Ehrlich II –2nd World Conference on Magic Bullets

Analysis of fish immune response through bacterial stimulation and its application to oral vaccination for the cultured marine fishes TAMARU Y^1^,2*, OHTSUKA M^1, MATSUDA T^1, KATO K^3, MANABE S^4, KURODA K^5, SANADA M^5, UEDA M^5 ^1Deprt. of Life Science, Grad. Sch. of Bioresources and ^2Venture Business Lab., Mie Univ., 1577 Kurimamachiya, Tsu, Mie 514-8507, Japan ^3Fisheries Lab. of Kinki Univ., 3153 Shirahama, Nishimuro, Wakayama 649-2211, Japan ^4Kan-on-ji Inst., The Research Foundation for Microbial Disease of Osaka Univ., 2-9-41 Yahata, Kanonji, Kagawa 768-0061, Japan ^5Dept. Appl. Biochem., Grad. Sch. of Agriculture, Kyoto Univ., Kitashirakawa-Oiwake, Sakyo, Kyoto 606-8502, Japan Background: The establishment of systems to display heterologous proteins on the cell surface of microorganisms is expected to be useful in the separation of produced polypeptides; the production of microbial biocatalysts, whole-cell adsorbents, and live vaccines; and the screening of modified or novel proteins. Thus, since cell-surface display is a promising approach in protein engineering, by using the cell-surface engineering system in S. cerevisiae, we tried to express the 380R antigen from red sea bream iridovirus (RSIV), which is one of famous viral diseases in the cultured marine fish. Methods: We have cloned the 380R antigen from RSIV and purified the recombinant 380R from Escherichia coli. 200ul of the purified recombinant 380R antigen (approx. 0.8mg/ml) was injected and the antiserum was collected from two rabbits. Western-blot analysis revealed that anti-380R antigen antibody immunoreacted with the recombinant proteins in E. coli. Next, we tried to detect the expression of the recombinant protein on the outside of the yeast cells by using the antibody. Results: The recombinant 380R antigen on the yeast cells was detected by immunofluorescence labeling methods. Finally, we tried to use whether the display system of arming yeast could be applied for oral vaccination in RSIV. RSIV infection test towards Oplegnathus fasciatus clearly indicated that the arming yeast carrying the recombinant 380R antigen immediately decreased mortality rate. Conclusions: Further work is needed to analyze the immunological system in fish and to show whether the arming yeast carrying the candidate for target antigens from RSIV and how oral vaccination works in the red sea bream. Therefore, we have used the zebrafish, an ideal model for developmental research that has now emerged as a valuable tool for immunological study.	Carbapenem –resistant Bacteroides isolated in Japan TANAKA K, GOTO T, WATANABE K Division of Anaerobe Research, Life Science Research Center, Gifu University, Gifu, Japan Background: Bacteroides fragilis group are one of the most clinically important group among anaerobic bacteria. Related infections are usually mixed infection with several anaerobic and aerobic bacteria. Most of the isolates in B.fragilis group produce ß-lactamase with a variety spectrum of substrate specificity and it may affect activity of the ß-lactams used. Besides ß-lactam resistant, B.fragilis group shows resistance to wide range of antimicrobial agents. On the other hand, initial chemotherapy for anaerobic infection is necessarily empiric because it takes time to define isolated anaerobes and their susceptibility. Because of the wide range of resistance in B.fragilis group, useful agents for empiric therapy are restricted. Carbapenem(Carb)s are useful and important agents for anaerobic infection. We studied on Carb resistant strains in B.fragilis group. Methods: Susceptibility of isolates was determined by agar dilution methods. Production of metallo-ß-lactamase (MBL) in Carb resistant strains was detected by enzyme activity or sodium mercaptacetic acid (SMA; Eiken, Tokyo, Japan) disk method. Presence of cfiA gene and IS were detected by PCR. Results: In ca.1200 strains isolated from 1987 to 1994, we detected 38 resistant strains (B.fragilis 23, non-B.fragilis 15). MBL production was detected in only 9 strains of B.fragilis. MICs of imipenem in MBL (+) strains were higher (≥ 64 µg/mL) than that of MBL (-) resistant strains. MBL (+) strains were resistant to other ß-lactams including cephamaycins and ß-lactamase inhibitor(BLI)/ß-lactam, reflecting substrate specificity of MBL. Most of MBL (-) strains (especially in non-B.fragilis) were rather resistant to other ß-lactams including BLI/ ß-lactam and cephamycins. Resistant rate to clindamycin in those strains were ca 80%, although the rate in whole B.fragiis group isolated in the same period was ca.30%. Among 120 strains isolated after 2000, three MBL (+) B.fragilis and 7 MBL (-) carbapenem less-susceptible strains (MICs of carbapenems; 4~16 µg/mL) were detected. Conclusions: 1) MBL (+) strains showed higher Carb MIC than that of MBL (-) Carb resistant. 2) MBL (-) type may isolate more often and distribute more widely in B.fragilis group than MBL (+) type. 3) MBL (-) type showed resistant to wide-range of antimicrobial agents. 4) Not only MBL (+) type but also MBL (-) type strains should be monitored.
Xylitol Resistance of Streptococcus mutans Appears To Be An Unexpected Benefit to The Human or Rodent Host TANZER JM^1, THOMPSON AS^1, WEN ZT^2 ^1Univ. Connecticut, Farmington, CT and ^2Louisiana State Univ, New Orleans, LA, USA. Background: S. mutans is the prime cariogenic bacterium. Xylitol, a sugar substitute, inhibits its glycolytic metabolism, transmission from mother to child, and induction/severity of caries. However, xylitol-resistant mutants arise in the mouths of habitual consumers. There are no genotypic data on these mutants and no reports on their cariogenicity (virulence). S. mutans mutants have been constructed that are defective in fructose transport and one of these is also defective in xylitol transport. The mutant of one fructose PTS system that can transport xylitol (fruI^-) is notably resistant to growth and glycolytic inhibition by xylitol, unlike either its wild type (WT) progenitor, other fructose transport mutants, or reference mutans streptococcal strains. Methods: We characterized the virulences of WT and engineered fructose transport-defective strains, one of which also fails to transport xylitol, using rats initially free of indigenous mutans streptococci. The rats ate a high sucrose diet 2000 supporting maximal cariogenicity. S. mutans UA159, its isogenic double crossover deletion mutants of fructose PTS (fruI^-), (fruCD^-), a double fructose PTS mutant (fruI^-/fruCD^-), and a sucrose phosphorylase mutant (gtfA^-) were simultaneously studied in TAN:SPFOM(OMASF)BR rats. Some rats were un-inoculated and some were inoculated with reference S. mutans strain 10449S. Results: In two separate experiments, all S. mutans strains heavily colonized the rats, however, the recoveries of the fruI^- mutant from sonicates of the rats’ teeth post mortem were decreased, by comparison with other mutants and its WT. Mutants defective in fruI^-, fruCD^-, and fruI^-/fruCD^- partially lost cariogenicity on enamel. But the fruI^- mutant especially lost ability to induce decay deep into the teeth. The control gtfA^- mutant did not lose virulence. Conclusions: Fructose transport via the PEP-dependent fructose PTS of S. mutans UA159 contributes to virulence in sucrose-fed rats, but there are site-specific mutation effects on the ability of this cell to colonize the teeth. An engineered fructose PTS mutant (fruI^-) that fails to transport xylitol, is resistant to growth and glycolysis inhibition by xylitol, and loses some of its ability to colonize the teeth and to cause deep lesions. These results have been reported in extenso by Tanzer et al, J Dent Res 85,369-73,2006. The results suggest that emergence of xylitol resistance among S. mutans strains colonizing habitual xylitol users may, in fact, be of benefit to the host. Supported by grants UCHC 4-04020 and NIH DE-12236.	Fasudil: the New ''Magic Bullet'' to Thwart Malaria Mortality? TAOUFIQ Z^1, 2, MAZIER D^1, 2, 3 ^1 : INSERM U511, Laboratoire d’Immunobiologie Cellulaire et Moléculaire des Infections Parasitaires, Paris, France ^2 : Université Pierre et Marie Curie-Paris6, UMR S511, Paris 75013, France ^3 : Groupe hospitalier Pitié-Salpêtrière, Service de Parasitologie-Mycologie, Paris, France Malaria, with more than 2 million deaths every year, remains a major world public health problem in terms of mortality. The onset of acute clinical manifestations of severe malaria, such as cerebral malaria (CM), occur rapidly and unpredictably, and may lead to coma and death within hours if left untreated. So far, severe malaria emergency treatments have mainly consisted of intravenous administration of antiplasmodial drugs, such as quinine or artemisinin. However, despite the high efficiency of those molecules in clearing the blood of parasites, 15-20% of lethality is still observed and a non-negligible proportion of survivors have malaria sequelae. Thus, current treatments that mainly rely on a ‘direct antiparasitic strategy’, are not sufficient and adjunctive anti-disease approaches are urgently needed. Life-threatening malaria is overwhelmingly a result of the protozoan parasite Plasmodium falciparum, which infects red blood cells and confers them the unique ability to adhere massively on endothelial cells. There is increasing evidence that endothelium plays a key role in the pathogenesis of those malaria cases. Amongst these data is the demonstration that adhesion of Plasmodium falciparum parasitized red blood cells triggers inflammatory, oxidative stress and apoptotic cascades within endothelial cells of vital organs (e.g. brain, lungs, kidneys). We have exploited co-culture models of infected erythrocyte/endothelium interactions. We demonstrated that P. falciparum adhesion activates the Rho-kinase signaling pathway, a response highly involved in various human vascular diseases. A Rho-kinase inhibitor, Fasudil (HA-1077), a drug already in clinical use for the treatment of human cerebral ischemic stroke, was tested for its capacity to ameliorate endothelial functions. When added concomitantly with parasites, Fasudil was shown to abolish both NF-kappaB activation and endothelial apoptosis. More importantly, we showed that Fasudil could restored endothelial barrier integrity after endothelial cells were exposed to to P. falciparum adhesion. This indicates that Fasudil may target efficiently endothelial pathogenic mechanisms even after severe malaria onset. Therefore Fasudil and and Rho-kinase inhibition-based strategies opens hopeful therapeutic venues in fatal malaria management.
The clinical role of the statins in surgical neurosciences TAPIA-PÉREZ JH ^1,2, 3,SÁNCHEZ-AGUILAR M ^1, TORRES-CORZO J G ^2, GORDILLO-MOSCOSO A ^1, MANDEVILLE P ^1, CHALITA-WILLIAMS J ^2, FIRSCHING R ^3 ^1 Universidad Autónoma de San Luis Potosí. San Luis Potosí, Mexico ^2 Hospital Central “Dr. Ignacio Morones Prieto”. San Luis Potosí, México ^3 Universitätsklinikum Magdeburg. Magdeburg, Germany Background: Statins are structural analogs of hydroxy-methylglutaryl Co A reductase, a restrictive enzyme in the cholesterol pathway.They are used for treatment of dyslipidemias and cardiovascular prevention. Animal studies show broad evidence of a neuroprotector effect. In humans is growing evidence of profit after subarachnoidal hemorrhage. After intracerebral hemorrhage (ICH) and traumatic brain injury (TBI) in humans, the evidence is scarce of yet. Methods: We conducted a blind randomized clinical trial of patients with TBI, Glasgow 913, and intracranial lesions by CT scan. Patients with previous disability, hepatopathy or myopathy, multisystemic trauma, prior treatment in other clinic, surgical lesion or in brainstem were excluded. Each patient was allocated to rosuvastatin (RVS) or placebo 10 days. Main outcome, amnesia and disorientation time were evaluated. In another trial a prospective series of patients with ICH was treated with RVS and compared with a retrospective control group (relation 1:3). Exclusion criteria were history of neoplasm or TBI 4 weeks previous, non-hypertensive causes of haemorrhage, brainstem hemorrhage, steroid use, cranial surgery, initial hydrocephalus, and NIHSS (National Institut Health Stroke Skale) >30. Mortality and functionality at the time of discharge from the hospital were evaluated. Results: The TBI study included 8 patients with RVS and 13 controls with similar basal characteristics. The use of RVS showed a reduction of amnesia time with an hazard ration of 53.76 (95% confidence intervals [CI], 1.581824.64). The ICH trial analyzed 18 patients with RVS and 57 controls with similar basal characteristics. The mortality rate in hospital was 1 (5.6%) with RVS and 9 (15.8%) in the control group. The odds ratios for a NIHSS >15 at discharge was 0.04 (95% CI 0.003-0.93). Conclusions: Statins may reduce amnesia time after TBI and improve outcome after ICH. Immunomodulation seems to be involved. Further trials are needed in order to confirm this positive relation.	Bait bullets controlling Bacillus anthracis: from spore inhibition to toxins neutralization and prevention of spore- and/or toxin-induced cell death TARASENKO O^1, SCOTT A ^1, RODRIGUEZ N ^1, ALUSTA P^1, SODERBERG L^2 ^1University of Arkansas at Little Rock, ^2University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA The potential use of glycoconjugates (carbohydrate-bearing polymers) is receiving considerable attention for biomedical and pharmaceutical applications. Among cellular targets by glycoconjugates, macrophages are ideal, since they play a central role in inflammation and act as reservoirs for microorganisms that are involved with deadly infectious diseases. In this context, we studied effects of glycoconjugates contributing to the inhibition of Bacillus spores and neutralization of toxins during phagocytosis. The effects of glycoconjugates were studied under the following three conditions, namely a) prior to, b) during, and c) following macrophage contact with B. anthracis recombinant toxins and spores during phagocytosis. Post-phagocytosis studies involved colony forming units, microscopic observation, macrophage viability, cytotoxicity, and Caspases release assays. Glycoconjugates promote inhibition of spores and neutralization of toxins by blocking spore– and/or toxins–induced macrophage cell death, while increasing their activation level. This results in higher phagocytosis rate of spores, toxins neutralization, and macrophage viability. Even after being bound to spores/ toxins on one side, glycoconjugates serve as chemoattractants for macrophages on the other side. Macrophages become more prone to adhere to glycoconjugate-coated spores/toxins, resulting in decreased lactate dehydrogenase and Caspases, in increased nitric oxide production, phagocytosis, and killing of spores and neutralizing toxins. The technique presented in this study may be helpful in finding glycoconjugates with bactericidal /antimicrobial and antitoxic properties. Acknowledgement: National Science Foundation EPSCoR RII Grant “Arkansas AASSET Initiatives” (#EPS-0701890) and the Arkansas Science & Technology Authority; SURF; 2008 American Society for Microbiology URF and UTF and McNair programs; Kathleen Thomsen Hall Charitable Trust Grants
Micronutrient Interactions in Health and Oxidative Stress Conditions AGTE VV, TARWADI KV Agharkar Research Institute, G.G. Agarkar Road, Pune 411004, India Background: The nutrition research during last two decades has evolved the newer dimensions of protective role of micronutrients in diseases related to oxidative stress and their interactions with each other. Methods: Linkages of micronutrients have been extensively studied by our group in foods as well as through in vitro systems and in animal and human models. Results & Conclusions: Riboflavin enhanced the bioavailability and uptake of food zinc and copper and had beneficial effect on iron and zinc utilization in weanling mice. Supplementation of nicotinic acid enhanced bioavailability of iron as observed through in vitro, animal and human studies. Apparent absorption of zinc in healthy adults was inhibited by thiamine and ascorbic acid and copper absorption was inhibited by phosphorus, calcium and niacin. In the human ileostomy model, dietary riboflavin had significant positive association while intake of iron and manganese showed weak negative associations with beta-carotene absorption from vegetarian meals. Factors influencing dietary and erythrocyte membrane zinc status in apparently healthy Indians were found to be phytic acid and zinc. Further, in a prospective study on supplementation of greens in healthy adults (40) multiple regression analysis indicated significant association of percent change in plasma zinc with intakes of zinc, riboflavin, iron, ascorbic acid, beta-carotene and copper. In a study on antioxidant potential of commonly consumed fruits and vegetables (96), ascorbic acid levels were associated with inhibition of lipid peroxidation (r = 0.32, p<0.05) and ferrous ion chelating ability (r = 0.43, p<0.01). In a cataract study, plasma TBARS, a measure of oxidative stress, showed a negative and highly significant correlation with lens iron(r=-0.53,p<0.0001) and positive association with lens opacity (r=0.30,p<0.05). Diabetic cataract patients exhibited inferior plasma levels of selenium, zinc, ceruloplasmin, superoxide dismutase and retinol as compared to cataract patients without diabetes. Further, multiple regression analysis indicated association of intakes of iron, beta-carotene, ascorbic acid, polyphenols and inositol hexa phosphate with plasma oxidative stress (p<0.01) and intakes of iron, ascorbic acid and inositol triphosphate with lens oxidative stress (p<0.01). Insulin secretagogue activity was found to be significantly associated with zinc levels and marginally associated with oxygen radical absorbance capacity, copper or manganese levels.	Structure and Antimalarial Activity of Immunomodulator P-MAPA Durán M^1, Marcato PD^1, Costa FTM^2, Durán N^1, Tasic L^1 ^1IQ, UNICAMP, Campinas, Brazil; ^2IB, UNICAMP, Campinas, Brazil. Background: Malaria is one of the world's most common diseases, caused by a four different parasites: Plasmodium vivax, P. falciparum, P. malariae and P. ovale, which is transmitted to humans by a female mosquito's bites. It is a public health problem today in more than 90 countries, inhabited by a total of some 2,400 million people - 40% of the world's population. Worldwide prevalence of the disease is estimated to be in the order of 300-500 million clinical cases while mortality due to malaria is estimated to be up to 3 million deaths each year, majority young children in Africa. The drug resistance has reduced the effectiveness of several commonly used antimalarials, such as chloroquine. Although the artemisinin, a relatively new drug derived from wormwood, has shown promise in treating drug-resistant strains, in some parts of the world, artemisinin drugs are used indiscriminately for self treatment of suspected uncomplicated malaria - so we can expect to see malaria forms resistant to artemisinin soon according to WHO. Methods: This work has as aim the investigations on immunomodulator P-MAPA, aggregated polymer of protein magnesium ammonium phospholinoleate-palmitoleate anhydride, isolated from Aspergillus oryzae, and relates its antimalarial activity. The 3D structure of protein part of P-MAPA and its antimalarial activity, as well as, the activities of P-MAPA´s micro- and nano- crystals in vitro, on Plasmodium falciparum, and in vivo in experimental infection models are our goals. Firstly, we are going to isolate, purify and characterize the protein part of P-MAPA that was shown to be small (16 kDa), arginine rich (35.2 %) and contain tryptophan (1.3 %). Therefore, its primary structure is going to be determined applying mass spectrometry, its secondary structure applying circular dichroism, while it is suggested that 3D structure elucidation can be achieved by fluorescence and nuclear magnetic resonance (NMR) techniques. Results: At the moment, the P-MAPA crystals have been tested and the strong bioactivity against malaria was observed in vitro. A part of our research is dedicated to new P-MAPA formulations and we have obtained the nano-crystals and are optimizing the two nanonization method’s conditions with aim to achieve as uniform as possible nanocrystals. Conclusions: Hopefully, the P-MAPA could be presented as a new antimalarial agent.
CYP1A1, GST Gene Polymorphisms and Risk of Chronic Myeloid Leukaemia TASPINAR M^1, ERDOGAN AYDOS S^1, COMEZ O^1, ELHAN AH^2, KARABULUT HG^3, SUNGUROGLU A^1 ^1Ankara University Faculty of Medicine, Department of Medical Biology, Ankara, Turkey ^2Ankara University Faculty of Medicine, Department of Biostatistics, Ankara, Turkey ^3Ankara University Faculty of Medicine, Department of Medical Genetics, Ankara, Turkey Background: Associations between polymorphisms for genes encoding enzymes involved in biotransformation of xenobiotics and susceptibility to several cancers have been shown in several studies. The aim of the present study is to investigate the influence of cytochromes P450 (CYP450) 1A1*2C and Glutathione S-transferases (GSTs) (T1 and M1) gene polymorphisms in susceptibility to chronic myeloid leukaemia (CML). Methods: The frequency of CYP1A1 Ile/Val alleles and of GSTT1 and GSTM1 homozygous deletions was examined in 107 patients with CML and 132 healthy controls by PCR and/or PCRRFLP methods using blood samples. Results: The frequency of CYP1A1 Val allele was found to be 19.2% in CML patients and 4.4% for controls, indicating that persons carrying this allele had an increased risk of CML (OR = 5.10, 95% CI: 2.60–9.97)(Table 1). The frequency of individuals carrying the GSTT1 null genotype was higher among CML patients (40.2%) compared to controls (19.2%) (OR = 2.82, 95% CI: 1.58–5.05; p <0.001). Therefore, GSTT1 present genotype may be a protective factor for CML. Although GSTM1 null genotype frequency was slightly higher in the patient group (44.9%) than in the controls (42.3%), this difference was not statistically significant (OR = 1.11, 95% CI: 0.66–1.86; p = 0.693). Individuals with GSTM1 null genotypes without the T allele have a 5.981 higher risk for CML than those who have the T allele (Table 2).