1.2nd Medical Clinic, Department of clinical Chemistry, UKE, Martinistraße 52, 2000 Hamburg 20, FRG.
2. Bernhard-Nocht Institute, Department of Virology, Bernhard-Nocht-Straßc 74, 2000 Hamburg 4, FRG.
3. DPZ, German Primate Center, Department of Virology and Immunology, Kellnerweg 4, 3400 Göttingen, FRG.
4. Technical University of Aachen, Depart ment of Clinical Chemistry and Pathobiochemistry, Pauwelsstraße 30, 5100 Aachen, FRG,
5. Laboratory of Tumor Cell Biology, National Cancer Institute (NCI), National Institutes of Health and Human Services (NIH), Bethesda, Maryland, USA.
* Parts of this study have been supported by the Karl und Veronika Carstens Stiftung im Stifterverband für die Deutschen Wissenschaften.
** Awarded with the "Henry Kaplan Award 1990 (Cell Biological Session)".

Introduction

The acquired immune deficiency syndrome (AIDS) is caused by an infection with the human immunodeficiency virus (HIV-1) [2 8]. CD4-positive lymphocytes were shown to be one major target in HIV-1 infections [9-10]. Apart of CD 4 + cell depletion, the functional impairment of the T -cell system also plays an important role in the progress of this disease [11,12,13]. Two distinct approaches to controlling HIV -1 infections have been explored so far, specifically, inhibition of the reverse transcriptase and inhibition of HIV -1 replication. For the first approach, inhibition of the virus replication, 3'-azido-3' deoxythymidine (AZT) [15] and its nucleoside analogues [16, 17], suramin and its derivatives [18], phosphonoformic acid [19], and antimoniotungstate [20] have been used. Inhibition of virus replication was demonstrated on the other hand using interferon-alfa [21, 22], AL 721 [23], D-penicillamine [24], amphotericin analogues [25], dextrane sulfate [36], chondroitine sulfate [36, 42], A varone [27], A varol [27], and synthetic oligonucleotides [26]. The need to obtain an effective principle for the treatment of AIDS prompted the search for selective and nontoxic antiHIV -1 agents even in medicinal plants. Some extracts with anti-HIV -1 properties have been isolated from medicinal plants of Chinese folk remedies [46], for instance, Altherantera philoxeroides [44], Viola yedoensis [45], and the chemically partially defined prunellin isolated from' Prunella vulgaris [43]. Most of these extracts and partially purified substances have shown in vitro anti-HIV-1 properties accompanied by some cytotoxic activities [43-46]. Lai et al. have reported a dose-dependent modification of the viral replication of HIV -1-infected CR 10, CEM, and U 937 cells by two defined extracts (PC 6 and PC 7) from the Japanese white pine (Pinus parvifloria Sieb. et Zucc.) [49]. In previous studies, extracts from Thuja occidentalis' L. (Arborvitae), another plant in the cedar/ pine family, were shown to be in vitro inhibitors of plant pathogenic viruses and human herpes simplex viruses (HSV-l strain) [34, 35]. In the present paper we are dealing with a new substance, the g fraction of thujapolysaccharides (TPSg), and its ability to modify HIV -1 replication in both human MT -2 and MT -4 cells as measured by determination of reverse transcriptase (R T) activity, cell growth (both MT -4 cell system), and the expression of HIV -1-specific proteins by indirect immunofluorescence (MT -2 cell system).

Materials and Methods

Virus and Cell Lines

The HIV -1 strain HTLV IIIb used for the MT -2 experiments was obtained from culture supernatants of virus-producing H 9 cells, as previously described [4]. MT -2 cells were maintained in RPMl 1640 (Gibco, Eggenstein, FRG) containing 15% fetal calf serum. MT -2 is a HTL V -l-preinfected human T -cellleukemia line and has been shown to be highly susceptible to infections with HlV -1 [28, 47]. MT -2 cells have been used as target cell lines for in vitro HIV-l infection experiments using indirect immunofluorescence assays [39]. H 9 cells used as the HlV -1 source for the MT -2 experiments were also maintained in RPMl 1640 (Gibco, Eggenstein, FRG) containing 15% fetal calf serum. This cell line was a kind gift from M. Popovic (NCl, Bethesda, Maryland, USA). MT -4 cells were kept in Click-RPMl medium (Biochrom, Berlin, FRG) containing 10% (v/v) complement-inactivated fetal bovine serum (Seromed, Berlin, FRG) and antibiotics. MT -4 cells are highly susceptible to in vitro HIV-l infections [39], too. For the in vitro infection experiments with MT -4 cells, the HTL V IIIb strain of HIV -1 was used. HIV-l has been generated on Jurkat cells as described in detail elsewhere [41]. Jurkat cells were also grown in RPMl 1640 medium (Gibco, Karlsruhe, FRG) with the supplements described above.

Virus Titration

For virus titration on MT -2 cells, cell-free supernatants were harvested from HIV1-infected H 9 cells. The virus titration was performed by indirect immunofluorescence. The quantitative determination of the infectious capability of the HIV -1 stocks was performed according to the method described by Kaerber et al. [31]. The HlV-1 preparations for the MT -2 experiments were shown to have a titer of 1 x 10 high 7 TClDso/ml. In the MT -4 system, a final infectious activity of lOO TCIDso for each well was used.

Indirect Immunofluorescence

For immunofluorescence experiments, both freshly HIV-l-infected and noninfected MT -2 cells were used and incubated for 12 days at 37C. For preparing the cell smears, HIV-l-infected and noninfected MT -2 cells were contrifuged for 10 min at 250 g. The supernatants were removed and the sediments resuspended in phosphate-buffered saline (PBS). Cell smears were performed on 10well multitest slides (Flow Lab., Meckenheim, FRG). The slides are air dried and fixed for 10 min in acetone at -20 C. A standardized HIV -l-positive human serum was used as reagent. Cell smears of HIV -l-infected and noninfected MT -2 cells were incubated for 60 min in a moist chamber at 37 CC with titrated serum of an AIDS patient (25 µ1/well; dilution 1 :20) [29,30]. HIV -1positive cells were visualized after incubation with FITC-conjugated goat antihuman immunoglobulin G (AHS Deutschland, Bereich Merz and Dade, Munich, FRG) for 30 min (25 µI/well; dilution 1 :200). As negative controls, sera of noninfected human individuals were used. The specific reaction was determined by fluorescence microscopical evaluation.

Determination of RT activity

Uninfected MT-4 cells or MT -4 cells infected with HIV -1 were treated with various concentrations of TPSg and incubated for 5 days under standard conditions. For the RT inhibition assay, HIV -1 was harvested from infected J urkat cells by centrifugation. The virus was then suspended in PBS at pH 7.2 and mixed with the same amount of ultrapure glycerol (Serva, Heidelberg, FRG). Different final concentrations of TPSg were examined in 50 mM Tris-HCI pH 7.8, mM dithiothreitol (DTT), 25 mM Mg2+, 30 mM KCI, 6% Triton X-100, 1 µg polyrC:oligodG, 9µM dGTP, 1µCi [32p]dGTP and lysed HIV-1. The R T assay was performed according to the procedure described previously [40, 42]. The influence of TPSg on virus production in infected MT -4 cells was monitored by R T activity in culture supernatants. The virus was prepared from the supernatants by centrifugation as described above and the RT assay was performed as shown.

[³ H ]Thymidine Incorporation

[³H]Thymidine incorporation experiments were performed according to standard procedures to measure HIV-1specific cytopathic effects on MT -4 cells [39]. The MT-4 assay was performed in 96-well micro titer plates as described previously [42].3 x 104 MT -4 cells/well were incubated with TPSg at 625 µg/ml, 62.5 µg/ml, 6.25 µg/ml and 625 ng/ml final concentrations, with or without HIV-l. The concentration of the infectious particles used was 100 TCID 50 for each well. Fresh Click-RPMI medium was added to each well 3 days after setup. 5 days after infection, 0.1 µCi [³H]thymidine (Amersham-Buchler, Brunswick, FRG; specific activity 185 GBq/mmol) was added to the cultures. The cells were harvested 20 h later on glass fiber filters (Whatman GFC, UK) using a Scatron cell harvester and dried. After addition of scintillation cocktail (PPO, POPOP, and toluene; Roth, Karlsruhe FRG) filters were counted in a ß-Iiquid scintillation counter. The results were expressed as the arithmetic mean in counts per minutes of triplicate determinations. As an alternative to the determination of the cellular DNA synthesis in the MT 4 assay, the cell growth was measured on day 5 after infection. Cell viability was assayed microscopically in a hematocytometer by trypan blue exclusion experiments and the R T activity was measured in the supernatant of the cultures.

Preparation of TPSg

Thujapolysaccharides, g-fraction (TPSg), from the Cupressaceae Thuja occidentalis L. (Arborvitae) was prepared as described in detail elsewhere (EPO 315182). TPSg was stored up to use lyophilized at -20 CC. TPSg was reconstituted in the appropriate cell culture media and was sterilized directly before use using 0.2 µm filter systems (Sartorius, FRG).

Results

The anti-HIV-1 activity and cytotoxicity of the polysaccharide fraction TPSg was examined in MT -2 and MT -4 cell culture systems. The ability of TPSg to inhibit the HIV -1-specific R Twas also examined. Finally, the 50% inhibitory concentration (IC5O) of TPSg on MT -4 cells was determined.

Protection of HIV -1-Dependent Cytopathic Effects by TPSg

TPSg inhibited HIV-l-dependent cell death at final concentrations of 625 µg/ml (Fig. 1). At this concentration TPSg was shown to be completely nontoxic for MT -4 cells, which had not been infected with HIV-l (Figs. 1, 2). This result was confirmed by comparing the cell growth of TPSg-treated infected and noninfected MT -4 cells (Fig. 2). These experiments were performed in triplicate and repeated three times.

Inhibition of H IV -1 Expression by TPSg

HIV -1-specific viral antigen expression was measured by indirect immunofluorescence. The inhibitory effect of TPSg was tested on freshly HIV-I-infected MT -2 cells. TPSg was shown to inhibit HIV-1specific antigen expression on freshly infected MT -2 cells in a dose-dependent manner (Figs. 3, 4). TPSg did not alter viral antigen expression at a concentration of 0.625µg/ml (99.6%+-0.5%). A significant reduction in HIV-1 antigens measured by immunofluorescence was observed at a concentration of 6.25 µg/ml 69.8%+-10.8% of HIV-1 infected MT-2 cells expressed HIV-1-specific antigens). Only 0.4% of all HIV-l-infected MT -2 cells counted (200 cells/slide) were shown to express HIV -1-specific antigens at final concentrations of 62.5 µg/ml, and an inhibition of 99.94%+-0.08% of HIV-1 expression was measured at the final TPSg concentrations of 625 µg/ml.

Fig.3. Indirect immunofluorcsccnce of freshly HIV-1-infected MT-2 cells. The cells were prepared as described in "Material and Methods." They were labelled with an anti serum against HIV -1 and FITC-conjugated goat anti-human IgG. This micrograph shows the non- TPSg-treated fresh]y H IV -1-infected MT -2 cells after 5 days of incubation. x 500

Inhibition of RT Activity by TPSg

As an additional approach, HIV -1 replication was determined by measuring R T activity in the supernatants of HIV -1infected MT -4 cells 5 days after infection. In uninfected MT -4 cells, no R T activity was detected in the culture medium after an incubation period of 5 days. In contrast to HIV -1-infected MT -4 cells not treated with TPSg, no R T -dependent dG MP incorporation was found in supernatants of infected MT -4 cells treated with final concentrations of TPSg of up to 62.5 µg/ml (Fig. 5). In addition, the inhibition of R T activity was measured with disrupted HIV-1. TPSg was found to be active against the enzyme (Fig. 6) with a IC5o of 300 µg/ml.

Discussion

Several authors have reported antiretroviral activities of plant extracts, for example, extracts of Prunella vulgaris [43], Alternanthera philoxcroidcs [44], Viola yedoensis [45], Gerardia savaglia [49] and some Chinese medicinal herbs [46, 50, 51]. Lai et al. have reported a dose-dependent modification of the viral replication of HIV-1-infected CR10, CEM, and U 937 cells by two defined extracts (PC 6 and PC 7) of the Japanese white pine (Pinus parvifloria Sieb. et Zucc.), a plant of the pine family [49]. Previously, extracts of Thuja occidcntalis L., another plant belonging to the cedar/ pine family, were shown to inhibit the cytolytic activity of herpes simplex virus

Fig.4. Indirect immunofluoresence of freshly HIV-1-infcctcd MT-2 cells treated with TPSg. The cells were prepared as described in "Material and Methods." They were labelled with an antiserum against HIV -1 and FITC conjugated goat anti-human TgG. This micrograph shows freshly HIV -1-infected MT -2 cells treated with TPSg 625 µg/ml after 5 days of incubation. x 500

Fig.5. R T activity in the supernatant of HTV 1-infected MT -4 cells treated with differentconcentrations of TPSg (median of three experiments). The cells were treated with final concentrations of TPSg of 625 ng/ml to625 µg/ml. The RT activity is expressed in picomoles dGMP incorporated into DNA

Fig.6. Inhibition of the RT activity of an HIV -1 lysate expressed in %. The 50% inhibitory dose of TPSg (ID5o) is extrapolated from the curve

type 1 and some plant pathogenic viruses in vitro [34, 35]. TPSg, a high molecular weigth polysaccharide fraction isolated from Thuja occidentali.s, was shown to be a compound with "immunomodulatory" properties. This compound was demonstrated to induce the proliferation of T -cells (CD4+) of the human peripheral blood [1, 37,48]. Furthermore, TPSg was shown to induce a different pattern of cytokines such as interleukin-1, interleukin-2, and interferon-y [32]. In the EALE/c system, TPSg was found to cause a modification in terms of upregulation of natural killer cell activity against Y AC-1 target cells [43]. These findings indicated possible antiviral properties of this compound. Hence, in this preliminary study, we have evaluated the antiretroviral potential of this compound. TPSg was found to inhibit the HIV1-dependent cell death of HIV -l-infected MT -4 cells at concentrations of 625 µg/ml. Additionally, it was shown to block the expression of HIV -l-specific proteins in freshly HIV-1-infected MT-2 cells in a dose-dependent manner, as judged by a 99.94% (99.6%) inhibition of the HIV -l-mediated specific immunofluorescences at a final concentrations of 625 µg/ml (62.5 µg/ml). TPSg completely blocks HIV-1 release into the culture supernatant at concentrations up to 62.5 µg/ml, as demonstrated by the lack of RT activity in the supernatants of HIV -1-infected MT -4 cells. Furthermore, TPSg blocks the R T of disrupted virus particles with an IC5o of 300 µg/ml. In the present paper, TPSg was demonstrated to be a compound with an inhibitory effect on both H1V-1 entry and HIV -1 absorption in both MT -2 and MT 4 cells. Even at high concentrations, it was shown to be nontoxic for MT -4 (Fig. 2) and MT -2 (data not shown) cells. Furthermore, it was demonstrated to be nontoxic for primary human leukocyte cultures (PEL), even at high concentrations [33]. In comparison with most of the plant extracts described above, TPSg therefore shows promising antiviral and immunomodulating properties. Since TPSg is only a partially purified natural product, isolation of the active principle(s) is required. This work is in progress. First hints in this direction were given by Hans et al. [38], who described the monosaccharide composition of sprouts and wood of the Arborvitae. Future investigations concerning this compound must rule out the possibility of its inducing autoimmune diseases and must show a lack of toxicity in vivo and mutagenicity in vitro. The present study might be a hint to further and more detailed investigations of the anti-HIV -1 properties of this compound. Whether TPSg might be of use in the therapy of primary and secondary immune deficiencies must be elucidated in further and more detailed investigations. The present study exemplifies the necessity of synergy of pharmacognostic research with molecular biology, clinical research, and immunology, to obtain new substances with significant immunomodulatory and antiviral properties.

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