Ehrlich II –2nd World Conference on Magic Bullets

Conclusions: Cytoplasmic protein complexome involved in CRO resistance of E. coli was achieved by proteomic methodologies based on combined 2-D native/SDS PAGE and Co-IP, far-Western blotting or His-tag pull down assays. Eight differential expressed proteins and three new protein complexes, TnaA-PflB, MalE-TalB and CysK-SodB were identified from the cytoplasmic fraction. Among these proteins, down-regulated of MalP, AtpD and PflB, up-regulated of SucC-SucD, which involved in modification of energy production and conversion, may play key roles related to resistance according to the antimicrobial susceptibility and carbohydrate metabolism analyses using genetic modified strains with gene deletion of the proteins. These findings provide new insights into the mechanisms of CRO resistance, and these key proteins can be the targets for development novel antibiotic compounds. In our knowledge, this is the first report on functionally altered complexome and energy metabolism modification to antibiotic-resistant bacteria. This work was sponsored by grants from National Basic Research Program of China (2006CB101807) and “863” project (2006AA09Z43Z).

Innovative Nanopharmaceutical Strategies for Neuronal Survival and Regeneration After Traumatic Brain Injury PENKOWA M University of Copenhagen, Faculty of Health Sciences, Section of Neuroprotection, Copenhagen, Denmark. Background: Metallothionein (MT) is a multipurpose cytoprotectant important for host defense responses, immunoregulation, and cellular survival. We have explored MT functions in the injured brain in vivo and tested the pharmaceutical effects MT. The aim is to develop treatment strategies for neurological patients. Methods: This study applied genetically modified mice with MT deficiency or overexpression along wildtypes. The mice were subjected to a focal brain injury and in the following weeks, they received i.p. injection of exogenous MT protein or placebo. Animals were sacrificed at different time points and brains were removed for histopathology, molecular analyses and global gene profiling. Results: In injured brain, MT inhibits inflammation by reducing macrophages, T lymphocytes and formation of proinflammatory interleukins, tumor necrosis factor-alpha, matrix metalloproteinases, reactive oxygen species and proapoptotic factors. MT enhances neuronal survival and brain repair processes including angiogenesis and neuroregeneration. The latter is due to activation and recruitment of resident neural stem cells and neuroglial precursor cells. Both endogenous and exogenous MT had neuroprotective actions. Peripherally injected MT is well tolerated and is detected in the brain within 1 h after i.p. administration. This transport of MT from peripheral tissues into the brain is likely facilitated by blood-brain barrier leakage caused by the traumatic injury, which transiently disrupts integrity and permeability of the blood-brain barrier. The intercellular MT signaling is mediated in part by megalin, a multiligand endocytotic receptor and by lipoprotein receptor-related protein-1 (LRP). The intracellular signaling of MT involves kinases, phosphorylation and dephosphorylation-mechanisms, G-protein coupled receptors, and zinc ion regulation, although the precise mechanisms downstream of MT remain to be fully established. Conclusions: The data demonstrate the importance of MT for neuroprotective and regenerative responses following traumatic brain injury. MT is an antiinflammatory and antiapoptotic factor, which promotes brain tissue repair and functional recovery. MT may provide a novel pharmaceutical strategy against brain trauma.
Adverse Reactions of Titanium-based Chemotherapeutic Agents on Male Reproductive Organs PEREIRA ML ^1, GARCIA E COSTA F^2 ^1Department of Biology, CICECO, Univ. Aveiro, Portugal; ^2CIISA, Faculty of Veterinary Medicine, Technical Univ. Lisbon, Portugal Background: Although the treatment efficacy of some titanium compounds as cytostatic agents on cancers is known, some side effects must be better estimated. Our previous studies have shown that titanocene dichloride, a metal-based antineoplastic complex, disrupted the blood-testis barrier in mice. Aims: 1) To compare the effects of two concentrations of titanocene dichloride on testis. 2) To evaluate sperm quality. 3) To estimate possible recovery of the lesions. Methods: Male mice (2 months old) were subcutaneously administered with 0.12mg/g and 0.15mg/g/body weight of titanocene dichloride per se, and sacrificed after 72h. Other groups were injected similarly and kept for 35 days without any treatment. Controls were also considered. Animals were sacrificed for organs removal. Testes were fixed in Bouin´s solution and prepared for histology. Caudal epididymal fluid was collected for sperm quality analysis using standard procedures. Observations and micrographs were made using a light Nikon AFX-Dx microscope. Results: A surviving rate of 100% was noted through the present study. However, histopathological studies have shown a considerable loss of germ cells after 72h, derangement of cellular organization within spermiogenesis of mice treated with the lowest dose. In comparison, the other group (0.15mg/g/body weight) showed more evidenced lesions. The sperm analysis from these groups of mice revealed a wide range of abnormalities in the concentration, motility, vitality, and morphology. After one cycle of spermatogenesis, a partial recovery of the lesions was denoted in the testis and seminal fluid from the group administered with 0.12mg/g of titanocene dichloride. However, no reversible effects were detected in the highest dosed group of animals. Spermiogenesis, and sperm quality were deeply affected. Conclusions: 1) These results confirm that titanocene dichloride impairs at least spermiogenesis. 2) The persisting abnormalities observed after the recovery period suggest that this fact may compromise male fertility. Future studies must address the biochemical and molecular approaches of the blood-testis barrier, for the understanding of the pathophysiological mechanisms leading to the testis and epididymis alterations.	The differential dose-dependence for pro-angiogenic and cytotoxical effects of new anticancer plant agent within different nanocarrier formulations PEREIRA LOPES EF^1, BARBOSA MR^1, STELLA CN^1, SANTOS WA^1, PEREIRA EM^1, POZZI FM^1, LEITE FB^1, GOMES LF ^1Universidade de São Paulo, Brazil New drugs for cancer have been tested with a promising success. Molecular plant components were far ahead aimed into the angiogenesis and anti-angiogenesis pathways. Those molecules were able to destroy the mitochondria of cancer cells, what made scientists focus on the cancer cells themselves, not considering the destruction of the blood feeding system to eliminate the tumor. Another point revealed as a difficulty was the poor capacity of these drugs to be administrated into an in vivo analyzing system, to prove other mechanisms of promoting the cancer cells death. The present paper deals with the construction of a nanocarrier system, in which a molecule of an anticancer plant agent was included. The effects of these new constructs were tested into the Chorioallantoic Membrane (CAM) in the chicken embryo model. A14 stimulates human umbilical vein endothelial cell (HUVEC) proliferation in the 1-10 micromolar range, and promoted cell death in the 1-10 milimolar range. It was also toxic to Murine melanoma B16F10 in 1-10 milimolar concentrations, even as proliferative effects were not observed. In vivo pro-angiogenic effect was observed in CAM, which presented increased number of blood vessel ramifications and capillary budding. The new vessels presented leakier and less organized than those from control membranes. When molecular drug carriers were employed, the pro-angiogenic A14 effects could be observed at nanomolar concentrations, either in cell cultures or in the CAM model. Accordingly, treatment with A14 or its formulation dramatically reduced the number and size of the disseminated B16F10 murine melanoma tumors in the CAMs of live eggs. It is proposed that not only direct toxicity against B16F10 cell, but also the drug effects upon angiogenesis were relevant to both decreasing tumor growth and inhibiting metastatic dissemination in the in vivo model of neoplasia.
Chemical agents in root canal therapy. Key of the success PÉREZ-HEREDIA M^1, GONZÁLEZ-RODRÍGUEZ MP^1, ARIAS-MOLIZ MT^1, NAVARRO-ESCOBAR E^1 , FERRER-LUQUE CM^1 ^1Univ. Granada, Spain. Background: The ideal irrigating solution or combinations of irrigating solutions at appropriate concentrations and pH, or the time of application of each one to achieve success in the cleaning and disinfection of the root canal is not known. Aims: To determine which irrigating agent cleans better the root canal after rotatory and manual instrumentation and which one is a decalcifying agent of the root canal dentin wall eventually. Methods: 80 human teeth were randomly divided into 8 groups. 4 groups were prepared with hand instrumentation and other 4 with rotary. The irrigating solutions were 2.5% NaOCl; 15% citric acid plus 2.5% NaOCl; 15% EDTA plus 2.5% NaOCl; and 5% orthophosphoric acid plus 2.5% NaOCl. Canal walls were observed with SEM, and photomicrographs were taken in three thirds to evaluate the cleaning ability. In order to study the decalcifying effect, two slices were cut from the cervical third of the root of 10 human incisors. Each slice was sectioned into 2 equal parts. Specimens were assigned to one of four groups (n=10) for immersion in 15% EDTA, 15% citric acid, 5% phosphoric acid or 2.5% NaOCl, during 5, 10, and 15 min. The concentration of Ca^2+ was measured by AAS. Results: At 5, 10, 15 min 15% EDTA (0.085±0.029; 0.094±0.028; 0.098±0.028) and 15% citric acid (0.075±0.019; 0.093±0.024; 0.099±0.027) extracted the largest amount calcium. The most rapid decalcification rate was with 15% EDTA. NaOCl extracted negligible calcium. Conclusions: Acid solutions with 2.5% NaOCl were more effective than 2.5% NaOCl in the root canal cleaning with both techniques of instrumentation. It is the same for decalcification, acid solutions produce decalcification of root dentin, with most calcium extracted during the first 5 min of action.	Increased Expression of Tumor-Specific Cyclin B1 Sensitizes Prostate Cancer Cells to Apoptosis Induced by Anti-Mitotic Drugs PEREZ-STABLE C^1, 2, REINER T^2, DE LAS POZAS A^1, GOMEZ A^3, PARRONDO R^1 ^1Geriatric Research, Education, and Clinical Center and Research Service, VA Medical Center, Miami, Florida ^2Division of Gerontology & Geriatric Medicine, Department of Medicine and Sylvester Comprehensive Cancer Center, University of Miami Miller School of Medicine, Miami Florida ^3South Florida VA Foundation, Miami, Florida Chemotherapeutic drugs ideally should take advantage of the differences between transformed and normal cells and induce apoptosis only in cancer cells. One such difference may be the overexpression of cyclin B1 protein in cancer cells, which is required for the proper progression through mitosis and is considered to be a tumor-specific antigen. We showed that treatment of human prostate cancer cells with the promising antimitotic drugs paclitaxel, docetaxel, or 2-methoxyestradiol (2ME2, a metabolite of estradiol) results in a rapid accumulation of cyclin B1 protein and an increase in cyclin B1 kinase activity, followed by induction of apoptotic cell death. Inhibition of cyclin B1 kinase lowers apoptosis induced by paclitaxel, docetaxel, and 2ME2. We hypothesize that a cancer-specific mechanism whereby these antimitotic drugs may exert anti-prostate cancer activity is the deregulated activation of cyclin B1 kinase during the cell cycle, leading to the induction of apoptotic cell death. Our results in human prostate cancer cells established a positive correlation between cyclin B1 protein and apoptosis induced by chemotherapy. There is minimal cyclin B1 and induction of apoptosis by chemotherapy in non-transformed cells. Stable overexpression of cyclin B1 in human LNCaP prostate cancer cells increases sensitivity to apoptosis, possibly by decreasing anti-apoptotic Bcl-2 and increasing pro-apoptotic p53. Experiments using siRNA and a dominant negative mutant of CDK1 to lower cyclin B1 protein and kinase activity in LNCaP cells showed a decrease in apoptosis. We then determined whether the combination of docetaxel and 2ME2 can increase apoptosis utilizing human prostate cancer cell lines and the FG/Tag (fetal globin promoter linked to SV40 T antigen) transgenic mouse model of androgen-independent prostate cancer (AI-PC). Cell growth, flow cytometry, and apoptosis assays demonstrated that the combination of low doses of 2-ME (0.5 mM) and docetaxel (0.1 nM) can inhibit growth, increase G2/M cell cycle block, and induce apoptosis more significantly than either drug alone in a variety of human prostate cancer cell lines. In the FG/Tag mice, a low dose combination of docetaxel and 2ME2 (2.5 + 75 mg/kg) significantly inhibited primary prostate tumors by 42% (P<0.02), despite each dose of the drug by itself having no effect. Reduction of primary prostate tumor weights required doses of docetaxel and 2ME2 that increased mitotic cell cycle arrest and increased apoptosis, as shown by flow cytometry and immunohistochemistry. In contrast, inhibition of proliferation or angiogenesis without an effect on apoptosis did not reduce the weights of primary prostate tumors. Docetaxel and 2ME2-mediated increase in mitotic block occurred only in cyclin B1 overexpressing prostate tumors and not in normal tissues (negative for cyclin B1). Conclusion: The ability of the antimitotic drugs paclitaxel, docetaxel, and 2ME2 to induce apoptotic cell death in AI-PC cells will probably one of the mechanisms required for an improved patient survival benefit. Our results suggest that despite its association with transformed cells, higher levels of cyclin B1 protein in prostate cancer may be a good prognostic marker for chemotherapy induced apoptosis.
Status of BCG Vaccine at the Beginning of the 21^st Century PESUT DP^1^,2 ^1Univ. of Belgrade School of Medicine, Belgrade, Serbia; ^2Research and Epidemiology Department, Institute of Lung Diseases and Tuberculosis, Belgrade, Serbia. Background: Since tuberculosis (TB) is declared global emergency by the World Health Organization, TB vaccine development is making rapid progress. Virtually hundreds of candidate vaccine antigens are currently passing through different phases of preclinical research and several new vaccine products are now in trials’ phases in humans to achieve the formulation of a superior TB vaccine. It was in 1921 when Mycobacterium bovis bacillus Calmette–Guérin (BCG) vaccine was administered for the first time, with success, to an infant born to a mother who died for TB and brought up by a grandmother suffering for TB. BCG vaccine is almost 90-year old now and the world still does not have anti-TB vaccine which would be better or with less adverse reactions than BCG is. The vaccine is able to protect newborns and infants from the most serious forms of TB – TB meningoenceplalitis and to prevent death of TB. Despite a number of shortcomings, BCG is likely to be administered for at least the next 5-10 years in protection against TB. Also known as a powerful immune modulator, it is used in many ways to treat other conditions. The aim of this presentation was to review the current status BCG vaccine, including the general view on the immunization policies, which differ among countries, to explain reasons for its variable efficacy and effectiveness, duration of protection against tuberculosis, cost/effectiveness studies and general plans on vaccination compared to the World Health Organization recommendations and national legislation. Particular issues like BCG vaccination in HIV-infected persons and immunization of TB high risk groups will also be presented together with cost-benefit models, which explored the utility of BCG in health care settings in the countries where BCG vaccine was not mandatory. The results of this latter encouraged a return to a policy of selective use of BCG among health care professionals. The International Union against Tuberculosis and Lung Disease suggested the criteria for the countries which would shift from routine BCG to a selective vaccination or its discontinuation. They are based on fulfilled TB epidemiological conditions in a particular setting.	Detemir Insulin in Type 1 and Type 2 Diabetes PETERSON GE Diagnostic & Critical Care Medicine, The Diabetes Center, 411aurel Street, Suite 3275, Des Moines, USA We have learned from our experience with the first long-acting insulin analogue, Insulin Glargine (Lantus) that a continuous dose of insulin is beneficial. This insulin relies on a less soluble insulin molecule, which becomes soluble when it interfaces with neutral pH. This basal insulin forms a depot subcutaneously and the insulin is slowly released. A revolutionary concept in insulin therapy was introduced in March 2006. Insulin Detemir (Levemir) is a clear, colorless, aqueous, neutral sterile solution, which protracts and maintains duration of action of up to 24 hours. Insulin Detemir (Levemir) is the first insulin that has a fatty acid side chain, which binds with albumin and protracts in a manner that provides a decrease in intra-patient as well as intter-patient variability. Clinical Pharmacology: Recombinant DNA technology has led to the development of novel insulin molecules with absorption and biological activity profiles that are similar to physiological insulin. The amino acids threonine in position B30 has been omitted, and a C14 fatty acid chain has been attached to the amino acid B29. Mechanism of action: Insulin Detemir exerts its action by binding to insulin receptors, lowering blood glucose by facilitating cellular uptake of glucose into skeletal muscle and fat. Insulin Detemir acts like all insulins inhibiting output of glucose from the liver. Insulin inhibits lipolysys, inhibits proteolysis and enhances protein synthesis. Pharmacokinetics/Pharmacodynamics: Insulin Detemir (Levemir) has a relatively flat action profile. Glucose clamp studies have demonstrated efficacy at doses from 0.1 Units/kg with a mean duration of action of 5.7 hours to 23.2 hours with doses of 0.4 Units/kg. The sampling ended at 24 hours and hence it is felt to have efficacy up to 24 hours when the clamp study was discontinued. A slow systemic absorption of insulin Detemir molecules from the injection site is due to the strong self-association of the drug molecules and albumin binding. Distribution: Likewise, due the albumin binding there is a slower, less variable distribution of the insulin Detemir molecules to peripheral target tissues. Since detemir is 98% albumin bound, binding studies were performed and found no clinically relevant interactions between insulin detemir and fatty acids or other protein-bound drugs. These insulin detemir molecules aggregate in a continuum from hexamer to di-hexamer and it is the monomer that binds with the receptor exerting its action. We report on the use of insulin detemir in Type 1 and Type 2 diabetes. Since insulin detemir (Levemir) can be given once or twice daily, lasts 16-24 hours, has a relatively flat action profile and does not require resuspension before injection it provides a greater flexibility of dosing regimens for the patients to follow. It is available in the ever popular FlexPen as well as vial. It should not be mixed with other insulins in the same syringe. It can be initiated on a Unit per kg basis as in Type 1 Diabetes. Generally, a unit per unit conversion is made comparing it with glargine, and 80% of the total NPH dose. Basal/Bolus therapy is usually initiated by spitting the insulins as half with 50% going to basal and 50% going to meal time insulin divided traditionally into three based on meal times. Initiating Levemir in Type 2 Diabetes is simpler. Generally patients are begun at 0.2 units/kg or 10 units daily with weekly titration. Some advocate sampling the average of three fasting blood sugars and increase the dose by 1 unit for every 40mg/dL blood glucose beginning at 180mg’dL. Through their own experience with basal insulin in partnership with their provider patients learn the merits of achieving A1C goals with a less risk of hypoglycemia. The greater flexibility of dosing and lower risk of hypoglycemia makes the initiation of this new insulin with greater ease. Recommendations and conclusions: Insulin Detemir is an extraordinary basal insulin that will provide patients with less variability and hence less hypoglycemia. The concern of weight gain to achieve tighter control of blood glucose levels has always been a concern. This unique insulin delivery to the peripheral tissues will achieve normalization of glucose levels with no weight gain or possibly weight loss. Patients will accept insulin therapy more readily with the expectation of less hypoglycemia and the potential weight loss.
High-dose Levofloxacin to Reduce Duration of Therapy and Slow Emergence of Resistance PETERSON J^1, NICHOLSON S^1 ^1. Ortho-McNeil Janssen Scientific Affairs, LLC, Raritan, NJ, USA. Background: The quinolone class of antibacterials was introduced more than 40 years ago with the accidental discovery of nalidixic acid as a by-product of the synthesis of the antimalarial compound, chloroquine. Naldixic acid had a narrow spectrum of antibacterial activity, poor absorption, and significant adverse events. Methods: Extensive pharmacologic and clinical development of the quinolone class eventually led to the discovery and development of the fluoroquinolone, levofloxacin. Results: With a broad spectrum of activity that targeted the most relevant respiratory pathogens; excellent pharmacokinetics including nearly complete absorption from the GI tract, broad tissue penetration, high serum concentrations and half –life suitable for once-daily dosing; levofloxacin was broadly adopted as the first true respiratory fluoroquinolone. Conclusions: Levofloxacin remains a potent and relevant antibacterial therapy today. Additional pharmacodynamic development of a high dose, short course treatment regimen of levofloxacin achieved the specific objectives of enhanced bacterial killing, reduced potential for emergence of bacterial killing, and has pioneered a paradigm shift towards short-course therapy of respiratory infections.	Tetracycline Biosynthesis: How to Overcome Nature's Potential in Developing Novel Anti-infectives? PETKOVIĆ H^1*, LEŠNIK U^1, HUNTER IS^2, RASPOR P^1 ^1University of Ljubljana, Biotechnical Faculty, Slovenia, ^2University of Strathclyde, Scotland, UK