Methods: We have designed hammerhead ribozymes against hTERT, the catalytic subunit of telomerase. We treated various breast and ovarian carcinoma cell lines with the ribozyme. Using isogenic systems of telomerase negative and hTERT-overexpressing fibroblast we demonstrate that telomerase protects cells from various stresses, including clinically relevant drugs such as topoisomerase I inhibitors. If telomerase is inhibited or withdrawn from tumour cells these cells have an increased sensitivity to apoptosis and DNA damaging drugs.
Results: We found that cells treated with anti-hTERT ribozymes go into fast apoptosis. Many other approaches using different agents such as anti-sense molecules, chemical inhibitors or dominant-negative mutants of hTERT rely on the effect of telomere shortening, which means that tumours with long telomeres have to go through many rounds of cell divisions until they finally end up with short telomeres and eventually go into apoptosis or senescence.
We believe that in our case, the withdrawal of telomerase functions in a telomere-length independent manner. We and other groups could show that telomerase has additional functions in addition to telomere maintenance. We could show recently that telomerase enters mitochondria upon stress and suggest that this could contribute to an increased resistance of cancer cells to apoptosis-inducing and DNA damaging chemo- or radio-therapeutic drugs.
Conclusions: Telomerase proves to be an important and exciting new target for the development of chemotherapeutic agents and anti-neoplasstic drugs.Telomerase-inhibitors, developed by other groups and companies are undergoing clinical trials at the moment and are recommended as new, highly specific anti-tumour agents.
Production of cystine rich peptides and protein in E. coli: hepcidin, the iron-regulating hormone
GAGLIARDO B1, FAYE A2, KUBAT N2 , JAOUEN M1, DESCHEMIN JC2, CANONNE-HERGAUX F3, VAULONT S2, SARI MA1
1Université PARIS DESCARTES, CNRS UMR 8601, 45 rue des Saints-Pères, 75006 PARIS, FRANCE. 2Université PARIS DESCARTES, CNRS UMR 8104 ,INSERM U567, PARIS, FRANCE. 3ICSN-CNRS, GIF/YVETTE, FRANCE
Background: Hepcidin is a liver produced cysteine-rich peptide hormone that acts as the central regulator of body iron metabolism. Hepcidin is synthesized under the form of a precursor, prohepcidin, which is processed to produce the biologically active mature 25 amino acid peptide. This peptide is secreted and acts by controlling the concentration of the membrane iron exporter ferroportin on intestinal enterocytes and macrophages. Hepcidin binds to ferroportin, inducing its internalization and degradation, thus regulating the export of iron from cells to plasma. The aim of this work was to develop a novel method to produce human and mouse recombinant hepcidins, and to compare their biological activity towards their natural receptor ferroportin.
Methods: Human and mouse hepcidins were produced in E coli as Thioredoxin fusion proteins. Upon cleavage peptides, were purified by HPLC and characterized. The biological activity was measured on macrophages, following the ferroportin degradation induced by hepcidin
Results: Human and mouse hepcidins, purified after cleavage from thioredoxin, were properly folded and contained the expected 4-disulfide bridges without the need of any renaturation or oxidation steps. Hepcidins were found to be biologically active, promoting ferroportin degradation in macrophages. Importantly, biologically inactive aggregated forms of hepcidin were observed depending on purification and storage conditions, but such forms were unrelated to disulfide bridge formation. Moreover this strategy was extended to the production and purification of pro-hepcidin, the 61 amino acids precursor of hepcidin. Prohepcidin was also found to contain the 4 disulfide bridges and able to generate biologically active hepcidin upon cleavage by furins (in vitro and in vivo).
Conclusions: Biologically active hepcidin and prohepcidin (its natural precursor) were produced in E coli. The thioredoxin fusion protein strategy allows correct formation of disulfide bridges in E. coli and circumvent the necessity for unfolfding/folding strategies that are necessary with other recombinant systems or synthetic peptides.
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Albendazole Sulphoxide Levels in Endemic Normals and Filariasis Patients from a Lymphatic Filariasis Endemic Region of India Administered with Albendazole Using Liquid Chromatography
SARIN R
IPE Global, 81/82 Mona Villa, Ekamra Marg, Forest Park Area, Bhubaneswar, Orissa, India
Background : Determination of albendazole ( ABZ ) and its metabolites, albendazole sulphoxide ( ABZSO ) and albendazole sulphone ( ABZSO2 ) in biological fluids is important to optimize dosage, length and frequency of therapy for the treatment of lymphatic filariasis. Aims: 1) To develop a simple and sensitive liquid chromatographic method for ABZSO determination. 2) To determine ABZSO in plasma of endemic normals. 3) To determine ABZSO in plasma of lymphatic filariasis patients following oral administration of 600 mg of ABZ.
Methods: A simple and sensitive reversed-phase isocratic high performance liquid chromatographic ( HPLC ) method for the determination of ABZ, ABZSO and ABZSO2 has been developed. The mobile phase consisting of acetonitrile–water–perchloric acid (70%) (30:110:0.06 (v/v/v)) was pumped at a flow rate of 0.80 ml/min on a 5 μm, reverse phase, Discovery® RPamide C16 column with UV detection at 290 nm. The calibration graphs were linear in the range of 0.05–1 μg/ml for ABZ, ABZSO and ABZSO2. The limit of quantification was 50 ng/ml for ABZ, 25 ng/ml for ABZSO and 30 ng/ml for ABZSO2. The within-day and day-to-day coefficient of variation averaged 4.98 and 6.95% for ABZ, 3.83 and 6.83% for ABZSO and 3.44 and 5.51% for ABZSO2, respectively. The mean extraction recoveries of ABZ, ABZSO and ABZSO2 were 79.25, 93.03 and 88.78%, respectively. The method was applied to determine the plasma levels of ABZSO in 10 healthy endemic normals and 10 lymphatic filariasis patients administered with ABZ during pharmacokinetic studies.
Results: The method is suitable for the separation and determination of ABZSO and ABZSO2 in a single chromatographic run. ABZSO attains peak plasma concentration of 362.50 ng/ml within 2-4 hours in endemic normals while peak plasma concentrations were 884.02 ng/ml in lymphatic filariasis patients within 2 hours following an oral administration of 600 mg of ABZ.
Conclusions: The method satisfies the criteria required for an assay required for human pharmacokinetic studies. The study shows good relation between dose, plasma concentration of ABZSO and time which has therapeutic significance for the treatment of lymphatic filariasis.
Keywords: Albendazole sulphoxide, lymphatic filariasis and liquid chromatography
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Evaluation of Ecotoxicological impact of xenobiotic contaminants in terms of cytochrome P450 induction in marine fishes
SARKAR A1*, VASHISTHA D1, LERAY M2, GIRARD A2, LEPRAT P2, KAISARY S1, D’SILVA C1
1 Marine Pollution assessment and Ecotoxicology Group, National Institute of Oceanography, Dona Paula, Goa, India
2 National Higher Engineering School of Limoges (ENSIL) 16 Rue d’Atlantis
Parc d’Ester Technopole BP 6804, 87068 Limoges Cedex, France
The use of molecular biomarker in marine pollution monitoring has gained enormous importance because of the increasing trend of contamination of the coastal environment by highly persistent organic pollutants. Most of these contaminant are being biomagnified through the food chain posing a serious threat to human health on environmental carcinogensis. Among the persistent pollutants polyaromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), polychlorinated dibenzo-dioxin (PCDDs), polychlorinated dibenzo-furans (PCDFs) are well known for their toxic potentials. The accumulation of these contaminants into the tissues of marine organisms are detoxified by the induction of cytochrome P450 enzymes. In order to assess the extent of ecotoxicological impact of xenobiotic contaminants on the marine environment the induction of cytochrome P4501A1 was studied in the edible marine fishes (Mugil cephalus, Sardinella longicep and Rastrelliger kanagurta) from the Arabian sea along the Goa coast in terms of hepatic EROD activities. The Goan coastal environment is greatly contaminated with oils because of extensive shipping activities pertaining to fishing trawler, tourist boats, cargo ships, passenger ships, iron ore carrier, burges etc. The oil found in the contaminated water, sediments and biota were composed of mostly high molecular weight polyaromatic hydrocarbons (PAHs) with wide variation (0.476–5.882ug/L in water, 1.342--5.104ug/g in sediments and 8.54–37.89ug/g in biota) along the coast of Goa. The variation of EROD activities in fishes were in the range of 0.4335–3.377 pmol/mg/min along the Goa coast clearly indicating the extent of contamination of the coastal water by xenobiotic compounds such as PAHs, PCBs etc. Apart from the oil contamination, the coastal water also received huge amount of industrial wastes containing various types of xenobiotic compounds from the peripheral industries. The enhanced hepatic EROD activities in edible marine fishes clearly provides early warning signal of environmental carcinogenesis as evident from the production of reactive oxygen species via cytochrome P450 enzyme induction leads to the formation of DNA adduct resulting into DNA strand breaks.
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Chitosan-stealth polymeric nanoparticles for mucosal insulin delivery
SARMENTO B1,2, TEIXEIRA J1, NEVES R1, MARTINS S1, FERREIRA D1
1Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Porto, Porto, Portugal
2Department of Pharmaceutical Sciences, CICS-Instituto Superior de Ciências da Saúde, Gandra, Portugal
The gastrointestinal uptake of proteins like insulin can be improved by association to nanoparticles, protecting insulin from degradation in the gastrointestinal tract, providing excellent penetration into the intestinal mucus layer and prolonging the retention of insulin in the absorption window. Further, nanoparticles are able to be taken up by the M cells of the Peyer’s patches, a type of lymphatic island within the intestinal tract that represent the major gateway through which nanoparticles may be absorbed. Novel preparation methods to produce biodegradable chitosan-stealth polymeric nanoparticles have been developed in our group in order to improve the oral insulin bioavailability and solve major problems related to the current formulations. PLGA, solid lipids, dextran sulfate and alginate have been used as nanoparticle matrix. Alone, and coated with chitosan, insulin-loaded nanoparticles have been administered to diabetic animal model. Chitosan, the most widely employed natural polysaccharide, is able to reduce the transepithelial electrical resistance and transiently opening tight conjunction between epithelial cells and to combine with anionic sialic acid residues of the intestinal mucosa due to mucoadhesive properties. The adhesion of chitosan at the site of insulin absorption may offer various advantages for its uptake. Our experiments have demonstrated the feasibility of these different nanoparticles sizing between 250 to 750 nm to encapsulate insulin, maintain its bioactive form, provide controlled insulin release under gastrointestinal simulated conditions and markedly enhance intestinal absorption of insulin following oral administration by lowering serum glucose levels. Moreover, we have confirmed through intestinal section and Caco-2 cell monolayer permeability studies the absorption of labeled insulin and internalization of the protein, more pronounced when nanoparticles were formulated with chitosan coating, emphasize the absorptive enhancing effect of this polymer. In this talk, new and previous data are being presented and compared, highlighting the advantages and weaknesses of each system, and perspectives of optimizing formulations towards an efficient oral or other potential mucosal insulin delivery carrier.
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Tumor Necrosis Factor-alpha Antibody Reduces Pain Related Behavior Induced by both Epidural Application of Nucleus Pulposus and Nerve Root Compression in Rats
SASAKI N1,2, KIKUCHI S2, KONNO S2
1Minamisouma City General Hospital, Fukushima, Japan; 2Department of Orthopedic Surgery, Fukushima Medical University School of Medicine, Fukushima, Japan
Background: Pain of lumbar disc herniation (LDH) can be induced by not only mechanical nerve root compression but also chemical factors such as cytokines involved nucleus pulposus (NP). Tumor necrosis factor alpha (TNF-alpha) plays an important role in the pathophysiology of LDH. The aim of this study is to evaluate if TNF-alpha antibody administered intravenously reduces the pain related behavior induced by application of NP or compression to the nerve root in rats.
Methods: Two experiments were conducted. Experiment 1: Left L5 partial laminectomy was performed and NP was applied to the L5 nerve root in 24 rats. The rats were divided into 4 groups. In 3 groups, anti-rat TNF-alpha antibody was intravenously administered immediately after, or 6 or 20 days after NP application. The fourth group was not treated with anti-rat TNF-alpha antibody (untreated rats). Experiment 2: Left L5 partial laminectomy was performed and stainless steel rod was inserted into the laminectomy hole to compress the L5 nerve root in 12 rats. The rats were divided into 2 groups. In one group, anti-rat TNF-alpha antibody was administered 6 days after the operation. In both Experiments, the withdrawal threshold of the plantar surface was determined 1 day before up through 28 days after the operation.
Results: Experimtent1: The withdrawal threshold of rats that had been treated with anti-rat TNF-alpha antibody immediately after or 6 days after, but not 20 days after, NP application, was significantly higher than that of the untreated rats. Experiment 2: The withdrawal threshold of rats treated with anti-rat TNF-alpha antibody was significantly higher than that of the untreated rats.
Conclusions: Anti-TNF-alpha antibody reduced allodynia induced by both NP application and compression to the nerve root. Late administration of anti-TNF-alpha antibody did not have an antiallodynic effect.
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Firing the “Magic Bullets” at Brain Tumors: From Bench-to-Bedside and Back Again
SATHORNSUMETEE S1, POUNGVARIN N1, RICH JN2
1Department of Medicine (Neurology), Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700 THAILAND;
2Departments of Medicine, Surgery, Pharmacology and Cancer Biology; Preston Robert Tisch Brain Tumor Center, Duke University Medical Center, Durham, North Carolina, USA
Malignant brain tumors are relatively rare but lethal cancers. The median survival of glioblastoma multiforme (GBM), the most common primary brain tumor in adults, is 12-15 months despite current state-of-the-art multimodal therapies. Recent elucidation of the molecular pathogenesis of GBM has led to a rational development of molecularly targeted agents (“magic bullets”) as a novel treatment venue against this devastating cancer. Monoclonal antibodies and low molecular weight kinase inhibitors are the most common classes of molecularly targeted therapeutics. Most clinical trials of these agents as monotherapies have failed to demonstrate survival benefit in unselected GBM patient populations. Several strategies have been developed to circumvent the poor response to first-generation targeted agents in GBM. Such strategies may include inhibition of multiple targets by either multi-targeted (“magic shotgun”) inhibitors or novel treatment combinations. Multi-modality combination of targeted agents with radiotherapy or chemotherapy may improve efficacy. Indeed, the most promising salvage regimen for progressive GBM at present seems to be the combination of a VEGF neutralizing monoclonal antibody, bevacizumab and chemotherapy, irinotecan. This regimen is associated with remarkable radiographic response rate and significant survival benefit in patients with recurrent GBM. Recently, we have identified tumor molecular profiles that may predict radiographic response and survival benefit in patients treated with this combination regimen. High tumor VEGF expression was associated with increased likelihood of radiographic response, while tumor hypoxia as measured by high expression of carbonic anhydrase-IX predicts poor survival outcome. Future development of these “magic bullets” for GBM will require advances in discovery and validation of new molecular targets, improvement of therapeutic delivery and identification of biomarkers of response or resistance. Subsequently, each patient may be treated with personalized “magic bullets” based on molecular or genetic signatures.
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Protective effect of plant polyphenols-containing azuki bean (Vigna angularis) on renal damage
SATO S1, MUKAI Y1, AND YAMATE J2
1Aomori Univ. Health Welfare, Aomori, Japan; 2Osaka Pref. Univ, Sakai, Japan
Background: The azuki bean has been widely cultivated and is one of the most important crops in Japan. Azuki beans contain proanthocyanidins, which are a group of polyphenols with remarkable radical scavenging activities. We investigated the effect of azuki bean seed coats (ABSC), which mainly contain proanthocyanidins and dietary fibers, on the infiltration of macrophages and the progression of renal fibrosis in two different rat models.
Methods: Expt. 1; The streptozotocin-induced diabetic Wistar male rats were divided into three groups with 0%, 0.1% and 1.0% ABSC diets. At 10 weeks, the macrophage appearance and degree of fibrosis in glomeruli were evaluated, and mRNA expression for monocyte chemoattractant protein-1 (MCP-1) were examined by quantitative RT-PCR. Expt. 2; Wistar male rats in different groups were treated with a saline (control), cisplatin (CDDP), CDDP + 0.5% ABSC diet, CDDP + 2.0% ABSC diet. At 5 weeks after the fifth CDDP injection, the macrophages appearance and the interstitial fibrotic areas were examined.
Results: Expt. 1; There was no difference in plasma glucose levels between diabetic rats treated with and without ABSC. The plasma levels of thiobarbituric acid-reactive substances in the ABSC-treated diabetic rats were significantly lower than those in the untreated diabetic rats. Histopathologically, the percentage of fibrotic areas in the glomeruli in the ABSC-treated diabetic rats was lower than in the untreated diabetic rats. Macrophages in the glomeruli and tubulointerstitium in the untreated diabetic rats showed a significant increase in number compared with the controls. In contrast, the number of macrophages in the ABSC-treated diabetic rats was smaller than that in untreated diabetic rats. MCP-1 mRNA expression increased 2.5-fold in the untreated diabetic rat kidney, while a lower level was observed in the ABSC treated diabetic rats. Expt. 2; Histopathologically, the fibrotic areas developed around the dilated or atrophic tubules in the corticomedullary junction in CDDP-treated rat kidney, whereas the extent and magnitude of the damage were reduced in the ABSC-treated rats. Macrophages in CDDP-treated rats showed a significant increase in number, compared with the control. The number of macrophages in CDDP-plus-ABSC-treated rats was significantly smaller than that in CDDP-treated rats.
Conclusions: These results suggest that ABSC suppress the increase of infiltrating macrophages in the damaged kidney, and may lead to the attenuation of the glomerular or tubulointerstitial fibrosis in these models.
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Possible role for human leukocyte antigen haplotypes in hepatotoxicity and/or pancreotoxicity associated with chemotherapy
SATO K1, TAKAGI H1, MORI M1
1Gunma University, Maebashi, Japan
Background: Drug-induced liver injury (DILI) and drug-induced pancreatitis (DIP) are major health problems worldwide and leading causes of acute liver failure or severe pancreatitis. In addition, DILI and DIP are the main reasons for postmarketing regulatory decisions including withdrawal. Currently, there are no reliable markers for the diagnosis of DILI or DIP. The identification of patients who are more susceptible to these unpredictable, idiosyncratic forms of adverse events is much needed. Genetic factors for susceptibility to DILI or DIP are receiving increasing attention.
Methods: Human leukocyte antigen (HLA) was analyzed by a standard microlymphocytotoxicity method in patients with DILI and/or DIP associated with chemotherapy to elucidate the immunogenetic predisposition of DILI and/or DIP.
Results: A case of terbinafine-associated fulminant hepatitis without pancreatitis showed HLA haplotype-A26, -A33, -B62, -B58, -DR9, and -DR13. A case of micafungin-associated DIP without hepatitis showed HLA haplotype-A26, -B44, -B54, -B60, -DR9, and -DR14. A case of trimethoprim-sulfamethoxazole-associated hepatitis and pancreatitis showed HLA haplotype-A2, -B24, -B56, -B62, -DR9, and -DR14. We previously shows that the exact same HLA haplotype-A33, -B44 and -DR6 is detected in a case of rofecoxib-associated pancreatitis and cholestatic hepatitis and case series of tiopronin (mercaptopropionylglycine)-associated intrahepatic cholestasis. Moreover case series of ticlopidine-induced hepatotoxicity are associated with the same specific HLA haplotype-A33. All taken together, HLA haplotype-A33 may also be important for DILI associated with chemotherapy. On the other hand, HLA haplotypes-DR were common between our two cases of pancreatitis, suggesting that HLA haplotypes-DR9 and -DR14 may be linked to DIP.
Conclusions: HLA haplotype-A33 and -DR9/-DR14 may be associated with DILI and DIP, respectively. Because our presented cases are all Japanese, it is unknown whether the racial difference is involved in the pathogenesis of DILI and/or DIP besides on HLA haplotypes. Further studies regarding HLA haplotypes in patients with DILI and/or DIP regardless of race are much needed to avoid unpleasant hepatotoxicity and pancreotoxicity associated with drug therapy including chemotherapy.
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Malaria vaccines for the better
SAUERWEIN RW
Dept. of Medical Microbiology, Radboud University Nijmegen Medical Centre . Nijmegen, The Netherlands
The most effective way to reduce disease an death from infectious diseases is to vaccinate susceptible populations. There is an urgent need for malaria vaccines with overwhelming scientific social and economical justification in particular in the perspective of increasing drug resistance against cheap and available drugs. However, immunity to malaria is considered hard to acquire and artificial induction of sterile protection in humans has until now only been achieved by inoculating radiation-attenuated sporozoites through >1000 infective mosquito bites. Because of the scientific and financial constraints clinical vaccine development has been slow over the past decade but has more recently accelerated. Presently there are more vaccine candidate near or in clinical development than ever. The most advanced recombinant protein vaccine will enter phase III later this year. Experimental human malaria infection (EHMI) is a powerful test for down-selection of vaccine candidates by testing efficacy under controlled conditions. We significantly improved EHMI by using RT-qPCR for parasite detection and introduction of a statistical model for parasitaemia. Moreover , we demonstrate that sterile protection can be induced markedly more efficiently by inoculation of intact sporozoites under cover of a blood-stage anti-malarial drug. We furthermore identify parasite-specific pluripotent effector memory T-cells producing IFN?, TNF? and IL-2 as promising novel immunological associates of protection. In conclusion, EHMI shows to be an excellent model for studies on immunity and protective efficacy. Our data support the development of a malaria vaccine based on whole parasites.
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