946 resultados para Ames assay


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Contract No. W-7405-eng-48.

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Mode of access: Internet.

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Includes index.

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"Project 16030 ELZ; Program element 1BA027."

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The study was a comparison of bioassay and HPLC analysis of artesunate (ARTS) and dihydroartemisinin (DHA) in plasma. ARTS and DHA in plasma samples from patients treated with ARTS were quantified by HPLC and expressed as DHA. DHA-equivalents in the same plasma samples were measured using a standardised parasite culture technique. DHA concentrations estimated by both methods were highly correlated (bioassay = 0.96 x HPLC + 11.0; r(2) = 0.92). At high concentrations ( > 12 000 nmol/l) bioassay sometimes overestimated DHA. Bioassay of active drug in plasma correlates well with specific chemical analysis by HPLC. ARTS and DHA appear to account for the total antimalarial activity in plasma after ARTS administration. (C) 2003 Elsevier Science B.V. All rights reserved.

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The aim of this study was to develop a simple, field-practical, and effective in vitro method for determining the sensitivity of fresh erythrocytic Plasmodium vivax isolates to a range of antimalarials. The method used is a modification of the standard World Health Organization (WHO) microtest for determination of P.falciparum drug sensitivity. The WHO method was modified by removing leukocytes and using a growth medium supplemented with AB(+) serum. We successfully carried out 34 in vitro drug assays on 39 P. vivax isolates collected from the Mae Sod malaria clinic, Tak Province, Thailand. The mean percentage of parasites maturing to schizonts (six or more merozoites) in control wells was 66.5% +/- 5.9% (standard deviation). This level of growth in the control wells enabled rapid microscopic determination (5 min per isolate per drug) of the MICs of chloroquine, dihydroartemisinin, WR238605 (tafenoquine), and sulfadoxine. P. vivax was relatively sensitive to chloroquine (MIC = 160 ng/ml, 50% inhibitory concentration [IC50] = 49.8 ng/ml) and dihydroartemisinin (MIC = 0.5 ng/ml, IC50 = 0.47 ng/ml). The poor response of P. vivax to both tafenoquine (MIC = 14,000 ng/ml, IC50 = 9,739 ng/ml) and sulfadoxine (MIC = 500,000 ng/ml, IC50 = 249,000 ng/ml) was due to the slow action of these drugs and the innate resistance of P. vivax to sulfadoxine. The in vitro assay developed in our study should be useful both for assessing the antimalarial sensitivity of P. vivax populations and for screening new antimalarials in the absence of long-term P. vivax cultures.

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The in vitro growth of erythroid colonies in the absence of erythropoietin, known as endogenous erythroid colonies (EEC) forms part of the diagnostic criteria for polycythaemia vera (PV). The availability of EEC culture in routine laboratory setting is limited as culture methods are technically demanding, difficult to standardize, expensive and laborious. In this study, we assessed the performance characteristics of a simplified method using ammonium chloride red cell lysis followed by culture on commercially available, batch-tested, methylcellulose media. Seventy-six patients were included; four were secondarily excluded on the basis of culture failure. Of the 14 patients with PV, 13 (93%) were positive for EEC on at least one occasion: 90% (nine of 10) of bone marrow and 67% (six of nine) of peripheral blood specimens were positive. All 30 patients with secondary polycythaemia (n = 12) or apparent polycythaemia (n = 18) were negative for EEC. The incidence of EEC in idiopathic erythrocytosis was 40% (eight of 28); 50% (five of 10) in those who met one of the minor criteria for PV and 17% (three of 18) in those who did not. We conclude that our EEC assay yield results comparable with that of more elaborate methods.

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Dendritic cells (DC) from distinct DC subsets are essential contributors to normal human immune responses. Despite this, reliable assays that enable DC to be counted precisely have been slow to evolve. We have now developed a new single-platform flow cytometric assay based on TruCOUN(TM) beads and the whole blood Lyse/No-Wash protocol that allows precise counting of the CD14(-) blood DC subsets: CD11c(+)CD16(-) DC, CD11c(+)CD16(+) DC, CD123(hi) DC, CD1c(+) DC and BDCA-3(+) DC. This assay requires 50 mul of whole blood; does not rely on a hematology blood analyser for the absolute DC counts; allows DC counting in EDTA samples 24 It after collection; and is suitable for cord blood and peripheral blood. The data is highly reproducible with intra-assay and inter-assay coefficients of variation less than 3% and 11%, respectively. This assay does not produce the DC-T lymphocyte conjugates that result in DC counting abnormalities in conventional gradient-density separation procedures. Using the TruCOUNT assay, we established that absolute blood DC counts reduce with age in healthy individuals. In preliminary studies, we found a significantly lower absolute blood CD11c(+)CD16(+) DC count in stage III/IV versus stage I/II breast carcinoma patients and a lower absolute blood CD123(hi) DC count in multiple myeloma patients, compared to age-matched controls. These data indicate that scientific progress in DC counting technology will lead to the global standardization of DC counting and allow clinically meaningful data to be obtained. (C) 2003 Elsevier B.V. All rights reserved.