On the bases of these in vitro results we also investigated the effect of antiretroviral therapy on the release and the expression of MMP-9 from circulating peripheral blood mononuclear cells (PBMC) from HIV-infected individuals. By using a sensitive fluorescent-activated substrate conversion (FASC) assay we demonstrated the presence of active MMP-9 in PBMC supernatants from HIV-infected patients naïve for antiretroviral therapy (ARV). By contrast, in both healthy donors and ARV-treated subjects, there was no MMP-9 net activity, indicating that MMP-9 was completely blocked by binding to its natural tissue inhibitor TIMP-1.
These results outline the possibility to use antiretroviral drugs and compounds with anti-inflammatory properties, which have been shown to inhibit MMP function, for the experimental treatment of neurological disorders in which the inhibition of MMP could have clinical benefits.
Inhibition of HIV-1 through an Innate Humoral Mechanism – a Potential for Vaccine Development LOBO PI, SCHLEGEL KH, YUAN W, TOWNSEND GC, WHITE JA
Center for Immunity Inflammation and Regenerative Medicine, Division of Infectious Diseases and International Health, and Myles H. Thaler Center for AIDS and Human Retrovirus Research, University of Virginia Health System, Charlottesville, VA 22908
Background: In prior studies we show that naturally occurring IgM anti-leucocyte autoantibodies (IgM-ALA) bind to certain leucocyte receptors e.g. CD3, CD4, CCR5 and CXCR4 but not to others e.g. CD8, CD28 and HLA. IgM-ALA inhibit T cell function, chemokine binding to receptors and chemotaxis of leucocytes. Using monoclonal IgM, derived form umbilical cord B cell clones, we show that <10% clones have IgM-ALA activity and <1% clones bind to CD4 indicating that binding of IgM to receptors is highly specific. Umbilical cord sera contained IgM-ALA but not IgG-ALA. We wanted to determine if IgM-ALA by binding to co-receptors, inhibits HIV-1 entry into cells.
Methods: IgM and IgG was purified from 10 normal and 25 HIV sera using size exclusion column chromatography and contaminating IgG from purified IgM was absorbed out.
Results: Physiological doses of purified individual IgM, but not IgG and not IgM pre-absorbed with leucocytes, from normal and HIV-1 sera inhibited (>95%) both X4 and R5 HIV-1 from infecting PHA+IL2 activated human PBL in-vitro and in vivo in human PBL-SCID mice. HIV-1 infectivity of PBL was also inhibited with human monoclonal IgM (2 clones) having anti-CD4 activity but not with IgM (6 clones) that lacked binding to leucocytes indicating that IgM mediated inhibition of HIV-1 is highly specific. We show that IgM inhibits HIV-1 attachment to core receptors as IgM inhibited syncytia formation (by >90%) and inhibited infection of R5 and X4 pseudo typed virus. IgM from certain individual HIV-1 sera were not inhibitory to some R5-HIV-1 viral strains and did not bind to CCR5 receptors indicating that certain HIV-IgM may lack antibodies reactive to strain specific co-receptor epitopes.
Conclusion: An innate humoral mechanism which is present from birth i.e. IgM-ALA, has a role in inhibiting HIV-1 viral entry into cells. Developing strategies to enhance in-vivo IgM-ALA e.g. through a vaccine, could prolong the asymptomatic state in HIV-1 infected individuals. This work has been published (J of Immunol, 2008. 180: 1769).
Multiple antibiotic resistance of heterotrophic bacteria from Siberian lakes as an indicator of anthropogenic influence on the ecosystems LOBOVA TI International Centre for Research of Extremal States of Organisms, Krasnoyarsk Scientific Center of Siberian Branch of Russian Academy of Sciences, Krasnoyarsk, Russia.
Background: Antibiotic usage in medicine practice and veterinary has led to dissimination of genes multiple antibiotic resitstance (MAR) among natural aquatic bacteria. Aimes: To examine the effect of anthropogenic impact on appearance of MAR by heterotrophic bacteria isolated from two lakes differing in anthropogenic impact level and bacterial plasmid profile.
Methods: Heterotrophic bacteria isolated from central and resort/shore-line parts of Lake Shira/Lake Shunet (Southern Siberia) were investigated. The method of replica plating was used for antibiotic sensitivity testing. The following antibiotics were used: amikacin (30 µg/ml), ampicilin (50 µg/ml), benzylpenicillin (10 µg/ml), cefotaxime (25 µg/ml), doxycycline (30 µg/ml), gentamicin (10 µg/ml), kanamycin (30 µg/ml), streptomycin (30 µg/ml). Isolation of plasmids was carried out by the method of alkaline lysis.
Results: The antibiotic resistance profiles of heterotrophic bacterial isolates recovered from two lakes were determined. Resistance was detected in at least one strain for seven of the eight antibiotics tested, the exception being amikacin. No bacteria with single antibiotic resistance were found. Resistance was more frequently observed among isolates recovered from within the proximity to a tourist resort (Lake Shira), or the shore-line (Lake Shunet) than comparative samples from the centre of each lake. Bacteria with multiple antibiotic resistance (resistant to 2 and more antibiotics) were checked on a presence plasmid in their cells. We found plasmids of varying both in sizes (from less than 2.3 to <23.1 kb) and their number (from 1 to four per cell). Plasmids of medium size (about 23.1) predominated in bacterial cells. The same plasmid profile was found for bacteria isolated from the central and from the resort parts of Lake Shira and these bacteria have the same antibiotic resistance pattern.
Conclusions: Multiple antibiotic resistance of heterotrophic bacteria studied is due to anthropogenic impact on the ecosystem and this property of bacteria can be used for ecological monitoring on the lakes. One of the possible reasons of the plasmid distribution among bacteria may be plasmid capture by bacteria under human activity conditions.
Specificity of multiple antibiotic resistance appearance by luminous bacteria inhabiting the Pacific and Indian oceans MEDVEDEVA SE1, ZAKHAROVA NV1, LOBOVA TI2, POPOVA LYU2 1Institute of Biophisics of SB RAS, Krasnoyarsk, 2Russia; International Centre for Research of Extremal States of Organisms, Krasnoyarsk Scientific Center of SB RAS, Krasnoyarsk, Russia.
Background: A level of pollution of World ocean by bacteria containing R-plasmids with genes encoding for multiple resistance to antibiotics (MAR) is determined by both density of population of seasides and presence on them cities and resorts. Reistance of luminous bacteria to high concentrations of antibiotics can be used as a marker of anthropogenic influence on sea ecosystems. Aimes: To study resistance of luminous bacteria to different antibiotics and to evaluate possible mechanisms of bacterial MAR.
Methods: Luminous bacteria (180 strains) belonging to the Photobacterium leiognathi, Photobacterium phosphoreum and Vibrio harveyi isolated from water and different sea inhabitatnts of the Pacific and Indian oceans were investigated. The method of replica plating was used for antibiotic sensitivity testing. Mechanisms of bacterial antibiotic resistance were investigated on molecular, cellular and population levels.
Results: About 90% of all strains studied appeared resistance to high concentrations (50-500 mkg/ml) of ampicillin and penicillin. Resistance of the strains to kanamycin and streptomycin ranged from 30 till 90%, to rifampicine, tetracycline and to doxycycline was no more than 50% and 15% correspondingly. Bacteria isolated from Indian Ocean were found to be resistant to doxycycline, and resistance of strains from the Pacific Ocean to this antibiotic was not exceeding 5%. Strains of P.phosphoreum were more sensitive to used antibiotics than other strains under the study. Strains of V.harveyi isolated from both oceans appeared the highest level of MAR.Strains of P.leiognathi isolated from Indian Ocean were resistant to 4-8 antibiotics while in the Pacific Oceans the same bacteria were resistant to 2-4 antibiotics.
Conclusions: The largest number of bacterial strains with MAR was found among free living of V. harveyi isolated fromcoastal zones of both oceans. The tendency to increasing of MAR was found of P.phosphoreum, P. leiognathi, V. harveyi. In the Pacific Ocean bacteria with MAR were isolated mainly from coastal zone, and in Indian Ocean through all water basin. Bacteria with high level of MAR were chosen as potential plasmid-bearing strains.
Identification of the cocaine binding site on the dopamine transporter LOLAND CJ1, BEUMING T2, KNIAZEFF J1, NEWMAN AH3, KATZ JL3, WEINSTEIN H2, GETHER U1 1Dept of Neuroscience and Pharmacology, Univ. of Copenhagen, Copenhagen, Denmark. 2Dept. of Physiology and Biophysics, Cornell Univ., New York, USA. 3Medications Discovery Research Branch, National Institute on Drug Abuse, Baltimore, USA
Background: Cocaine is a widely abused substance with psychostimulant effects attributed to inhibition of the dopamine transporter (DAT). Currently, there is no medical treatment against cocaine addiction. Interestingly, analogues of benztropine (BZTs) are less effective than cocaine as behavioral stimulants, despite having similar or higher affinity and selectivity for the DAT than cocaine. In fact, some BZTs have shown to antagonize the effect of cocaine in rea behavioural models.
Methods: We use molecular docking models dopamine, cocaine and BZTs to assess the binding site of the compounds in the DAT. The models were validated experimentally using systematic mutagenesis of the involved residues and studyint the effect on substrate affinity, We also assessed the correctness of the docking model of the cocaine analogue, CFT, by the engineering of a zinc binding site and using chemical cross-linkers.
Results: The models of dopamine, CFT and BZTs show almost complete overlap with only a few residues shown to be unique for either compound. Systematic mutagenesis of the residues proposed to be involved completely validated the models. Trapping of the radiolabeled cocaine analog [3H]CFT in the transporter, either by cross-linking engineered cysteines or with an engineered Zn2+-binding site that was situated extracellularly to the predicted common binding pocket also produced results in agreement of the docking model. In particular, the orientation of Tyr156 in TM3 showed marked differences between the models: In the model for dopamine and BZTs, a hydrogen bond is formed between the OH-group of Tyr156 and Asp79 in TM1. In contrast, docking of CFT causes a disruption of this H-bond. Disruption of this interaction (Y156F) resulted in several fold decrease in affinity for dopamine and BZTs but had no effect on the affinity for CFT.
Conclusions: Our data show the molecular basis for the competitive action of cocaine at DAT. We also demonstrate in DAT a unique binding mode for cocaine, which unlike substrates and BZTs, produce a conformational rearrangement of the binding site that disrupts a stabilizing OH-bond between Tyr156 in TM3 and Asp79 in TM1.
Antisecretory Factor (AF) - an inducible antisecretory and antiinflammatory protein LONNROTH I, JOHANSSON E, LANGE S
University of Goteborg, Sweden.
Background. The Antisecretory Factor (AF) is a 41 kda protein which affects ion/water transport and inflammation in the small intestine.The endogenous plasma level of AF is increased by enterotoxins and by certain food constituents such as hydrothermally processed cereals. The active site of AF is a 16-peptide (AF16) at position 36-51. We here show that the peptide also exerts effects on the central nerve system.
Methods. Intracranial pressure was measured in rats with a fiber optic transducer positioned at the posterior parts of the lateral ventricle in the brains. An elevated pressure was induced by infection with Herpex simplex virus causing encephalitis (HSE); 25 g of AF16 (n=10) or vehicle alone (n=22) was administrated intranasally twice daily. In order to measure in vitro effects of AF16 nerve cell membranes from Deiters cells were mounted in microchambers and 36Cl- permeability studied.
Results AF16 rescued all rats with HSE; in contrast 90 % of animals given vehicle alone died. The effect of AF16 was probably due to its capacity to reduce intracranial pressure since a single dose of the peptide reduced the pressure to normal. In vitro pmol levels of AF16 was shown to counteract the out to in 36Cl- permeability in nerve cell membranes; in contrast AF peptides lacking the active sequence had no effect.
Conclusion The results suggest that AF affects ion/water transport not only in the gut but also in the central nerve system illustrating once more the so called gut/brain axis.
Inintracavitary-administered Nimotuzumab labeled with 188Re in adult recurrent high-grade glioma. CASACÓ A, LÓPEZ G, FERNÁNDEZ R, TORRES L, PERERA A, BATISTA J, LEYVA R, PEÑA Y, AMADOR Z, GONZÁLEZ A, ESTUPIÑAN B, COCA M, HERNÁNDEZ A, PUIG M, IGLESIAS M, HERNÁNDEZ A, RAMOS M, RODRÍQUEZ L, SUAREZ N.
Radioimmunotherapy (RIT) may improve the management of malignant gliomas. Nimotuzumab is a humanized monoclonal antibody directed against epidermal growth factor receptors. Three patients with anaplastic astrocytoma (AA) and 8 with glioblastoma multiforme (GBM) were intended to be treated with 3 mg of mAb labelled with 10 or 15 mCi of 188Re. In patients treated with 10 mCi (n = 6) transitory worsening of pre-existing neurological symptoms were observed. Two patients treated with 15 mCi (n = 4) developed early severe neurological symptoms and one also developed late severe toxicity (radionecrosis). In the group treated with 10 mCi, 1 GBM patient died in progression 6 months after the treatment, 2 patients (1 GBM and 1 AA) developed stable disease during 3 months. One GBM patient had partial response for more than 1 year and 2 patients (1 GBM and 1 AA) were asymptomatic and in complete response after 3 years of treatment. Maximal tolerated dose of the radioimmuno-conjugate 188Re-Nimotuzumab was 3 mg of the h-R3 labelled with 10 mCi of 188Re.The radioimmuno-conjugate showed a high retention in the surgical created resection cavity and the brain adjacent tissues with a mean value of 85.5% of the injected dose one hour post-administration. This radioimmunoconjugate may be relatively safe and a promising therapeutic approach for treating high grade gliomas.
Virostatics: a new class of immunomodulators with dual antiviral and cytostatic properties to inhibit viruses and protect the immune system from hyperactivation during chronic infections LORI F1 1ViroStatics srl, Sassari, Italy.
Background: Chronic infections are characterized by continued stimulation of the immune system, often resulting in variable degree of hyperactivation with consequent partial or complete immune exhaustion, as it is the case for HIV/AIDS.
Methods: We have characterized drugs or combination of drugs carrying both antiviral and cytostatic properties, and named them virostatic drugs. In particular, we have analyzed anti-HIV compounds, and screened them for antiviral and antiproliferartive properties both in activated and quiescent lymphocytes. VS411, a fixed combination of two drugs, is the leading compound.
Results: VS411 has successfully completed a bioavailabilty Phase I study. Formulation has been chosen and VS411 is presently in Phase II development program. Previous academic Phase II studies have shown that VS411 is effective at reducing viral load in HIV infected individuals. A second generation of virostatic drugs is undergoing a preclinical program in our laboratories.
Conclusions: Virostatic drugs represent a new family of antivirals designed not only to suppress viruses but also to preserve the immune system from chronic damage. VS411 is is a fixed combination of virostatic drugs undergoing Phase II clinical studies that appears to be safe, well tolerated, and effective at reducing viral load.
AdCD40L Cancer Vaccine – From Experimental Models to Clinical Application LOSKOG A1, LINDQVIST C1, FRANSSON M1, MANGSBO S1, WANDERS A2, GARDMARK T2, MALMSTROM PU2, and TOTTERMAN TH1.
1Uppsala University, Uppsala, Sweden. 2University Hospital, Uppsala, Sweden.
Background: Cancer vaccines are merging as novel treatment options for cancer patients. The first line of vaccines encountered obstacles such as tumor immune escape. Novel strategies have been explored that aim to both activate anti-tumor immunity as well as hamper the regulatory mechanisms exhibited by the tumor. The objective for our tumor vaccine program was to evaluate the efficacy of AdCD40L therapeutic vaccination for solid tumors such as bladder cancer.
Methods: Adenoviral vectors were used to transfer the immunostimulatory gene CD40L into tumors. Preclinical evaluation of tumor eradication and development of anti-tumor immunity was made in our murine bladder cancer model as well as in both murine and human culture systems. Clinical evaluation was made in patients with bladder cancer in a clinical phase I/IIa trial. Tumor immunity upon therapeutic vaccination was investigated with techniques such as CBA, quantitative PCR, proliferation assays, and flow cytometry.
Results: In a series of publications we have demonstrated that AdCD40L can cure highly aggressive bladder cancer in experimental models. In these models, AdCD40L was shown to efficiently activate tumor-specific immunity by maturing dendritic cells, stimulating Th1 cytokines and activating cytotoxic T cells. Further, the levels of T regulatory cells and suppressive cytokines such as IL10 and TGFb were decreased which may be crucial for the anti-tumor effect. In recently obtained unpublished animal data, local AdCD40L administration into tumor-positive bladders eradicated both bladder tumors and distant lung metastases. Currently, a clinical evaluation is performed. Phase I is completed (n=5) and AdCD40L vaccination was safe. No side effects have been documented. Routinely, bladders are removed from patients with high-grade malignancy to avoid incurable metastases. After AdCD40L therapy no high-grade tumor cells could be detected in the cystectomized bladders. Further, T regulatory cells were reduced after treatment in compliance to our experimental models.
Conclusion: AdCD40L cancer vaccination seems to eradicate high-grade malignant tumor cells and is a promising candidate for therapeutic vaccination of both local and disseminated malignancy.
Effects of Theranekron (alcoholic extract of Tarantula cubensis) in treatment of Foot- and- mouth disease (FMD) lesions in cattle LOTFOLLAHZADEH S1, MOHRI M2, MOKHBER DEZFOULI MR1 1 University of Tehran, Faculty of veterinary Medicine, Tehran, Iran; 2 Ferdowsi University, Faculty of Veterinary Medicine, Mashad, Iran.
Background: Foot- and- Mouth disease (FMD) is an acute viral disease of all cloven- footed animals. In enzootic countries where a slaughter policy is not in force, treatment of infected animals is recommended. Theranekron (Richterpharma, Austria) contains the whole extract from spider tarantula cubensis.In the present study therapeutic effect of Theranekron on foot- and- mouth disease in cattle was investigated.
Methods: During an outbreak of FMD in cattle in Iran, fifty infected cattle (in early stage of disease) treated with single subcutaneous injection of Theranekron (treatment group). Fifteen infected animals at the same time chose and treated as follow: daily injection of an anti inflammatory drug, broad spectrum antibiotic and daily dressing of lesions with a mild disinfectant for three to five days. Clinical examination and observation of lesions in infected animals (treatment and control groups) was carried out in 24, 48 and 72 hours and also one and two weeks after treatment.
Results: Rectal temperature (fever) in Theranekron injected group subsided to normal range after 24 hours of injection, and mean values of rectal temperature in treatment group during study was significantly lower than animals in control group (p< 0.05). in table the results of treatment in two groups on oral mucosal lesions of infected cattle has been appeared.
A significant difference between median value of oral lesions in treatment and control
groups were showed in 24 hours and days 2, 3 and 7 after treatment by Mann- whitney test (P<0.05).
Day
Group
Before Treatment
24 hours next
2 days next
7 days next
14 days next
NO
Control
1
(1- 1)
1
(1- 2)
2
(2- 1)
3
(4- 3)
4
(4- 4)
15
Treatment
1
(1- 1)
2
(5- 2)
3
(4- 3)
4
(4- 4)
4
(4- 4)
50
1) Blister , 2) Burst blister with hyperemia , 3) Reepithelialization of lesions
4) Complete healing, 5)Reabsorption of vesicles without bursting
Appetite in treatment group had been returned to normal after 48 hours and in control group lasted for 4 days.
Conclusion: From results of present study it appears that Theranekron is a very efficient drug for treatment of FMD cases in cattle especially because of its anti inflammatory and healing properties and convenient in use (single injection) compare to the routine treatment.
Cardioprotective Anthracycline PKC Activators for The Treatment of Drug-Resistant Tumors LOTHSTEIN L, HOFMANN PA, ISRAEL M, SWEATMAN TW
University of Tennessee Health Science Center Memphis USA
Successful cancer chemotherapy is often limited by cellular drug resistance, which requires dose escalation for circumvention, and by systemic adverse effects, which limit the cumulative dose of drug administered. Typical of these dual limitations are the anthracycline antibiotics, such doxorubicin (DOX), whose cytotoxicity is blocked by multiple resistance mechanisms, including the expression of multidrug transport proteins (MRP-1, P-gp), anti-apoptotic proteins (Bcl-2, Bcl-XL), proliferative proteins (NF-kB, Bcr-Abl kinase), and by p53 protein dysfunction. DOX efficacy is also limited by well-characterized and often irreversible cumulative cardiotoxicities linked to the generation of reactive oxygen species (ROS) by the anthraquinone ring.
In response to these therapeutic impediments, we have developed a novel class of anthracyclines, represented by N-benzyladriamycin-14-valerate (AD 198) and N-benzyladriamycin-14-pivalate (AD 445). AD 198 and AD 445 are markedly more lipophilic than DOX and, consequently, circumvent efflux by multidrug transport proteins. Unlike DOX, AD 198 and AD 445 localize in the cytoplasm and do not target DNA but, rather, bind to the C1b (diacylglycerol-binding) domain of protein kinase C (PKC). Drug-mediated PKC-delta activation triggers rapid apoptosis in proliferating cells through a novel mitochondrial-dependent pathway and in a manner that circumvents the anti-apoptotic effects of Bcl-2 and Bcl-XL expression. Since apoptosis is triggered rapidly and without the requirement of cell cycle arrest, enhanced proliferative signaling or p53 dysfunction do not block AD 198/AD 445-mediated apoptosis.
Despite the retention of an anthraquinone ring, AD 198 is non-cardiotoxic in the Bertazzoli (chronically-dosed) mouse model. This correlates with the activation of PKC-epsilon by AD 198 and enhanced cardioprotective signaling in cardiomyocytes, which protects the heart from reperfusion injury following induced ischemic or from high-dose DOX-induced injury in an ex vivo perfused heart model. Our studies suggest that AD 198 and AD 445 may provide improved therapy for drug-resistant tumors without concern for dose-limiting cardiotoxicities associated with conventional anthracyclines or with cardioprotection when administered in combination with potentially cardiotoxic antitumor agents.
Proposed antiangiogenic agents with mechanisms that prevent tumor cell survival and resistance to cytotoxic therapies LOUNSBURY, KM1, HALE, SA1, HATLE, K2, RINCON, M2 Departments of 1Pharmacology and 2Medicine, University of Vermont, College of Medicine, Burlington, VT, USA
Background: The transcription factor hypoxia inducible factor-1α (HIF-1α) facilitates cell survival and angiogenesis through transcriptional activation of multiple target genes including vascular endothelial growth factor (VEGF). HIF-1α has also been linked to increased drug resistance through expression of the ABCB1 drug transporter, preventing intracellular drug accumulation. We recently found that loss of methylation-controlled J protein (MCJ) correlates with increased expression of ABCB1 and increased resistance. Others have also shown that inhibition of glycogen synthase kinase 3β (GSK3β) promotes drug resistance and that HIF-1α contains consensus sites for phosphorylation by GSK3β. Because MCJ regulates protein translation/degradation and HIF-1α is primarily regulated by proteasomal degradation, we tested the hypothesis that HIF-1α is negatively regulated by MCJ through sites phosphorylated by GSK3β.
Methods: This study used MCF-7 breast cancer cells, SKOV-3 ovarian cancer cells and HEK 293 cells. ShRNA expression constructs were used to reduce MCJ, and overexpression of GSK3β was accomplished by constitutively active GSK3S9A. HIF-1α levels were determined by Western blot, transcription was measured by quantitative PCR, and protein interactions were identified by co-immunoprecipitation.
Results: Inhibition of MCJ expression or GSK3β activity resulted in increased HIF-1α expression, VEGF transcription and drug resistance. Conversely, overexpression of constitutively active GSK3β led to both a decrease in hypoxia-driven HIF-1α protein levels and VEGF transcription. Interaction between GSK3β and HIF-1α was detected by co-immunoprecipitation. Mutation of the putative GSK3β phosphorylation sites within HIF-1α promoted an increase in protein level and a decrease in ubiquitination of the HIF-1α mutant protein.
Conclusions: This study illustrates a mechanism in which posttranslational modification by GSK3β attenuates HIF-1α possibly through MCJ interaction, ubiquitination and proteasomal degradation, thus decreasing angiogenic potential and preventing drug resistance. We propose the future study of GSK3β activation by means of PI3 kinase inhibitors as therapy to prevent angiogenesis and drug resistance in cancer cells.
A Potential Anti-Cancer Drug From a Plant Extract, Tillandsia recurvata LOWE H1, BRYANT JL2 1Environmental Health Foundation; 2University of Maryland (IHV) School of Medicine
Background: Most successful cancer drugs have been derived either directly or indirectly from plant materials. Jamaica is fortunate to have 84 of the 120 recognized medicinal plants of the world. One of these plants, Tillandsia recurvata has demonstrated potent anti-cancer activities both invitro and invivo. The following specific activities were done: 1) Isolation and purification of bioactive compound from this plant extract; 2) The anti-cancer properties were tested invitro and invivo activities of this isolated compound; and 3) Assessed through preliminary findings one of the mechanisim of action of this compound
Methods:Tillandsia recurvata was collected in Jamaica. The fresh plant material was dried in steam oven at 70°C then macerated and then extracted with cold MeOH. Various fractions were then isolated be colume comotography. Subfractions were then processed and tested via bioguided fractionation. This was follwed by using advanced separation technology (HPLC, supercritical fluied chromatography and capillary electropheresis. This was followed by LC/MS and finally NMR). This study involves the testing of the isolated fractions against 5 different histogenic tumors invitro using the trypan blue exclusion invitro assays. We also utilized the 3H Thymadine incorporation assay. The five tumor cell lines used in these assays were the following: 1) melanoma; 2) prostate; 3) breast; 4) Kaposi sarcoma and 5) b-cell lymphoma. The extract was tested invivo against the above tumor cell lines. The invivo studies included 10 mice per group and using the crude form of the extract at 10mg per mice per day were for 7 days. This was done for each of the above tumor cell lines compared with controls with normal saline treatment. The purified compound is now currently being produced at ground levels to test invivo.
Results: Utilizing the bioassay-guided fractionation process, the bioactive moiety was isolated at 98% purity. This purified compound was tested invitro and demonstrated to be higly effective at a rate of 95% to 100% cell kill in the invitro assays of 5 different histogenic tumor cell lines. The invivo studies were equally as impressive utilizing the crude extract. All tumors responded to the treatment by reducing the tumor size from .4mm X .4mm to almost non existent on gross examinations of all the above tumor cell lines. On histology 90% to 95% of the tumors were undergoing cell death. Using immunohistochemical staining we were able to determine that the cell death was due primarily to induced apoptosis. No toxic signs or systems were observed in any of the invivo studies.
Conclusions: This newly extracted compound demonstrated a significant anti-cancer properties. Preliminary studies from this newly isolated compound indicates that this compound may serve as an excellent new anti-cancer drug.
Authors’ disclosure statement This compound has been patented.
A New Molecular Mechanism of Action of a Leading Chemotherapeutic Drug—Cisplatin and Its Novel Applications LU QB, Nguyen J, Wang CR, Chan P
University of Waterloo, Waterloo, ON, Canada.
Background: There is pressing need for mechanistic understanding of action of existing anticancer drugs at the molecular level, which can, in turn, lead to molecular-mechanism-based design of new anticancer drugs. Despite its great success in treating certain cancers, cisplatin as the most effective chemotherapeutic drug has severe toxic side effects and both intrinsic and acquired resistance. Such drawbacks have even prompted the call to discontinue the therapeutic applications of cisplatin-like anticancer drugs. One outstanding problem is that the precise mechanisms of action of these anticancer drugs remain elusive. Aims: 1) To obtain a true understanding of the molecular mechanism of action of cisplatin. 2) To optimize cisplatin chemotherapy. 3) To apply our mechanistic understanding of cisplatin to developing new effective cancer therapies, including combinations with radiotherapy and photodynamic therapy.
Methods:Time-resolved ultrafast (femtosecond) laser spectroscopy is the most powerful technique for real-time observations of molecular reactions since it uses laser flashes of such short duration down to the time scale on which the reactions actually happen femtoseconds (fs) (1fs= 1015 second). The molecular mechanism of anticancer drugs revealed by this technique is examined with biomedical methods such as DNA damage and cell death measurements.
Results: An extremely high reactivity of cisplatin with electrons and a new electron transfer mechanism of the cisplatin-DNA interaction have been discovered. Furthermore, we have revealed the molecular reaction mechanism of the combination therapies of low-dose cisplatin with radiotherapy and photodynamic therapy. Based on our mechanistic understanding of cisplatin, new molecular regulators have been developed to enhance the therapeutic effects and to reduce the side effects.
Conclusions: 1) The therapeutic effectiveness of cisplatin is closely related to its high reactivity with electrons. 2) This finding can be utilized to improve the chemotherapy with cisplatin and to develop new combination therapies for effective treatment of cancers.
References: 1. Q.-B. Lu et al., Molecular Pharmaceutics4, 624(2007). 2. Q.-B. Lu, Journal of Medicinal Chemistry50, 2601(2007).
Authors’ disclosure statement: Financial support from the Canadian Institutes of Health Research (CIHR) is acknowledged.
MHC Molecules as Antigen Receptors? LÜ NQ Jiangsu Family Planning Research Institute, Nanjing, Jiangsu 210036, China.
The speculation that immunologically reactive haptens must be those attached to carriers’ immunodominant epitopes immediately suggests a clearer mechanism by which the mysterious hapten-carrier phenomena are generated. In a specified T-B cell cooperating response against an antigenic determinant, T and B lymphocytes specifically recognize this specified determinant of the same antigen molecule in two different ways and in different circumstances. The B cell recognizes an antigen by the preliminary antigen receptors on the cell’s surface, at the time it is still intact. In contrast, the T cell recognizes the hidden structure of this epitope in specific amino acid sequences that are first recognized by MHC class I and II molecules on the surface of APCs. This analysis revealed that only a small number of regions, called epitopes or determinants, in a protein antigen are immunogenic and capable of stimulating humoral and cellular immune responses. The program of immune response against an antigen must be similar to the program of fertilization, resulting in one cell, one antibody. Since the transfer of tissue between individuals is rare in Nature, graft rejection cannot be the primary function of MHC proteins. The concept that immunoglobulins on B cell membrane are antigen receptors has been challenged and may be replaced by the new hypothesis that all antigen presenting cells use their MHC class II molecules as receptors for antigen recognition to initiate humoral immune response. This is in view of the fact that no MHC II molecule has yet been found in T cells. This hypothesis supplies a single explanation for the anamnestic immune response. Alternatively, antigen presenting and processing is necessary for the anamnestic response. Furthermore, it must be MHC molecule that acts as a specific “enzyme-like molecule” by unknown mechanisms with the aid of some cofactors inside the antigen presenting cells, and protects antigen from complete degradation and allows them to emerge as peptides. In developing effective vaccines against hypervariable viruses, and effective drugs for immunosuppressive therapy for transplantation, there is still a lot to learn from the hapten-carrier phenomena.
TGF-: A primary tumor suppressor gene involved in leukemogenesis LUCAS PJ1, MCNEIL N1, HILGENFELDE1, ECKHAUS MA1, RIED T1, GRESS RE1 National Institutes of Health, Bethesda, MD, USA
Background: Tumorigenesis has been show to be a multistep process. Initial genetic mutations are often associated with dysfunctional growth regulation and are often followed by alterations in tumor suppressor gene function allowing unchecked cell cycle progression, and by genomic instability, additional genetic mutations responsible for tumor metastasis. Aim: To study the importance of TGF-, a critical immune suppressive cytokine, on T lymphocyte cell growth.
Methods: This study included 150 mixed gender transgenic (Tg) mice, which overexpress a dominant negative TGF- II receptor (DNRII) using a human CD2 promoter to target T lymphocytes. Forty-eight homozygous, sixty-five heterozygous DNRII Tg mice and twenty control mice were monitored by periodic differential blood counts, which were used to determine disease state. Five homozygous and five heterozygous mice were selected for chromosome analysis studies and 3 additional mice were used for adoptive transfer studies.
Results: DNRII Tg mice developed a CD8 lymphoproliferative disease marked by increased expression of CD44 and IL-2R. Homozygous mice developed disease, as determined by high white blood counts (WBC) more rapidly than heterozygous mice (37 vs 53 weeks). Lymphomas exhibited multiple chromosomal abnormalities, as determined by spectral karyotype analysis, including aneuploidy, deletions, and translocations as is shown in the table. Several lymphomas were adoptively transferred to irradiated recipients and shown to be capable of transferring the tumor.
ID#
Alleles
Aberration
2020
1
ta(1;14); t(11;15) [6]b
60
1
42,XXY [8]; 40,XY [2]; t(4;13 or 5)[4]; +5[8]c [10]
5787
2
40,XY; t(4;5) [5]; t(14;3) [4] [12]
5664
2
38-40,XX [3]; X -X[2]†; -8[2] [5]
a Translocation
b Number of chromosomes with aberration/total studied at end of line
C Addition (+) or deletion (-) of chromosome
Conclusions: Dysregulation of the TGF- pathway in T cells results in abnormal growth in the CD8 T cell subset resulting in a lymphoproliferative disease followed by leukemia/lymphomas as demonstrated by WBC, chromosomal abnormalities and ability to transfer to syngeneic host, suggesting that TGF- is acting as a primary tumor suppressor gene in CD8 T lymphocytes.
The gut, the forgotten metabolic organ: the story of testosterone and dextromethorphan. LUEDTKE D The liver is known as the major site of first pass extraction. Although a lot of efforts have been done to characterize the metabolic behavior of the intestine, the contribution of this organ to first pass extraction is often ignored. Recent studies have indicated that the small intestine contributes significantly to the first pass metabolism of many drugs in human, e.g. cylosporine, nifedipine, midazolam, diltiazem and verapamil.
Therefore, we have investigated the profile of CYP P450 proteins via Western blot analysis along the entire length of the intestine in the rat. We identified CYP3A and CYP2B1 as the major expressed CYP P450 isoforms in the rat upper intestine, while CYP2C6 and CYP2D1 dominate the lower parts of the intestine (ileum/colon). Furthermore, the expression data were compared with functional data using two well known CYP P450 substrates testosterone and dextromethorphan. Testosterone is predominantly metabolized by CYP3A and CYP2B1, while dextromethorphan is a CYP3A and CYP2D1 substrate. The data show how the intestinal metabolism might affect/limit absorption and, hence, how relevant the intestinal metabolism might be for the first pass extraction of orally administered drugs.
Specific Inhibitors of the Cyclic Nucleotide Phosphodiesterases PDE2 and PDE4 Overcome In Vitro and In Vivo Angiogenesis KERAVIS T, LUGNIER C CNRS UMR 7175,Université Louis Pasteur de Strasbourg, France
Background: Cyclic nucleotide phosphodiesterases (PDEs) play a key role, downstream receptor activation, in intracellular signalling by selectively hydrolyzing cyclic nucleotides that serve as second messengers in a number of cellular pathways. Eleven PDE isozyme families (> 100 proteins) have been characterized and are differentiated by their substrate specificity, their tissue, cell and subcellular distributions, and also by their short-term and long-term regulations. Altogether, the complexity of these famillies allows a fine and compartmentalized regulation of cyclic nucleotide levels. Among these famillies, PDE2, which hydrolyzes both cAMP and cGMP and whose the cAMP hydrolysis is stimulated by cGMP, and PDE4, which specifically hydrolyses cAMP, represent the main isozymes in human umbilical vein endothelial cells (HUVECs). Angiogenesis is defined as the formation of new blood vessels from pre-existing ones. Since angiogenesis plays a major role in tumor development induced by tumoral vascular endothelial growth factor (VEGF) secretion, nowadays anti-angiogenic efficient therapeutical approaches are developped mainly at the receptor level.
Methods: Herein, by using an in vitro angiogenesis model (HUVECs), an in vivo angiogenesis model (chicken embryo chorioallantoic membrane; CAM), and an in vivo tumorisation model (tumor growth induced by BF16/10 cells in 20 C57BL/6N mice), we show that the combination of PDE2 (EHNA) and PDE4 (RP73401) inhibitors overcome angiogenesis.
Results: Our studies show that VEGF-induced HUVECs proliferation and migration is associated with PDE2 and PDE4 upregulations (mRNA, proteins and activities) and that PDE2 and PDE4 inhibition increases cAMP level, inhibits cell migration and proliferation and also inhibits VEGF-induced cell cycle progression at the level of ERK phosphorylation, cyclin D1 expression. Similar studies performed with delphinidin (a grape polyphenol which inhibits PDE2 and PDE4), show that delphinidin inhibits in vitro (HUVECs) and in vivo (CAM) angiogenesis. Tumor growth treated with EHNA+RP73401 is reduced by 30% (P=0.014).
Conclusions: It clearly appears that targetting VEGF-upregulated endothelial PDE2 and PDE4 is a new and original strategy to overcome the intracellular signaling dysfunction induced by VEGF stimulation which should induce less side effects.
Prevention of Inflammatory Leukocyte Adhesions for Treatments in Acute Sepsis Condition by Anti-CD18 sFv Single-Chain Antibody LUK JM, WONG KF
Dept of Surgery, The University of Hong Kong, Pokfulam, Hong Kong SAR, China
Background: Leukocyte integrin (CD11/CD18 heterodimer) plays a major role in immune cell trafficking. Translocation of leukocytes from blood circulation to extracellular space in injured or infected tissues is essential to immune surveillance and defense against invading pathogens. However, in certain pathological conditions like systemic inflammatory response syndrome (SIRS) and bacterial sepsis, excessive infiltrated leukocytes could cause multiple organ failures. Aims: (i) to generate anti-CD18 scFv agent that could block leukocyte adhesion to injured wounds and (ii) to evaluate its efficacy on preventing inflammatory leukocyte migration in rodent sepsis model.
Methods: A phage display library was generated from splenocytes of BALB/c mice hyperimmunized with human CD18 βA antigen. Phage scFv antibodies were screened for binding activity against the CD18 antigen, and the clone was isolated and selected by three rounds of panning exercises. Antigenic specificity of the anti-CD18 scFv was determined by flow cytometry, immunohistochemistry, Western blot, and immunoprecipitation. This study also assessed the therapeutic efficacy of the purified anti-CD18 scFv (0.8 mg/kg, i.p.) compared with placebo control (each group, n = 5) in murine cecal-ligation puncture (CLP) model. We also determined the animal survival, leukocyte infiltration into liver and lung organs.
Results: A high affinity of anti-CD18 scFv phage was isolated, and subsequently produced in large quantity in E. coli. The purified scFv agent was shown to bind specifically to the CD18 antigen as shown by immunoprecipitation and Western blot. The scFv also stained in FACS analysis the Jurkat T lymphocytes, but not the Jurkat mutant lacking the CD18. In the CLP animal model, one bolus of anti-CD18 scFv, but not the placebo, could significantly reduce the severity of leukocyte infiltration into lungs and livers in the treated animals, as revealed by immunohistochemical staining of CD45+ cells. Furthermore, the scFv-treated animals revealed significantly low levels of circulating TNF-α and IL-6 pro-inflammatory cytokines. Of importance, the anti-CD18 scFv could demonstrate survival advantage in the CLP-inflicted mice.
Conclusions: 1) The scFv agent is shown to bind CD18 antigen and the eptiope mapped to the betaA domain. 2) Anti-CD18 scFv significantly blocks leukocyte infilitration into major organs (liver, lung) and attenuates the release of proinflammatory cytolines (TNF-α, IL-6) in systemic sepsis conditions. 3) Animals treated with the scFv could survive longer compared with those from the placebo group in the CLP model. Collectively, our findings suggest that blocking the CD18 βA domain by anti-CD18 scFv promises to be an effective approach for therapeutic intervention of leukocyte-mediated tissue damage.
Authors’ disclosure statement: This study is supported by the Research Grants Council of Hong Kong and has been filed for US Patent (No.: 61/080,558).
according to registration: Lukawska M Amidinoanthracyclines – Perspectives to Promising Modification of Known Anticancer Drugs WASOWSKA-LUKAWSKA M1, OSZCZAPOWICZ I1, AND OSZCZAPOWICZ J2 1Institute of Biotechnology and Antibiotics, Warsaw, 2Chemistry Department, Warsaw University, Warsaw, Poland
Background: Anthracylines such as daunorubicin, doxorubicin and epidoxorubicin are commonly used antitumor drugs with wide spectrum of activity in human cancer. However, their clinical effectiveness is limited by several important factors, including dose-dependent toxicity and cardiotoxicity. Moreover the resistance of tumor cells to this drug remains also one of the major clinical problems. The attempts reported in the literature to solve these problems are based mainly on the modifications of the structure of these antibiotics by introduction of various substituents at different sites of their molecules.
In the course of our study on the properties of anthracyclines and their derivatives we have set the hypothesis, that the one of the main factors responsible for their toxicity is the presence of the primary amino group (–NH2) at the position 4' of the daunosamine moiety and that its transformation into the trisubstituted amidino group (–N=CR'–NR''R'') may cause two effects: the decrease of toxicity of anthracyclines and additionally the increase of their antiproliferative activity.
Methods: To check this hypothesis 40 amidinoanthracyclines, were synthesized. To throw some light on the structure-activity relations the compounds in this novel family of the anthracyclines are divided into 4 analogous series, namely derivatives of daunorubicin, doxorubicin, epidaunorubicin and epidoxorubicin containing the same set of 10 amidino groups. This enabled to show the influence of the structure of amidino group and aglicone on activity.
Results: Significant support for the above mentioned hypothesis is provided by the results of biological tests such as toxicity (LD50), cardiotoxicity, and antiproliferative activity against 10 different cell lines. It is found that, most of obtained amidinoanthracyclines display lower toxicity and some of them much higher antiproliferative activity. Moreover almost all of the tested compounds exhibit the possibility to overcome the drug resistance barrier of cancer cells.
Conclusions: Obtained results indicate that the transformation of the primary amino group in anthracyclines into trisubstituted amidino group may appear as the most promising way to obtain new anticancer drugs, among which a real "magic bullet" can be found.
Anti-HIV activity of lectins from marine invertebrates MOLCHANOVA V1, LI W2, CHIKALOVETS I1, WANG J-H3, ZHENG Y-T3, LUKYANOVP1 1Pacific Institute of Bioorganic Chemistry, Vladivostok, Russia; 2Dalian Fisheries University, Dalian, China; 3Kunming Institute of Zoology, Kunming, China.
Background: Role of HIV envelope carbohydrates and target T-cells glycocalix is very important for understanding of mechanism of virus invasion processes. Aims: To check anti-HIV effect of next lectins: Gal/GalNAc-specific (CGL) from mussel Crenomytilus grayanus, Gal-specific (CVL) from sea worm Chaetopteris variopedatus, GlcNAc- (DTL) and GlcNAc/GalNAc-specific (DTL-A) from colonial ascidia Didemnum ternatanum,mannan-binding (SVL-1) and GlcNAc-specific (SVL-2) from sea worm Serpula vermicularis.
Methods: The data were obtained in experiments in vitro on C8166 T-lymphoblastoid cell line and HIV-1IIIB virus. The lectins cytotoxic concentration (CC50) for C8166 cells was calculated from data of the colorimetric MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. An effective concentration of lectins necessary for 50% inhibition of the HIV-1 replication (EC50) was determined as level of p24 viral antigen in cell lysates by Enzyme-Linked ImmunoSorbent Assay (ELISA). The effective concentration of lectin causing 50% inhibition of syncytium formation (EC50) between the C8166 cells and chronically infected cell culture H9/HIV-1IIIB was calculated.
Results: All lectins investigated were nontoxic for cells practically. They inhibit virus replication and syncytium formation in C8166 cells in different dose-dependent manner. DTL is more effective as HIV invasion blocker, it has maximum antiviral index (83,333).
1EC50 is an effective concentration in the ELISA. 2EC50 is an effective concentration in syncytium formation test. 3CC50 is a cytotoxic concentration in the MTT assay. 4Antiviral index (AI) is the CC50/EC501 ratio.
Conclusions: 1) The lectins investigated block virus replication and syncytium formation in target T-cells. 2) DTL has more effective anti-HIV activity. 3) Other CVL and SVL-2 are promise as anti-HIV drugs too.
The work was supported in part by grant of President of Russian Federation for scientific schools 2383.2008.4
Cytochrome P450 and Gene-activating Agents – Cholesterol Elimination and Regression of Atherosclerosis LUOMA PV Institute of Biomedicine, Pharmacology, FIN-00014 University of Helsinki, Finland
Background: Principal cardiovascular disorder responsible for the global rise in mortality is atherosclerotic vascular disease. Our original studies in the 1970s linked drug-caused gene activation and the induction of cytochrome P450 (P450) with elevated plasma levels of apolipoprotein AI (apo AI) and HDL cholesterol (HDL-C), powerful indicators of a reduced risk of atherosclerotic disease.
Methods: This presentation clarifies the effects of P450-enzymes and gene-activating agents on cholesterol homeostasis, the atherosclerotic process, prevention and regression of atherosclerosis, and the manifestation of atherosclerotic disease, particularly coronary heart disease (CHD), the leading cause of death in the world.
Results: Several compounds upregulate genes acting in cholesterol elimination such as apo AI, ABC (ATP-binding cassette) transporters and P450s. P450s including CYP7A1, CYP27A1 and CYP46A1 generate hydroxycholesterols which mediate the activation of cholesterol-eliminating mechanisms (see 1-3).
The progress in studies on cholesterol regulation has greatly stimulated the search for new agents with potential to regress atherosclerosis (1-3). Many xenobiotics and natural compounds activate – via for nuclear receptors including LXR, PPAR and PXR – mechanisms which eliminate excess cholesterol. The antiatherogenic effects of many compounds including statins, fibrates and cholestyramine are mediated through the actions of P450s. Rosuvastatin therapy which effectively reduced LDL-C and apo B and raised HDL-C and apo AI resulted in a significant regression coronary atherosclerosis. Data from statin trials revealed that HDL-C elevation by more than 7.5 % together with effective LDL-C lowering resulted in the most profound regression of atherosclerosis. The increases in HDL-C levels were found to be an independent predictor of a beneficial outcome with statin therapy (2,3).
Conclusions: P450-enzymes are essential in the maintenance of cholesterol homeostasis. Effective gene-activating agents upregulating cholesterol-eliminating mechanisms regress atherosclerosis and reduce the occurrence of fatal and non-fatal CHD and other cardiovascular events.
1. Pharmacol Toxicol 1997;81:57; 2. Ann Med 2007;39:359; 3. Eur J Clin Pharmacol 2008;64
Safety of Etanercept in Elderly Subjects With Rheumatoid Arthritis LURATI A1, SCARPELLINI M, RE KA, MARRAZZA MG
Rheumatology Unit, Fornaroli Hospital Magenta italy
Objective: To report side effects (SEs) seen in a clinical cohort of patients >65 years old with Rheumatoid Arthritis (RA) treated with the TNFα blocker Etanercept and to compare the AEs rate with patients ≤ 65 years old.
Methods: Subjects with RA that started anti TNF therapy with Etanercept from November 2005 to February 2008 and referring to our Rheumatology Unit were included in this study and prospectively followed. Safety profile points included incidence rates of all side effects (SEs), defined as: adverse events (AE), serious AE (SAE, defined as a AE that required the permanent discontinuation of the therapy, malignancies and deaths), infective AE (IAE
Results: Seventy three patients were enrolled: 26 (23 females, 3 males) aged >65 years, 47 (40 females, 7 males) aged <65 years. Mean disease onset age: 57.4±1.7 years in patients ≤65 years old, 60.4±3.2 years in patients >65 years old (p>0.05). Mean age at beginning of TNF blocker was 57.8±6.9 yrs in patients ≤65 years old and 68.2 ±3.2 yrs in patients >65 years (p<0.05). The duration of the anti TNF treatment was 19.9±16.9 and 18.6±6.3 months, respectively (p>0.05). In the whole population 29 SEs were observed (6AE, 17 IAE, 6 SAE), and led to temporary/permanent withdrawal in 38.4% of cases.
The rate of patients presenting SEs was 38.46% (AE 10%, IAE 50%, SAE 40%) of >65 years old versus 36.17% (AE 26.3%, IAE 63.1%, SAE 10.5%) of ≤ 65 years old patients (p=0.408) (table 1). The survival curves of these two groups were no significantly different (log rank test of Mantel Cox p=0.267)
Conclusions: In clinical experience, TNFα inhibitors are well-tolerated overall, SAEs are rare, and their risk-benefit profile strongly favours benefit. Although, long-term safety data still need to be established. Severe infections, including TB and sepsis, have been reported. Etanercept has been well-tolerated and safe overall, also in elderly patients, confirming the good safety profile of this TNF blocker
