbial competitions, which are termed bacteriocins. Bacteriocins from lactic acid bacteria and from enterobacteria (termed microcins) exert potent bactericidal activities with minimal inhibitory concentrations in the nanomolar range. In the context of urgent need for novel antibiotics, underlined by the increasing antibiotics-resistance problem, bacteriocins and microcins may offer new and promising strategies. Results: Nisin, a 34-residue lantibiotic bacteriocin, which is currently used as a food preservative, exerts a potent antibacterial activity by binding to lipid II, the essential precursor of cell wall synthesis, thus forming stable pores in membrane bilayers [1]. Microcin E492, a hydrophobic and anionic 84-amino acid peptide endowed with a C-terminal siderophore post-translational modification, targets the inner membrane of sensible bacteria. Microcin J25, a 21-residue cyclic peptide with a unique “lasso” three-dimensional structure inhibits RNA polymerase. Despite their low structural homology and different killing mechanisms, these two microcins have a similar import pathway into sensible bacteria that uses siderophore receptors responsible for iron import in bacteria [2]. In order to identify the structural regions involved in the recognition/translocation step and the killing mechanism, we are studying the structure-function relationships for these microcins. Conclusions: The mechanism of bactericidal activity used by microcins involves: i) recognition by outer membrane receptors used for vital functions in bacteria, which have been piratized by microcins; ii) translocation of the antibacterial peptide into the periplasm; iii) bacterial killing that involves either interaction with the inner membrane to form a toxic structure or passage through the inner membrane to interact with a cytoplasmic target. Microcins and bacteriocins exhibit complex, subtle and clever mechanisms of bactericidal activity that pave the way for the design of novel antibiotics. References: Hsu STD et al. Nat Struct Mol Biol 2004, 11, 963-7. Duquesne S et al. Nat Prod Rep. 2007, 24, 708-34.