Feb 24, 1988 - presence of glycyrrhizin sulfate, polysaccharide Krestin, dextran sulfate, ribofuranan sulfate, and lentinan sulfate was indistinguishable from ...
Vol. 26, No. 6
JOURNAL OF CLINICAL MICROBIOLOGY, June 1988, p. 1229-1232
0095-1137/88/061229-04$02.00/0 Copyright © 1988, American Society for Microbiology
Rapid Screening Method with a Cell Multisizer for Inhibitors of Human Immunodeficiency Virus-Induced Cell Fusion In Vitro HIDEKI NAKASHIMA, AKIKO TANABE, TADAFUMI S. TOCHIKURA, AND NAOKI YAMAMOTO* Department of Virology and Parasitology, Yamaguchi University School of Medicine, 1144 Kogushi,
Ube, Yamaguchi 755, Japan Received 8 December 1987/Accepted 24 February 1988 Cultivation of MOLT-4 and MOLT-4/HIVHTLV-IIIB cells induces syncytium formation very efficiently and is an appropriate model for evaluation of various substances which might inhibit human immunodeficiency virus (HIV)-induced multinucleated giant cell formation in vitro. We attempted here to quantify the grade of the syncytium formation by using a cell multisizer. The size distribution pattern of the cocultivated cells in the presence of glycyrrhizin sulfate, polysaccharide Krestin, dextran sulfate, ribofuranan sulfate, and lentinan sulfate was indistinguishable from that of cocultured cells grown in the absence of inhibitors. However, the pattern of cocultured cells without an inhibitor was quite different from that of MOLT-4 or MOLT-4/HIVHTLV IIIB tells alone. Moreover, the size distribution pattern of cocultured cells after treatment with two nucleoside analogs, 3'-azido-2',3'-dideoxythymidine and 2',3'-didehydro-2',3'-dideoxythymidine, which were known to inhibit cell-free but not cell-to-cell infection, was similar to that of cocultured cells without an inhibitor. These data are well correlated with the fusion index which was reported previously. Application of the cell multisizer is very quantitative for evaluating the syncytium formation induced by HIV and providing a simple ahd rapid screening for anti-HIV substances, especially for virus-inducèd cell fusion.
Human immunodeficiency virus (HIV) (1, 2, 5, 11) is the etiological agent associated with the acquired immune deficiency syndrome (AIDS) and its related disorders. Since the identification of HIV, many attempts have been made to develop agents that may be useful for the prevention and therapy of AIDS. It is generally accepted that HIV infect CD4-positive human T cells by either cell-free infection or a cell-to-cell fusion process, both of which lead to the loss of CD4-positive helper/inducer T cells. Thus, it is essential to develop systems to monitor cell-free infection and the HIVinduced cell-to-cell fusion process to search for anti-HIV agents. A trypan blue dye exclusion method, an indirectimmunofluorescence technique, and measurement of reverse transcriptase activity of cUlture fluids have been used to assess the inhibition of HIV-induced cytopathic effects, the expression of virus-specific antigens, and the replication of virus in the infected cells, respectively. A plaque assay for HIV which uses MT-4 cells which we have developed (4) is also very useful for evaluating the anti-HIV effect of manry substances. However, there has been no effective assay system for screening agents which inhibit HIV-induced giant cell formation up to now, because the extent of giant cell formation is highly variable and thus difficult to quantify. In this communication, we present a novel and quantitative screening method which uses a cell multisizer to evaluate substances which inhibit HIV-induced giant cell formation. MOLT-4 and MOLT-4/HIVHTLVIIIB cells were adjusted to a concentration of 5 x 105 cells per ml with RPMI 1640 medium supplemented with 10% fetal calf seruni and antibiotics, cultured for 24 h at 37°C in a CO2 incubator, and then monitored with the cell multisizer (Coulter Electronics Ltd., Luton, England) to determine the distribution of cell sizes (differentiation of particles). The size distribution curve of MOLT-4 cells was almost the same as that of MOLT4/HIVHTLV-IiB cells, and the percentage of particles with a diameter ranging between 9 and 20 ,um was 97.3 and 96.5% *
in MOLT-4 and MOLT-4/HIVHTLV cells, respectively. The proportion of particles with these size distributions was considered to be normal. When MOLT-4 and MOLT4/HIVHTLV-IIIB cells were mixed in a 1:1 ratio, adjusted to a final cell density of 5 x 105/ml, and cultured for 24 h, multinucleated giant cells were observed. The proportion of cells ranging in diameter between 9 and 20 ,m decreased to 82.8%, and that of cells with a diameter of more than 20 p.m increased to 17.2% instead (Fig. 1). An increase in the number of particles with a diameter of less than 9 ,um was also recognized with time after coculture, indicating an increase in the amount of cell debris, most probably due to destruction of cocultured cells. Next, we examined the dose-response effect of a potent inhibitor of HIV fusion, dextran sulfate (DS) (10), in the cocultivation of MOLT-4 and MOLT-4/HIVHTLV cells. Although the cell proportion having a diameter of more than 20 j±m was 30.5% in the drug-free control, it was decreased in a dose-dependent manner-9.2, 12.1, 12.4, 15.8, and 18.5% in the presence of 50, 10, 5, 1, and 0.5 ,tg of DS per ml, respectively (Fig. 2). When 100 ,ug of DS per ml was present in the coculture, the percentage of cells with a diameter of more than 20 ,um was only 3.6% and the size distribution pattern was indistinguishable from that of MOLT-4 cells alone (Fig. 3). The inhibitory ratio (IR) was calculated as follows: IR = [1 - (percentage with cell size >20 ,um in test well/percentage with cell size >20 ptm in control well)] x 100. Ik is linearly related to the log of the dose of DS (Fig. 4). Previously, we demonstrated that the fusion index (FI) was correlated with the inhibition of multinucleated giant cell formation, which was calculated as follows: FI = (number of viable cells in control MOLT-4 well/number of viable cells in test well) 1.0 (T. S. Tochikura, H. Nakashima, A. Tanabe, and N. Yamamoto, Virology, in press). The IR was also correlated with this FI very nicely (r value, 0.968) (Fig. 4). The value of FI increased with decreasing concentrations of DS; e.g., 0.01, IIIB
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FIG. 3. Size distribution pattern as determined by using the cell multisizer (top) and as determined on the basis of morphological changes (bottom; original magnification, x200) at 24 h after coculture with various known anti-HIV substances. The concentration of each substance was as described in the text. GLS, Glycyrrhizin sulfate; PSK, polysaccharide Krestin; RS, ribofuranan sulfate; LS, lentinan sulfate.
(DHT) (3), respectively; that of the drug-free control was 17.2%. Since this concentration of AZT inhibits fresh cellfree HIV infection completely, this result apparently shows that AZT does not inhibit the HIV-induced syncytium formation (9). However, in the presence of anti-HIV substances which have inhibitory effects on not only cell-free infection but also cell-to-cell infection, such as glycyrrhizin sulfate (1 mg/ml) (8), polysaccharide Krestin (0.5 mg/ml) (12), DS (100 ,ug/ml), ribofuranan sulfate (100 ,tg/ml) (10), and lentinan sulfate (100 ,ug/ml) (O. Yoshida, H. Nakashima, T. Yoshida, Y. Kaneko, I. Yamamoto, T. Uryu, K. Matsuzaki, and N. Yamamoto, submitted for publication), the percentages of cells with diameters of more than 20 ,um were only 5.7, 5.5,
3.6, 5.7, and 3.7%, respectively (Fig. 3). IRs of the cocultured cells in the presence of these compounds were 11.0, 9.0, 66.9, 68.0, 79.1, 66.9, and 78.5 in the presence of AZT, DHT, glycyrrhizin sulfate, polysaccharide Krestin, DS, ribofuranan sulfate, and lentinan sulfate, respectively. FIs of the cocultured cells in the presence of these compounds were again correlated with the IR of each culture-1.57, 1.84, 0.62, 0.52, 0.01, 0.23, and 0.01, respectively. We extended our study to screen antiviral agents against HIV-induced multinucleated giant cell formation to perform the test more quickly and objectively by application of the cell multisizer. Inhibition of giant cell formation by effective compounds was well characterized by the cell multisizer in comparison with that of ineffective agents such as AZT and DHT. Therefore, the application of the cell multisizer should be very useful for the screening of substances which inhibit HIV-induced giant cell formation.
2-1
We thank N. Kobayashi for this laboratory for helpful suggestions. This work was supported by Grants-in-Aid for Cancer Research from the Ministry of Education, Science, and Culture and the Ministry of Health and Welfare, Japan.
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LITERATURE CITED 1. Barre-Sinoussi, F., J. C. Chermann, F. Rey, M. T. Nugeyre, S. Chamaret, J. Gruest, C. Dauguet, C. Axier-Blin, F. VezinetBrun, C. Rouzioux, W. Rozenbaum, and L. Montagnier. 1983. Isolation of a T-lymphotropic retrovirus from a patient at risk for acquired immune deficiency syndrome (AIDS). Science 220: 868-871. 2. Coffin, J., A. Haase, J. A. Levy, L. Montagnier, S. Oroszlan, N. Teich, H. Temin, K. Toyoshima, H. Varmus, P. Vogt, and R.
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4. 5.
6.
7.
8.
NOTES
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Motoki, M. Ito, S. Shigeta, T. Mori, and N. Yamamoto. 1987. A new anti-human immunodeficiency virus substance, glycyrrhizin sulfate; endowment of glycyrrhizin with reverse transcriptase-inhibitory activity by chemical modification. Jpn. J. Cancer Res. (Gann) 78:767-771. Nakashima, H., T. Tochikura, N. Kobayashi, A. Matsuda, T. Ueda, and N. Yamamoto. 1987. Effect of 3'-azido-2',3'-dideoxythymidine (AZT) and neutralizing antibody on human immunodeficiency virus (HIV)-induced cytopathic effects; implication of giant cell formation for the spread of virus in vivo. Virology 159:169-173. Nakashima, H., O. Yoshida, T. S. Tochikura, T. Yoshida, T. Mimura, Y. Kido, Y. Motoki, Y. Kaneko, T. Uryu, and N. Yamamoto. 1987. Sulfation of polysaccharides generates potent and selective inhibitors of human immunodeficiency virus infection and replication in vitro. Jpn. J. Cancer Res. (Gann) 78: 1164-1168. Popovic, M., M. G. Sarngadharan, E. Read, and R. C. Gallo. 1984. Detection, isolation, and continuous production of cytopathic retroviruses (HTLV-II1) from patients with AIDS and pre-AIDS. Science 224:497-500. Tochikura, T. S., H. Nakashima, K. Hirose, and N. Yamamoto. 1987. A biological response modifier, PSK, inhibits human immunodeficiency virus infection in vitro. Biochem. Biophys. Res. Commun. 148:726-733.
