Antiviral Effect Of Lamivudine, Emtricitabine, Adefovir Dipivoxil, And Tenofovir Disoproxil Fumarate, Administered Orally Alone And In Combination, To Woodchucks With Chronic Woodchuck Hepatitis Virus Infection MENNE S1, BUTLER SD1, GEORGE AL1, TOCHKOV IA1, ZHU Y2, XIONG S2, GERIN JL3, COTE PJ3, TENNANT BC1 1Cornell University, College of Veterinary Medicine, Department of Clinical Sciences, Ithaca, NY, USA; 2Gilead Sciences, Inc., Durham, NC, USA; 3Georgetown University Medical Center, Department of Microbiology and Immunology, Washington, DC, USA
Adefovir dipivoxil (ADV) and tenofovir disoproxil fumarate (TDF) are nucleotide analogs that inhibit replication of wild-type hepatitis B virus (HBV) and lamivudine- (3TC) resistant HBV in patients, including patients co-infected with the human immunodeficiency virus (HIV). Combination of ADV or TDF with other nucleoside analogs is a proposed strategy for managing antiviral drug resistance during treatment of chronic HBV infection. The antiviral effect of oral administration of ADV (15 mg/kg/day) or TDF (15 mg/kg/day), alone or in combination with 3TC (15 mg/kg/day) or emtricitabine (FTC; 15 mg/kg/day) against chronic woodchuck hepatitis virus (WHV) infection was evaluated in groups of 5 woodchucks for 48 weeks. Dosages were chosen due to their efficacy in previous treatment studies in woodchucks. Once-daily treatment with the combination of ADV+3TC (4 survivors) or of TDF+FTC (4 survivors) significantly reduced serum WHV viremia from pretreatment level by 6.2 and 6.1 log10, respectively, as determined by dot-blot hybridization and real-time PCR (P<0.01). Additional findings include treatment with TDF+3TC (4 survivors, 5.6 log10), ADV (4 survivors, 4.8 log10), ADV+FTC (1 survivor, 4.4 log10), TDF (3 survivors, 2.9 log10), 3TC (5 survivors, 2.7 log10), and FTC (5 survivors, 2.0 log10). Individual woodchucks across all treatment groups also demonstrated pronounced declines in serum WHV surface antigen as determined by ELISA. Characteristically, these woodchucks also had declines in hepatic WHV replication and hepatic expression of WHV antigens as determined by Southern and northern blot hybridization or immunohistochemistry, respectively. Most woodchucks had prompt recrudescence of WHV replication following drug withdrawal, but individual woodchucks across treatment groups had sustained effects. No signs of toxicity were observed for any of the drugs and drug combinations administered. In conclusion, oral administration of 3TC, FTC, ADV, and TDF, alone and in combination was safe and effective in the woodchuck model of chronic HBV infection.
Arsenic Trioxide Induces Apoptosis Preferentially In B-CLL Cells Of Patients
With Unfavorable Prognostic Factors Including Del17p13 MERKEL O1, HEYDER C1, ASSLABER D1, HAMACHER F1, TINHOFER I1, HOLLER C1, STÖCHER M1, PROKESCH A2, PAAR C2, SCHEIDELER M2, TRAJANOSKI Z2, GREIL R1 1Laboratory for Immunological and Molecular Cancer Research,.IIIrd Medical Department with Hematology, Medical Oncology, Hemostaseology, Rheumatology and Infectiology of the Paracelsus Medical University Salzburg, Salzburg, Austria, 2Institute for Genomics and Bioinformatics, Graz University of Technology
In the last decade, arsenic trioxide has been used very successfully to treat acute promyelocytic leukemia (APL). Much less is known about the effectiveness of arsenic trioxide in other neoplastic disorders. We report here that after 18 h in vitro treatment with 4 µM arsenic trioxide, 75 ± 18 % of B-CLL cells (n = 52) underwent apoptosis. Importantly, B-CLL cells harboring a deletion of chromosome 17p13, which predisposes to fludarabine resistance and has been identified as an important negative predictor of clinical outcome, were more susceptible to arsenic trioxide toxicity than cells lacking this aberration. Furthermore, unfavorable risk profiles such as unmutated IgVH status, high CD38 expression and prior treatment were associated with significantly higher sensitivity of B-CLL cells to arsenic trioxide. Arsenic trioxide also preferentially killed B-CLL cells as compared to B-cells from healthy age-matched controls. Molecular analysis revealed that basal superoxide dismutase activity was positively correlated with the pro-apoptotic activity of arsenic trioxide pointing to a role of reactive oxygen species in cell death induction. The high activity of arsenic trioxide in B-CLL cells from high-risk patients makes it a promising drug for high-risk and/or fludarabine-refractory B-CLL patients.
Gene Expression In Brain And Kidney In Response To Aluminum In Drugs MESHITSUKA S Tottori University Institute of Regenerative Medicine and Biofunction, Yonago, Japan
Background: Aluminum (Al) has been used in various drugs such as analgesics, antacids and vaccines as well as the domestic uses. It has been believed that the absorption rate of Al from the intestine is quite low and the absorbed Al is excreted rapidly into urine. However, it is not known whether Al remains in the body in the case of the intake of Al-containing drugs. Al has been linked to such diseases as dialysis encephalopathy, osteomalacia, 2-microglobulin-associated amyloidosis, amyotrophic lateral sclerosis and parkinsonisms-dementia in the Kii Peninsula and Guam. Recently the mechanism of adjuvant of Al in immune system has been reported. Therefore, the influence of Al on gene expression in relation to diseases has been interested in.
Methods: Al in human urine was analyzed by graphite furnace atomic absorption spectrometry with a Zeeman background correction. The gene-expression in mice was widely screened by differential display analysis. The corresponding genes were identified by sequencing. Gene expression was confirmed by RT-PCR and the corresponding proteins were identified by Western blotting in cultured cells and animals.
Results: The absorption rate of Al from the intestine was very low, but it was shown that the consecutive intake of Al-containing drugs exhibited extremely high excretion of Al in urine for days through the examination of volunteers. As a result it was confirmed that Al remained for a long period and circulated in the body. Al did not enter the brain in a normal condition, but in vitro astrocytes revealed apoptotic cell death, in which the gene expression and protein production in mitochondria did not change but those of endoplasmic reticulum altered. It was found that Al caused up-regulation of several gene-expression in kidneys. In further experiments it was confirmed to increase the renin production in response to relatively low concentration of Al.
Conclusions: Al remains for a long period and circulates in the body. Even at a very low concentration of Al the cultured cells revealed the ER stress as the alteration in gene expression. Particularly, it is pointed out that a chronic exposure of Al may be a cause of essential hypertension due to the up-regulation of renin.
Psychoncology And Psychoneuroendocrinoimmunology (PNEI): Relation Between The Anticancer Immunity And The Psychospiritual Status Of Cancer Patients MESSINA G1, LISSONI P, BRIVIO F
1Psychological Service, Policlinico Maggiore Hospital, University of Milan
Department of Oncology, San Gerardo Hospital, Monza, Milan
It is known that the psychospiritual status may influence tumour growth and the prognosis of the cancer. Only with the development of PNEI it has been possible to characterize the neuroimmunochemical mechanism responsible for the influence of the psychospiritual condition on tumour growth, through the modulation of the immune system. The anticancer immunity is stimulate by IL-2 and IL-12 whereas it is inhibited by IL-10 and IL-6. It has been observed that the evidence of low of IL-6 an IL-10, low number of lymphocytes and T-helper lymphocytes and an increase number of T reg is associated with the poor prognosis. Within the great number of the psychological variable, the spirituality has to be considered as different from the psychological profile. According to the PNEI discoveries we have performed several studies to investigate the psychological profile of cancer patients in relation to their psychoneuroendocrinoimmune status. The psychological profile was investigate by the Rorschach’s test and the spiritual status was analyzed by special spiritual test. We are performed five main studies, by obtaining the following results: 1)low number of lymphocytes and T-helper lymphocytes in patients with suppression of spiritual and sexual sensitivity; 2)low efficacy of IL-2 immunotherapy and low lymphocytes response to IL-2 in patients with anxiety and or loss of sexual identity; 3)low number of lymphocytes associated with alteration in the circadian rhythm of cortisol and hypercortisolemia in patients with low spiritual profile; 4)abnormally high percent of T -regulatory lymphocytes in patients showing self-punishment status; 5)lack of surgery induced-hyperprolactinemia in operable breast cancer patients with suppression of the maternal behaviour. This results would suggest the possibility to investigate the psychoneuroimmune basis of the overall psychological profile in cancer patients. Further studies by investigating the brain endocannabinoid system through the detection of the blood concentration of the main endocannabinoid agent anandamide will clarify the neurochemical alteration responsible for the progressive loss of the pleasure in neoplastic disease.
Anticancer Gold Compounds: Mechanistic Insights MESSORI L METMED Laboratoy, Department of Chemistry, Univ. Florence, Florence, Italy
Background: Gold compounds are a class of metallodrugs, of potential great interest for cancer treatment. During the past two decades a large variety of gold(I) and gold(III) compounds were shown to manifest relevant antiproliferative properties in vitro against selected human tumor cell lines qualifying themselves as excellent candidates for further pharmacological testing. On turn, mechanistic studies pointed out that the interactions of cytotoxic gold compounds with DNA are generally far weaker than those of platinum drugs implying a different mode of action. The main goal of our investigation is to provide new insight into the possible mechanisms of action of this variegate family of cytotoxic drugs.
Methods: A variety of methods were employed to disclose the mode of action of gold drugs. Several gold compounds were prepared according to classical methods of coordination chemistry and extensively characterized in the solid state and in solution. Their biological properties were evaluated both at the molecular and the cellular level. Their effecs on selected protein targets were investigated. In vitro cytotoxic properties toward a wide panel of human tumor cell lines were measured.
Results: A certain number of gold compounds, in most cases in the oxidation state +3, were prepared and characterised that manifested very pronounced cytotoxic effects in vitro. For most of them potent inhibition of thioredoxin reductase was established. It is proposed that thioredoxin reductase inhibition triggers cell apoptosis through a mitochondrial pathway. Morevover, extensive data have been collected on the cellular effects in vitro of a panel of gold compounds and specific insight into their respective mechanisms of action has been achieved.
Conclusions: Gold(III) compounds constitute a novel family of cytotoxic drugs of great interest as potential anticancer agents. Their cytotoxic effects are mediated by a variety of molecular mechanisms that are deeply different from those of platinum compounds.
The Knowledge And Expectations Of Parents About The Role Of Antibiotic Treatment In Upper Respiratory Tract Infection (URTI) – A Survey Among Parents Attending Tertiary Care Institution With Their Sick Child Premaratna R, Rajindrajith S, Mettananda KCD, Balasooriya H, Fonseka J, Randeny S, de Silva HJ
Faculty of Medicine, University of Kelaniya, Ragama, Sri Lanka
Introduction: Parents’ knowledge, attitudes and expectations of antibiotics have been identified as causes for antibiotic misuse in paediatric practice.
Methods: Parents accompanying their sick children with an URTI, attending at the out patients department and Professorial Paediatric unit of Colombo North Teaching Hospital, Ragama were interviewed by four qualified doctors for their knowledge, attitudes and practices during similar illness. The knowledge of antibiotics was assessed using pictures and samples of antibiotics.
Results: 235 parents (230 females) mean age 31.9 years (SD: 7.33) participated in the study. The level of education in the population was; below grade 5: 11 (4.7%), Grade 5-10: 142 (60.4%), grade 10-12: 71 (30.2%) and higher education: 11 (4.7%). Of the 235, 201 (85.1%) identified antibiotics as a component of their treatment. However only 11 (4.7%) knew they were against bacterial infections; 212(90.3%), 189 (80.8%), 176 (75%), 165 (70.4%), 130 (55.4%), 77 (32.8%) and 55 (23.6%) identified them as treatment for either or combinations of cough, fever, phlegm, cold, sore throat, ear ache and headache. While 116 (49.3%) thought that above treatment was important for cure of illness 119 (50.3%) thought them to be important for early recovery. The expectation of an antibiotic for an URTI (always, 75%, 50%, 25% and never) was 28 (12%), 39 (17%), 23 (10%), 119 (51%) and 23 (10%) respectively. Twenty (8.5%) had requested an antibiotic when it had not been prescribed; 12(60%) from the pharmacy and 8 (40%) from the doctor. Of the 235, 172 (73%) claimed to complete the full course of treatment while 18 (7.8%) keep the excess antibiotics for future use.
Conclusions: The knowledge, expectation, demand and self medication of antibiotics seems to be lower among parents in our population compared to European populations.
Bone Marrow Stromal Cells Attenuate Sepsis And Sepsis-Induced Acute Kidney Injury (AKI) Via A Novel Mechanism Of Action 2NEMETH K, 1LEELAHAVANICHKUL A, 1DOI K, 1HU X, 3WEISS JM, 2ROBEY PG, 1YUEN PST, 1STAR RA, 2E MEZEY 1Renal Diagnostics and Therapeutics Unit, NIDDK, NIH; 2NIDCR, NIH, Bethesda, MD; 3 NCI, NIH, Frederick MD, USA
Background: Acute kidney injury (AKI) in septic shock is associated with a high mortality in ICU patients. Bone marrow stromal cells (BMSCs), which are often referred to as mesenchymal stem cells, have been shown to improve outcome in a number of different animal injury models by altering inflammation, apoptosis, and necrosis. In this study we set out to determine whether BMSCs can attenuate the severity of sepsis-induced AKI.
Methods: Sepsis was induced in C57/BL6 mice using cecal ligation and puncture (CLP). BMSCs were obtained from the bone marrow of 6-8 week old mice.
Results: Intravenous administration of BMSCs (106 cells/mouse) immediately before CLP surgery resulted in significantly longer survival and improved kidney, liver and pancreatic function. Such organ protection was not obtained with either hematopoietic stem cells or necrotic BMSCs. Searching for a mechanism of action we found that 24 hs after CLP the serum levels of proinflammatory cytokines (TNF-, IL-6), peritoneal and kidney vascular permeability, splenic apoptosis and blood bacterial count were significantly reduced in BMSC-treated animals vs controls; the level of IL-10, an anti-inflammatory cytokine, was not affected. Six hours after their injection, fluorescently-labeled BMSCs were detected mostly in the lung. By prelabeling BMSCs with quantum dots and later performing immunostaining with a macrophage marker (Iba-1) we found the BMSCs adjacent to macrophages in the lung. Some were in the spleen, and rare cells were seen in the kidney; 24 hours after they were injected, we found few BMSCs in any organ. The positive effect of BMSC treatment was still present in Rag-/- mice; in NK cell depleted mice, and in IFN--/- mice, suggesting that B,T and NK cells, and IFN-do not play a significant role in mediating the effects of the BMSCs On the other hand, macrophage depletion or pretreatment with IL-10 or IL10 receptor antibodies eliminated the beneficial effects of BMSCs in the sepsis model.
Conclusion: Our results suggest that BMSCs may act on tissue macrophages resulting in enhanced production of IL-10, a potent anti-inflammatory cytokine.
Novel Antineoplastic Agent – Peptide Conjugates As Drug Delivery Systems For Targeted Chemotherapy MEZŐ G1, BAI KB1, SZABÓ I1, MANEA M2, ORBÁN E1, GAÁL D3, BŐSZE S1 1Research Group of Peptide Chemistry, Budapest, Hungary; 2Laboratory of Analytical Chemistry, Univ. Konstanz, Konstanz, Germany; 3National Institute of Oncology, Budapest, Hungary
Background: Receptor-mediated tumor-selective drug targeting by peptides might be an efficient tool in cancer therapy. The combination of appropriate drugs with a targeting moiety that recognizes tumor-specific or overexpressed receptors on tumor cells might lead to highly efficient chemotherapeutic agents.
Aims: 1) To develop new conjugates containing antineoplastic agents and peptides with own biological activity. 2) To study the structure-activity relationship in vitro. 3) To optimize the in vivo treatment of the selected compound.
Methods: Flow cytometry was used with FITC-labeled annexin-V and PI stain, to discriminate intact cells from apoptotic and necrotic cells (MonoMac6) after treatment with the oligotuftsin-Mtx conjugates and Mtx (10-8-10-6 M). In vitro antitumor activity was studied by MTT assay on MCF-7 human breast and C26 murine colon carcinoma cell lines. Treatments (i.p.) of 5-7 mice/group with free Daunorubicine (1x5 mg/kg or 5x2 mg/kg) or Dau-GnRH-III oxime conjugate (1x15 mg/kg or 5x5 mg/kg) were carried out on day 7 followed by repetition every second day in case of lower doses. The conjugate was applied in 2x15 mg/kg on days 4 and 7 or 7 and 10 after tumor transplantation.
Results: 1) Methotrexate-conjugate had cytotoxic effect. 2) Anthracycline-GnRH-III conjugates with different linkages had the following IC50 values (M): ester (0.8) < hydrazone (1.5) < oxime (3.9) < amide (>100) on MCF-7 cells, while somewhat higher on C26 cell line. 2) Dau-GnRH-III oxime conjugate had antitumor activity and it was not toxic up to 15 mg Dau content/kg body weight. The best result was ~50% tumor growth inhibition and 33% increase of survival time to the control on C26 tumor bearing mice in case of treatments on days 4 and 7 after tumor transplantation.
Conclusions: 1) Efficient drug delivery into monocytes and macrophages was obtained by the application of tuftsin-like carriers. 2) The type of ligation had an influence on the antitumor activity. 3) Dau-GnRH-III oxime conjugate prevented the toxic side effect of Dau even at a concentration higher than the lethal dose of the drug. Significant decrease of the tumor growth and increased survival time was determined in case of treatments with Dau-GnRH-III.
