Results: All subjects demonstrated large numbers of CFU's on media without triclosan. By contrast, supragingival plaque bacteria were substantially inhibited by TCN (p<0.001). Inhibition with 7.5 and 25 µg/ml TCN ranged from 97.3-100% and 99.6-100%, respectively. For each TCN concentration, ANOVA was used to compare the percent susceptible bacteria between each study period. Over the 17-year duration, no differences in microbial susceptibilities were observed between each period for either concentration of TCN (p>0.28). Regression analyses for each TCN concentration indicated no alterations or trends in efficacy (p values of 0.208 and 0.71 for the 7.5 and 25 µg/ml TCN, respectively) over time.
Conclusions: The data demonstrates the significant efficacy of triclosan on supragingival plaque bacteria over a 17-year period with no change in antimicrobial susceptibility. Since study participants were not selected based on the use of any oral hygiene formulation, the results indicate that triclosan is efficacious on supragingival dental plaque from a variety of subjects. These results demonstrate that triclosan represents a safe and effective agent for dental plaque bacteria.
Recombinant Gas Vesicles: A Novel Display/Delivery System for Peptide Antigens (SIV)
SREMAC M1, STUART ES1
1Univ. Massachusetts, Amherst, USA
Background: The goal of this research was to: 1) Extend demonstration of evoked immunologic memory in the absence of an exogenous adjuvant to diverse gene sequences by testing recombinant gas vesicles (r-GV) displaying peptides encoded by different SIV genes (tat, rev or nef). 2) Evaluate the resulting r-GV for specific antigenic and immunogenic capabilities.
Methods: The generation of transformed Vac- (minus), halobacterial mutant strain SD109 lacking the GV gene cluster, used a series of recombinant plasmids. Aliquots of isolated wt-GV and r-GV were electrophoresed using SDS-PAGE gels and the SIV peptides expressed as part of the r-GvpC protein identified with monkey anti-SIV/SHIV sera using Western blots. Mouse anti-SIV sera raised against SIV-DNA insert sequences were generated by immunizing BALB/c mice. To test immunogenecity four month-old mice (3 mice per each gene segment) were injected initially using low dose immunization (1-3μg total protein), boosters then used 50 μg total proteins per mouse. Sera were collected at 2 and 4 weeks after primary immunization and 4 weeks following the first booster. After the 2nd booster, sera were collected at 2, 4, 8, 12 and 17 weeks. Mice were re-immunized and sera collected 10 days later. The animals retained for a total of 43 weeks post-re-immunization. All sera were subsequently adsorbed and assayed by ELISA.
Results: Each of the three different exogenous DNA sequences had been stably retained in the recombinant gvpC gene. The transformations with each of the selected recombinant plasmids were successful and had conferred a functional Vac+ status. Antisera from mice immunized with r-GV and also monkey anti-SHIV sera each recognize the chimeric r-GvpC protein.
Conclusions: 1) Different recombinant gvpC-SIV genes will support the biosynthesis of chimeric GvpC fusion proteins and generate functional organelles. 2) Monkey antibody elicited by in vivo infection with SIV recognizes these expressed SIV sequences as SIV peptides. 3) Test of antiserum elicited by immunizing mice with r-GV demonstrated notable and long term antibody titers. Continued presence of elevated antigen specific antibody is a feature critical to immunization based protection. The observed level of humoral responses and the maintenance of elevated responses to encoded peptides are consistent with the suggestion that in vivo there may be natural and slow release of epitopes over time.
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Plumbagin: A Candidate for Targeted Anticancer Therapy
SRINIVAS GOPALA1, ASOKE BANERJI2, MADHAVAN RADHAKRISHNA PILLAI3, PRIYA SRINIVAS3
1Sree Chitra Tirunal Institute for Medical Sciences &Technology, Thiruvananthapuram, India; 2Centre for Biotechnology, Kollam, India; 3Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram, India
Background: There are emerging reports on plumbagin documenting evidence of antitumor function in experimental systems. It mainly operates through modulation of redox potential mediated cellular function, particularly affecting the DNA repair pathway. Earlier studies show that plumbagin known to generate ROS and has been found to inhibit the activity of topoisomerase II (Topo II) through the stabilization of the Topo II-DNA cleavable complex. On the basis on these data, we presumed that BRCA1 blocked cells may be more sensitive than the corresponding control cells against plumbagin. Our expereiments with BG-1 ovarian cancer cells indeed proved this.
Methods:.BG-1 ovarian cancer cells antisensly blocked for BRCA1 was the cell line used for this study.
Results: The dose of plumbagin needed to kill 50% was 5.1 M in the control cells and 2.68 M for the BRCA1 blocked cells indicating that the latter was about two fold more sensitive to plumbagin than the wild type cells. Plumbagin was also found to generate reactive oxygen species (ROS) in cultured cells. Plumbagin can bind to the active site of ER- inducing ER- 46 kDa truncated isoform, which was found abundantly preempted in the cytoplasm compared with a 66-kDa full-length isoform. The truncated isoform is known to inhibit classical ER- signaling pathways. SiRNA transfected cells for ER- exhibited lower cytotoxicity upon plumbagin treatment than the control-transfected cells. We have done molecular mechanism of action of plumbagin in BRCA1 blocked ovarian cancer cell line by suppression subtractive hybridization and microarray.
Conclusions: This throws light on the fact that plumbagin may have a chemotherapeutic potential as an anticancer agent in BRCA1 defective breast/ ovarian cancer patients.
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Acetylsalicylic Acid: an Immune-Modulator in HIV Infection
STANCZUK GA1 and SIBANDA EN2
1, 2 Medical School, University of Zimbabwe, P.O. Box A 178, Avondale, Harare, Zimbabwe
Background: Management of AIDS in a country when more than 80% of population lives in deep poverty is a challenge, which no clinician would like to experience. To begin with lack of water and sanitation, not to mention nutritional depravation makes Anti-retrovirals an impossible choice. Acetylsalicylic Acid, being an inhibitor of Nuclear Factor-kappa beta (NF-), may prove to be a cheap and relatively safe immune-modulator.
Methods: We randomized 20 drug naïve HIV-positive men and women into placebo and 1,2g of Acetylsalicylic Acid. All participants were supported by nutritional program with three meals per day. Baseline CD4 count, viral load (VL), and plasma levels of HIV-p24 antigen, Tumour Necrosis factor-alpha (TNF-), Interleukin -2 (IL-2), IL-4 and IL-6 were measured 3-monthly for 12 months.
Results: We observe a decrease in the median CD4 count in the placebo group from 267 to 221 cells/mL and an increase in CD4 count in Acetylsalicylic Acid group from 364 to 437 cells/mL (p=0.015). The mean VL increased in placebo by 0.46 log compare with Acetylsalicylic Acid by 0.15 log. Participants with detectable p24 antigen increase from 19 to 71% in placebo. The p24 antigen remained negative in all who started off negative in Acetylsalicylic Acid arm. Mean plasma TNF- levels were unchanged in the placebo but decreased from 11.31 pg/mL to undetectable in all in Acetylsalicylic Acid group. IL-6 levels decreased in the placebo arm but increased in the Acetylsalicylic Acid arm. IL-2 levels decreased in all but one in placebo; there was a decline from 7.64 to 5.67 pg/mL in the Acetylsalicylic Acid group. IL-4 increased from 59 to 186 pg/mL in the treatment arm.
Conclusions: This pilot study shows a beneficial increase in CD4 cell count following ingestion of Acetylsalicylic Acid. There is a lesser impact on the viral load. The cytokine studies that show a decrease in TNF- and IL-2 and increases in both IL-4 and IL-6 suggest that the effects are in part cytokine mediated. We believed there is an urgent need to investigate these findings in a larger population.
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Rabies in Bats and Protective Treatments of Humans in Europe
STANTIC PAVLINIC M1, HOSTNIK P2
1Institute of Public Health of Ljubljana, Zaloška 29, 1000 Ljubljana, Slovenia; 2Veterinary Faculty, Department on Virology, University of Ljubljana, Gerbičeva 60, 1000 Ljubljana, Slovenia
Background: Surveillance of rabies in bats across Europe during the last 50 years revealed rabies infection in bats in many European countries and also some human cases after exposure to rabid bats. Humans should be treated against rabies pre-exposure and post-exposure. There is a need to protect some professions and travellers as well. According to WHO, rabies control needs to be adapted on the rabies surveillance in each country and rabies pre-exposure and post-exposure treatment of humans needs to assume bat rabies as well. Goals: 1) Find out if post-exposure vaccination against rabies is implemented in particular countries; 2) Find out if preventive treatments of bat researchers and cavers are used as well.
Methods: Questionnaire was dispatched by mail to contributors of the Rabies Bulletin Europe from European countries. We asked questions concerning recommendation for post-exposure treatment after a bat bite and for pre-exposure treatment of bat researchers and cavers.
Results: 15 countries across Europe participated in the study: Austria, Belgium, Croatia, Czech Republic, Denmark, Estonia, Finland, France, Iceland, Netherlands, Norway, Poland, Serbia, Slovenia, Switzerland and United Kingdom. Post-exposure treatment is more frequently practiced (13 of 15 countries) than pre-exposure treatment of bat researchers (11 of 15 countries involved in the study). Pre-exposure treatment of cavers is practiced only in Czech Republic, France and Poland.
Conclusions: 1) Distribution of European lyssa viruses in Europe is not well understood because only few countries have suitable monitoring of these infections; 2) Growing knowledge concerning bat rabies and availability of successful treatment with rabies vaccine and immunoglobulin is interesting to medical doctors, veterinarians, cavers and bat researchers; 3) Reasons for actualizing recommendation concerning treatment against bat rabies are the changing world with large tourist movement and investigation of epidemiological importance of emerging infectious diseases as well.
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New Medicines, New Problems: Understanding the Failure of the Phase I Clinical Trial of Tgn1412
STEBBINGS R, EASTWOOD D, FINDLAY L, BURNS C, POOLE S, THORPE R
Biotherapeutics Group, National Institute for Biological Standards and Control (NIBSC), Potters Bar, Hertfordshire, United Kingdom
In 2006 a near-fatal “cytokine storm” occurred in six healthy volunteers during the phase-I clinical trial of TGN1412, a therapeutic superagonistic CD28-specific monoclonal antibody, signalling a failure of pre-clinical safety testing. Subsequently we established that TGN1412 could stimulate a “cytokine storm” in vitro but only if correctly presented to human cells by immobilisation onto plastic or when added in the presence of endothelial cells. These novel procedures would have predicted the toxicity of this superagonist and are now being applied to emerging immunotherapeutics and to other therapeutics that have the potential to act upon the immune system. Data from these novel in vitro procedures suggests that the dose of TGN1412 given to human volunteers was close to the maximum immunostimulatory dose. In contrast to humans we found that TGN1412 is not superagonist for cells from the non-human primate model used for safety testing. A “cytokine storm” was not seen during pre-clinical in vivo safety testing as non-superagonistic CD28-specific monoclonal antibody is not pro-inflammatory for human or non-human primate cells.
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A Common Molecular Basis For Ryanodine Receptor Dysfunction Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT) And Malignant Hyperthermia (MH) Provides Opportunities For Novel Therapeutic Strategies
STEELE DS1, IKEMOTO N2, YANG Z1
1Univ. of Leeds, Leeds, UK; 2Boston Biomedical Research Institute, Boston, USA
Background: Missense mutations in RyR2 underlie the condition CPVT, which is associated with cardiac arrhythmias during exercise or stress. CPVT mutations occur in 3 clusters, corresponding to the N-terminal, central and the pore-forming regions of RyR2 (or regions 1, 2 & 3 respectively). A similar clustering of RyR1 mutations occurs in the skeletal muscle disease MH. Recent evidence suggests that (i) mutations in regions 1 and 2 occur within interacting domains and (ii) region 3 is actually another pair of interacting domains. If this hypothesis is correct, it should be possible to mimic the effects of specific mutations using RyR peptides, which act competitively to disrupt the interdomain interactions. Here we report the effects of a peptide (DPc10), which binds competitively to region 2 of RyR2, thereby disrupting interactions between regions 1 and 2.
Methods: Rats (Wistar, 200g) were humanely killed in accordance with UK legislation and ventricular myocytes isolated enzymatically. Cells were permeabilized with saponin (5 µg/ml) and perfused with a mock intracellular solution containing (mM) ATP 5, phosphocreatine 10, HEPES 15 (free Ca2+ 200 nM, Mg2+ 1 mM. pH 7.1, 21oC) Solutions also contained fluo-3 (10 μM), allowing changes in cytosolic [Ca2+] to be detected using confocal microscopy.
Results: In permeabilized ventricular myocytes, 50 µM DPc10 induced (i) a transient increase in resting Ca2+ spark frequency (ii) a sustained increase in the RyR mediated Ca2+ leak, which could be revealed by inhibition of the SR Ca2+ pump and (iii) a decrease in the cytosolic [Ca2+] threshold for spontaneous Ca2+ waves. However, a similar peptide (DPc15-mut) containing a disease mutation (A2474S) linked to CPVT had no effect on RyR2 function.
Conclusions: 1) The decreased threshold for spontaneous SR Ca2+ release induced by DPc10 is likely to be proarrhythmic and therefore consistent with the disease phenotype. 2) The absence of effect of DPc10-mut can be explained if the disease mutation impairs the ability of the peptide to interact competitively with region 1. 3) The ability to mimic the effects of disease mutations in normal cells using targeted peptides provides an opportunity for the development of novel therapeutic strategies.
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Data review on Medicines utilization and expenditure in Serbia from 2004-2007
STEFANOVIC D1, SOLAROVIC T1, RADONJIC V1
1Medicines and Medical Devices Agency of Serbia (ALIMS), Belgrade, Republic of Serbia
Aim: The aim of this paper is to provide the review of the total consumption as well as financial means spent for medicines in Serbia from 2004-2007. Generally over the past decades, medicines have made a major contribution to improving the health status of patients. At the same time, pharmaceuticals expenditure has increased rapidly. Spending on medicines is outpacing economic growth. In respect of Serbia, it has a population of around 7.5 million and a tradition of a public health care system financed through a social health insurance. The Republic Institute for Health Insurance (RIHI) is financed by social insurance contributions raised on individual salaries (12, 3% of gross salary) and shared equally by employers and employees. Prescription drugs account for about 11% of the RIHI spending on health care.
Method: ALIMS is authorised (by the Law on Medicines and Medical Devices "The Official Gazette of the Republic of Serbia", 84/2004 and 85/2005-the other law) for collecting and processing data on medicines marketing and consumption. Data on medicines marketing from 2004-2007 were gathered from obliged entities and processed by the DDD/1000 inhibitant/day methodology and ATC classification. Financial analysis was done as well.
Results: It was established that the total medicines marketing in Serbia in 2004 was €340 million, in 2005 it was €380 million, in 2006 it was €510 million and in 2007 it was €690 million. Since the total DDD has been around 1000, the consumption data have shown that one drug has been used by each inhabitant every year in Serbia. In all four years, medicines for cardiovascular disorders (group C) have been at the first place. By processing consumption data for group C as DDD, from 2004-2007 consumption was 331, 334, 388 and 400 DDD/1000 inh./day, respectively.
Conclusion: In era of ageing population and rising health care costs, analyses and statistics on medicines market make an important indicator in health care. Combined with epidemiological data, it can provide a scientific contribution to the efficient, efficacious and safe use of medicines. Health care professionals and policy makers seek for implementation of effective pharmaceutical policies that would support further health improvements by introducing new and more effective medicines, whilst containing pharmaceuticals expenditure.
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Dipeptidyl peptidase 4 inhibitors: a new class of oral agents for the treatment of type 2 diabetes mellitus
STEFANOVIC V
University of Nis, and Serbian Academy of Sciences and Arts, Belgrade, Serbia
Type 2 diabetes mellitus is a major health problem in the 21st century. New therapies are needed to control metabolic abnormalities, and also to preserve β-cell mass and to prevent loss of β-cell function. Glucagon-like peptide 1 (GLP-1) is a drug candidate which potentially fulfils these conditions. Mice lacking dipeptidyl peptidase IV are protected against obesity and insulin resistance.
Several DPP-4 inhibitors are in clinical development; these are orally active and increase levels of active GLP-1, which in turn increases insulin secretion and reduces glucagon secretion and thereby lowers glucose levels. Most experience exists for sitagliptin and vildagliptin, which both have a long duration of action, allowing once-daily administration.
DPP-4 inhibition is an efficient treatment of type 2 diabetes, both as monotherapy and combination therapy. Because of its efficiency, safety, and tolerability in association with the oral mode of administration, it is expected that DPP-4 inhibition will be a first-line treatment of the early stage of type 2 diabetes, particularly in combination with metformin or thiazolidinediones.
The DPP-4 inhibitor vildagliptin improves insulin sensitivity and β-cell function, leading to improved postprandial glycemia in subjects with IFG, who are known to have β-cell dysfunction. Thus, vildagliptin may prevent progression to diabetes in high-risk subjects.
Incretin mimetics are a new class of pharmacological agents with multiple antihyperglycemic actions that mimic several of the actions of incretin hormones. Exenatide is the first incretin mimetic approved for clinical use. Exenatide therapy can be considered as an alternative to insulin in patients with treatment failure on metformin monotherapy or on metformin together with a sulfonylurea.
Conclusion: The long-term consequences of DPP-4 inhibition on ß-cell function and the durability of glucose lowering achieved with sustained DPP-4 inhibition require careful clinical assessment. It seems prudent to pursue additional detailed studies of the biological role(s) of DPP-4 and the consequences and safety of highly selective DPP-4 inhibition in experimental and clinical models of diabetes.
Therapy for diabetes will probably not alter radically in the next few years unless long-term data demonstrate other advantages over metformin and insulin. However, agents modulating GLP-1 are likely to be playing a major role in combating the world-wide burden of diabetes in the 21st century.
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Which ABC-transporters should we target in leukemia?
STEINBACH D
University Children’s Hospital Ulm, Eythstrasse 24, 89075 Ulm, Germany, Phone No.: +49 (0) 731 50057126, FAX No.: +49 (0) 731 50057485, Daniel@Steinba.ch
ABC-transporters are a large family of proteins involved in active transport across biological membranes. Some members of this family cause drug resistance in malignant diseases via ATP dependent drug efflux from malignant cells. This phenomenon was intensively analyzed in leukemia.
ABCB1 (P-g) and ABCG2 (BCRP) were shown to be associated with poor response to chemotherapy in adult acute myeloid leukemia (AML). Both proteins confer resistance to a wide range of chemotherapeutic drugs in vitro. Therefore, they represent possible therapeutic targets. In pediatric AML, this is the case for ABCG2 but not for ABCB1. In acute lymphoblastic leukemia (ALL), both proteins appear less relevant with the probable exception of ABCB1 in adult patients.
ABCC3 (MRP3) has a strong prognostic impact in AML and ALL independent of age group. However, ABCC3 does not cause much drug resistance in vitro. Therefore, it remains to be elucidated whether its correlation with poor response to therapy is causative or just an epiphenomenon.
ABCA3 might be an additional cause of drug resistance in AML. It is associated with in vitro drug resistance and response to therapy. Interestingly it causes drug resistance via intracellular drug sequestration instead of drug efflux from the malignant cell.
Specific inhibitors of ABC-transporters can sensitize leukemic cells to chemotherapy. For some types of leukemia it would be desirable to develop drugs that inhibit a set of ABC-transporters.
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The Pathogenesis of Autoimmune Diseases: New Possibilities for Drug Development
STENBERG P, ROTH EB
Hospital Pharmacy, Malmö University Hospital, S-205 02 Malmö, Sweden
With their overall prevalence of 5-8 %, the approximately 80 defined autoimmune diseases comprise a major burden to public health. They have many common features and are increasingly recognized as a group of related illnesses that should be studied collectively as well as individually. Unfortunately, efforts to prevent or treat these conditions are hampered by a limited knowledge of the pathogenesis. In 1997, a German group demonstrated that the antigen of the biomarker EMA (endomysial antibodies) in coeliac disease (CD) is a calcium-dependent thiol enzyme, transglutaminase 2 (TG2). This important discovery opened up an exciting field of research aimed at a better understanding of the pathogenesis of CD. Progress has already improved the opportunities for laboratory diagnostics, and hopefully, new ways of treating and preventing CD will become available. This presentation will highlight some of the intriguing mechanisms of the pathogenesis of CD, such as the structure of the neo-antigen, the involvement of calcium and zinc, and the effects of CD-antibodies on TG2 activity.
In another common inflammatory autoimmune disease, rheumatoid arthritis (RA), antibodies against peptide-bound citrulline constitute a specific biomarker. The enzymes catalyzing citrullination, the peptidylarginine deiminases (PADs), share many features with the TGs. Moreover, auto-antibodies against PAD can be detected in RA. Thus, in two major T-cell restricted autoimmune diseases, antibodies can be detected against a posttranslationally modified protein as well as against the calcium-dependent thiol enzyme responsible for the substrate modification. Reflecting the progress in the field of CD, this presentation will also focus on the fascinating mechanisms which may be involved in the pathogenesis of RA. Moreover, the many pitfalls due to dubious laboratory practice will be addressed together with the great potential when a fundamental biological mechanism is understood at the molecular level.
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