Ehrlich II –2nd World Conference on Magic Bullets

Intraluminal Cefotaxime – Heparin Lock’ Placement in the Primary Prevention of Hemodialysis Catheter-Related bloodstream Infections among the Elderly and Diabetics
SAXENA AK¹, PANHOTRA BR², AL-HAFIZ A^2, SUNDARAM DS², NAGUIB M², ABU OUN BA^2
1Al-Rahba Hospital- Johns Hopkins Medicine International, Abu Dhabi, United Arab Emirates (UAE), ² King Fahad Hospital and Tertiary Care Center, King Faisal University, Al-Hasa, Eastern Province, Saudi Arabia
Background: Tunnelled-cuffed catheters (TCCs) are often used in the elderly and diabetic end stage renal disease (ESRD) patients, to carryout hemodialysis (HD). Cmplications like infection and thrombosis reduce the life-span of TCCs. Aim: To investigate the efficacy of cefotaxime - heparin ‘lock’ in primary prevention of thrombotic and infectious complications and enhancement of TCC’s survival in the elderly and diabetic ESRD patients.

Methods: Prospective, randomized double-blind clinical trial I- TCCs (n=119, placed among 113 elderly patients requiring long-term HD were randomized to either group-I having TCCs (n=59, placed in 58 patients) ‘locked’ with cefotaxime (10mg/mL) and heparin (5000 U/mL) or group-II with TCCs (n=60, placed in 55 patients) having catheter-restricted filling of heparin (5000 U/ ml) alone.

Prospective, randomized double-blind clinical trial II - TCCs (n=109, placed among 96 diabetic patients requiring long-term HD were randomized to either group-I having TCCs (n=51, placed in 49 patients) ‘locked’ with cefotaxime (10mg/mL) and heparin (5000 U/mL) or group-II with TCCs (n=58, placed in 47 patients) having catheter-restricted filling of heparin (5000 U/ ml) alone. The incidence of catheter- thrombosis, CRBSI, percent catheter survival and the patient mortality - were statistically compared using Kaplan-Meier survival analysis between the two groups in each trial.

Results: Trial I - Elderly patients with intraluminal cefotaxime / heparin lock on cumulative survival analysis test showed higher thrombosis–free TCC survival (84.7% vs. 63.3%, P=0.021), infection-free survival (68.7% vs. 31.3 %, P <0.001) and infection and thrombosis-free survival (65.0% vs. 35.0 %, P=0.006) at 365 days in group I compared with group II. Trial II- Diabetic patients with intraluminal cefotaxime / heparin lock, on survival analysis showed a superior thrombosis–free (86.3 vs. 63.8 %, P=0.023, log rank), infection-free (72.9 vs. 27.1 %, P = 0.004, log rank) and thrombosis and infection -free TCC survival (78.4 vs. 37.9 %, P=0.001, log rank) at 365 days besides having significantly lower incidence of CRBSI (3.68 vs. 1.56 episodes /1000 catheter-days, P < 0.0001) and CRBSI-related mortality (23.4 vs. 9.8 %, P= 0.015), compared with heparin-alone group.

Conclusions: Intraluminal cefotaxime-heparin ‘locks’ safely and effectively enhance the life-span of TCCs by lowering the incidence of thrombotic and infectious complications, among the elderly and diabetic ESRD patients.

AntiJEd: A possible therapeutic and immunomodulatory drug for Japanese encephalitis SAXENA SK^1 ^1Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500007 (AP), INDIA Background: AntiJEd, a low molecular weight dithiol, has been described as an immunomodulator and modifier of diverse biological actions in human and animal models and has also been shown to be effective in several diseased conditions. Therefore we studied the therapeutic aspect of AntiJEd in providing inhibition of Japanese encephalitis virus (JEV) infection. Methods: Groups of mice were inoculated with JEV (10^2 LD50; i.c.), followed by administration of various doses of AntiJEd (10-100 mol/kg, i.p.) or placebo, daily or alternate day. Controls consist of mice that either received normal mouse brain suspension (= mock-inoculation) or were treated with various doses of AntiJEd (10-100 mol/kg) alone. Mice were observed for survival for 21 days and Average Survival Time (AST) was calculated. The data were analyzed using Student’s t-test and P value (two tailed) of <0.05 was considered significant. Results: AntiJEd tested at various doses (10-100 mol/kg) revealed that administration at low concentration (10 mol/kg; i.p.) on alternate days prolonged the average survival time (AST) of mice infected with lethal dose of JEV (10^2 LD50, i.c.) and delayed progression of the disease. The low dose also provided >80% survival in sub-clinical (10^5 LD50, i.c.) JEV infection. Administration of AntiJEd to JEV-infected mice enhanced the inducible nitric oxide synthase (iNOS) activity in brain and level of serum tumor necrosis factor- (TNF-). We have recently demonstrated the production of nitric oxide (NO) via induction of iNOS activity is meditated by circulating macrophage-derived factor (MDF), which may be responsible for the delayed progression of the disease. AntiJEd mediated inhibition of JEV is believed to involve the augmentation of protective role of MDF as evidenced by the observation that pretreatment with anti-MDF antibody significantly decreased the AST of mice and together with the inhibition of iNOS activity. Interestingly, AntiJEd alone did not stimulate iNOS and TNF- in mock-infected normal mice. Conclusions: Collectively our data suggest that AntiJEd may be a possible anti-JEV therapeutic agent as it provides inhibition of JEV infection probably by inducing the iNOS activity and TNF- and delays the progression of disease, though more work is needed to explore its role in JEV infection.
Breast and Prostate Tumor Cell Destruction by Genetically Altered Salmonella with Preferential Targeting of Mitochondria SCHATTEN H^1, ZHONG Z^1, KAZMIERCZAK RA^2, DINO A^2, KHREIS R^2, EISENSTARK A^2 ^1University of Missouri, Columbia, MO 65211, USA ^2Cancer Research Center, Columbia, MO, 65201, USA Background: Genetically modified Salmonella typhimurium strains present an attractive novel treatment for cancer, as Salmonella preferentially replicate within tumors and destroy cancer cells without causing septic shock that is typically associated with wild-type S. typhimurium infections. Different Salmonella strains have been used with varying results and the mechanisms by which Salmonella exploit their host cells may vary for different strains. Strain optimization is therefore important. Here we present data that show S. typhimurium strain VNP20009 and CRC1674 destroying human breast (MCF-7) and prostate (PC-3M) cancer cells by preferentially targeting mitochondria. Methods: S. typhimurium-infected PC-3M and MCF-7 cancer cells were analyzed with fluorescence and immunofluorescence microscopy at 20min, 4hrs, 8hrs, and 24hrs after inoculation and fixed with 3.7% paraformaldehyde. Rhodamine-phalloidin was employed to stain microfilaments, FITC-anti-tubulin antibody to stain microtubules, and DAPI to stain DNA. Transmission electron microscopy (TEM) was performed at the same time points after fixation in 2.5% glutaraldehyde in 0.1M HEPES containing 0.2% tannic acid. YFP-mitochondria transfected mouse 3T3 cells were used to study the effects of Salmonella infestation on mitochondria in live cells. Results: Our results show incorporation of VNP20009 and CRC1674 strains into Salmonella-containing vacuoles (SCVs) formed within host cells and gradual destruction of mitochondria within PC-3M and MCF-7 cells with complete loss of cristae at 24 hrs of inoculation. YFP-mitochondria transfected mouse 3T3 cells showed decreased mitochondrial fluorescence intensity after inoculation. The nucleus did not appear affected by either VNP20009 or CRC1674 strains within 24hrs. Conclusions: Our data show that genetically modified S. typhimurium (VNP20009 and CRC1674) destroy PC-3M prostate and MCF-7 breast cancer cells with obvious destruction of mitochondria while the nucleus does not appear affected.	Depending on PKC-theta Expression, the Novel PKC Activator PEP005 can Either Increase or Decrease Apoptosis of Hemopoetic cells LEE WY, HAMPSON P, SALMON M, LORD JM, SCHEEL-TOELLNERD School of Immunity and Infection, College of Medical and Dental Sciences, University of Birmingham, Birmingham B15 2TT, UK Background: Broad range activators of PKC isoforms induce apoptosis in many cancer cell lines. In T cells, however, activation of PKC can be a survival signal and a mitogen. We have been investigating the underlying mechanism explaining the difference between T cells and other hemopoetic cell lines in their response to PKC activation. Methods: Activated CD8^+T cells rapidly enter apoptosis when deprived from survival factors such as IL-2 or IL-15. We cultured human resting and activated CD8+ T cells in the presence and absence of IL-2 and PEP005 and investigated the effects of PEP005 on cell-survival and proliferation. Furthermore we studied the effect of PEP005 on activation and expression of factors involved in regulation of apoptosis such as NFkB, PKCθ as well as Bcl-2 family members BIM, Bcl-xl, Bcl-2 and Mcl-1. We compared the response of CD8+ activated T cells to PEP005 to that of promyeloid cell line HL-60, which does not express PKCθ. Results: We found that PEP005, a novel PKC activator, can replace cytokines as a survival signal. In freshly isolated naïve or central memory T cells, PEP005 inhibits apoptosis and induces proliferation. We demonstrate that the survival effect depends on activation of PKCθ. Expression of this PKC isoform is largely restricted to T cells and myocytes. Our findings suggest that incubation of T cells with PEP005 inhibits apoptosis through activation of NFkB downstream of PKCθ. It also involved the downregulation of the proapoptotic Bcl-2 family member BIM and upregulation of its anti-apoptotic counterparts Mcl-1 and Bcl-xL. Conclusions: We conclude that PKCθ expression determines if PKC activation increases or decreases the rate of apoptosis in haemopoetic cells. Authors’ disclosure statement (not counting towards the character count): This work was supported by the University of Birmingham, the Arthritis Research Campaign, the EU (FP6) and Peplin inc.
Rimonabant, First CB1 Receptor Antagonist, a Potential Magic Bullet to Reduce Cardiometabolic Risk in Patients with Abdominal Obesity SCHEEN AJ University of Liege, Liege, Belgium Background: Abdominal obesity is associated with type 2 diabetes and dyslipidaemia, leading to an increased risk of cardiovascular disease. The endocannabinoid system is overactivated in presence of abdominal obesity, which contributes to increased cardiometabolic risk. We describe the clinical results obtained with rimonabant, the first selective cannabinoid type 1 (CB1) receptor antagonist, in overweight and obese patients. Methods: Rimonabant (5 and 20 mg) was first evaluated in the RIO programme: RIO-Europe, RIO-North America and RIO-Lipids in non-diabetic patients, RIO-Diabetes in patients with type 2 diabetes on monotherapy with sulfonylurea or metformin. Rimonabant 20 mg was further evaluated in type 2 diabetic patients treated with diet alone (SERENADE) or with insulin (ARPEGGIO), in abdominally obese patients (ADAGIO) and in patients with coronary atherosclerosis (STRADIVARIUS). Results: Rimonabant 20 mg, compared to placebo, consistently induced greater reductions in body weight, waist circumference, blood pressure, triglycerides, metabolic syndrome, insulin resistance and inflammation markers, and greater increases in HDL cholesterol and adiponectin concentrations. In addition, in the diabetic population, rimonabant 20 mg was associated with a greater reduction in glycated haemoglobin (HbA1c) levels whatever the baseline therapy. Almost half of the metabolic effects of rimonabant were beyond weight loss. Favourable metabolic effects observed after 1 year persisted after 2 years. Rimonabant also reduced intra-abdominal and liver fat (ADAGIO). Rimonabant 20 mg for 18 months was associated with a non significant reduction in percent coronary atheroma volume, but a significant reduction in total atheroma volume (STRADIVARIUS). Adverse events more frequently reported with rimonabant were gastrointestinal, neurologic and psychiatric in nature, contraindicating the use of rimonabant in case of depression. Conclusion: In both non-diabetic and diabetic overweight/obese patients, rimonabant 20 mg/day remarkably improved the cardiometabolic profile via pleiotropic central and peripheral effects. Whether CB1 receptor antagonism might be considered as a “magic bullet” capable of reducing the incidence of cardiovascular events in this high risk population is currently evaluated in the large CRESCENDO outcome prospective trial.	Sodium and Diffusion MRI as Biomarkers of Initial Tumor Response to Therapy in Rodents SCHEPKIN VD^1, LEVENSON CW^2, BREY WW^1, FIGUEIROA SM^2, GOR’KOV PL^1, CHENEVERT TL^3, REHEMTULLA A^3, ROSS BD^3 ^1National High Magnetic Field Laboratory/FSU, Tallahassee, FL, USA; ^2Florida State University, Tallahassee, FL, USA, ^3University of Michigan Medical School, Ann Arbor, MI, USA Background: The finding that, during therapy, tumors’ sodium content correlates with corresponding alterations of water diffusion is attracting keen attention in the efforts to understand and further develop surrogate MRI biomarkers for tumor therapy. It is especially noteworthy that changes in both sodium and diffusion take place in the first days after therapy. Both types of imaging are able to detect a heterogeneity of tumor response and have potential to predict future individual tumor responses. Methods: Rat 9L gliosarcoma cells were implanted intra-cranially or subcutaneously in male Fisher 344 rats (weight ~ 120 g). In ~14 days after tumor implantation, animals were subjected to a single ip BCNU chemotherapy with two different doses (13 & 26 mg/kg). Each 2-3 days, tumor growth, together with 3D sodium MRI and diffusion map were detected following the treatments. The previous experiments were performed using 9.4T MRI scanner and the latest results were acquired using 21T magnet. Results: The overview of sodium/diffusion MRI studies of rodent glioma and 9L subcutaneous tumors will be discussed, including the latest data acquired using the ultra high magnetic field of 21.1T available at NHMFL. Representative MR images of sodium and diffusion map acquired before and seven days after BCNU therapy (26 mg/kg) demonstrate dramatic increases in tumor sodium and ADC. At 21T, a unique sodium resolution of 1µL was attained (Fig. 1).  A B C D Fig. 1. In vivo sodium and diffusion map MRI of rat glioma before (A, B) and 7 days after BCNU chemotherapy (C, D), respectively, for the same animal and slice position. Conclusions: Sodium MRI and proton diffusion exhibit early and strong correlated responses in rodent models during tumor treatments equally in brain and subcutaneous tumors. Both methods revealed a decreased response if therapy doses were decreased or if tumor acquired an additional resistance. The ultra high field experiments at 21T allowed a record high resolution for sodium and demonstrate a unique sensitivity of sodium MRI to tumor therapy. These two imaging modalities can be valuable biomarkers for individual evaluation of in vivo therapeutic cellular changes and for developing new drugs for tumor therapy.
Targeting of nitric oxide-donors to the phagosomal compartment of macrophages and the release of NO mediate killing of intracellular Leishmania parasites within the host cell SCHLEICHER U^1, VALDEZ CA^3, MAMMATO R^2, EL-GAYAR S^1, KEEFER LK^3, BOGDAN C^1 ^1Institute of Clinical Microbiology, Immunology and Hygiene, University Clinic of Erlangen, Germany; ^2 Institute of Medical Microbiology and Hygiene, University Clinic of Freiburg, Germany; ^3 Laboratory of Comparative Carcinogenesis, National Cancer Institute at Frederick, U.S.A. Leishmania are intracellular protozoan parasites. After their transmission to mammalian organisms they mainly reside in the phagosomes of macrophages, which either serve as host cells or - after activation - as antileishmanial effector cells. Efficient control of Leishmania in these cells requires the enzyme inducible nitric oxide synthase (iNOS), which is expressed following activation of infected macrophages by interferon (IFN)-gamma and/or tumor necrosis factor (TNF) and metabolizes L-arginine into citrulline and nitric oxide (NO). NO is known as reactive radical with potential toxic effects. Here, we present data that clearly support the idea that NO itself or reactive substances generated by NO represents the metabolite responsible for the killing of the parasites. First, the addition of a NO-scavenger to Leishmania (L.) major-infected macrophages stimulated by interferon (IFN)-gamma and TNF led to a significant increase of parasite proliferation. Second, transwell and co-culture experiments with infected iNOS knockout (ko) macrophages and IFN-gamma/LPS-stimulated wildtype (wt) macrophages revealed that the destruction of Leishmania in the iNOS ko cells was dependent on the close proximity of iNOS wt macrophages, indicating that unstable and reactive NO mediated the kill. And third, we were able to generate NO-donors targeted to the phagosomal compartment by endocytosis that finally induce killing of the intracellular L. major parasites within the vacuoles without toxic effects on the host cells. These prodrugs of NO are characterized as O^2-glycosylated diazeniumdiolates consisting of mono- or disaccharides (e.g. N-acetyglucosamine) that are recognized and transported by the mannose receptor (MR) of macrophages and glycosylated diazeniumdiolate ions that, in their free form, spontaneously release molecular NO at physiological pH. Depending on the attached sugar the O^2-glycosylated diazeniumdiolates were more or less stable under physiologic conditions (pH7.4), but macrophages were shown to be capable of metabolizing the compounds resulting in the release of NO. This macrophage-induced accelerated hydrolysis of the glycosides was sufficient to exert potent antiparasitic effects indicating the potential utility of O^2-glycosylated diazeniumdiolates for delivering therapeutic levels of NO to intracellular pathogens resident in infected macrophages. Together, these data led us to conclude that iNOS-dependent killing of intracellular Leishmania parasites depends on direct toxic effects of NO and pharmacological supplementation of the macrophages’ NO reserves might restore the immune system’s capacity to contain and eliminate these pathogens.	A Role for Macroautophagy in Antiestrogen Resistance SCHOENLEIN PV^1 , BARRETT JT^2, SAMUUELS T^1, BIEBERICH E^1 ^1Medical College of Georgia, Augusta, Ga., ^2Watson Clinic, Lakeland, Fl Background: Adjuvant antiestrogen therapy of estrogen receptor positive breast cancer is effective for approximately 70% of patients, while 30% of patients either do not respond or develop resistance to the antiestrogen being administered. Typically, antiestrogen treatment of breast cancer cells leads to a cytostatic response, with only a small percentage of cells actually dying due to the cytotoxic action(s) of the antiestrogen. We hypothesized that breast cancer cells surviving antiestrogen therapy undergo the catabolic process of macroautophagy and that macroautophagy facilitates the development of antiestrogen resistance. Methods: To test our hypothesis we have utilized several breast cancer models. Molecular, biochemical, histological and imaging methods were used to identify the induction of macroautophagy and the autophagosome-the functional organelle of macroautophagy, breast cancer cell proliferation, and death in response to hormonal therapy. Autophagosome function was monitored by analyzing the levels of two proteins key to the function of the autophagosome- the lipidated form of light chain 3, designated LC3 II, and p62. In addition, long-lived protein turnover was assayed as a measure of autophagosome function. Results: We have determined that antiestrogen therapy induces macroautophagy and that blockade of autophagosome function can convert the cytostatic action of antiestrogen treatment to a cytotoxic (death promoting) action. Small molecule inhibitors of autophagosome function as well as siRNA knockdown of macroautophagy-inducing genes were shown to induce apoptosis in breast cancer cells. Importantly, we were able to demonstrate that macroautophagy facilitates the development of antiestrogen resistance by utilizing a step wise drug selection protocol in which breast cancer cells were exposed to incremental doses of 4-hydroxytamoxifen. Conclusions: Macroautophagy is now being appreciated as a major survival mechanism in cancer cells under a variety of stress conditions. Our studies now uniquely demonstrate that macroautophagy facilitates the development of antiestrogen resistance. Our current studies are aimed at identifying small molecule inhibitors of autophagosome function, such as chloroquine, that may have potential use as an adjuvant to antiestrogen therapy in clinical trials.
High Throughput Screening for In Vitro Toxicity Screening: A Gradual Acceptance of New Test Methods SCHOONEN WGEJ, WESTERINK WMA, HORBACH GJ Pharmacology, Schering-Plough, Oss, The Netherlands Background: Early in vitro toxicity screening within the pharmaceutical industry has several advantages. A large number of compounds can be tested with small amounts of compound at a low cost price. Moreover deselection can take place at an early stage. The focus in this review is on genotoxicity, cytotoxicity, nuclear receptor activation as well as phase I and II enzyme competition assays. Results: For genotoxicity, enzyme mutation and SOS repair are studied with Salmonella in Ames II (Xenometrix) or Vitotox^TM (Thermo) assays. Chromosomal aberrations are studied with a RAD54 promoter activation in yeast with Green Fluorescence Protein (GFP) Greenscreen (Gentronix) or ß-galactosidase Radarscreen (ReMynd) as read-outs. The Vitotox^TM assay is a predictive substitute for the full Ames test, while the RadarScreen assay is one for chromosomal aberration and micronuclei tests. For cytotoxicity, human liver HepG2 cells are used to determine glutathione depletion (Monochlorobimane), calcein uptake (Calcein-AM), mitochondrial failure (Alamar Blue, O2 consumption Luxcell), DNA proliferation (Hoechst 33342), and radical oxygen species activation (Dichlorofluorescein) or NRF2 Responsive Element mediated luciferase activation. These assays predict cytotoxicity towards a confidence level of 75%. Nuclear activation can be induced for the Arylhydrocarbon Receptor (AhR), Pregnane X receptor (PXR), Constitutive Androstane Receptor (CAR), Retinoic Acid Receptor α and ß (RARα and ß), Thyroid Receptor α (TRα), Liver X Receptor α (LXRα) or Farnesoid X receptor (FXR) in human liver HepG2 and/or H4IIE cells. This can be measured by Q-PCR or by the use of specific substrates for the individual cytochrome P450 enzymes. Competition assays for Cytochrome P450 and UDP-glucuronosyltransferase are available and show good predictivity for human enzymes.