Anti-rheumatic compound aurothioglucose inhibits tumor necrosis factor-{alpha}-induced HIV-1 replication in latently infected OM10.1 and Ach2 cells

Katrina E. Traber1, Hiroshi Okamoto1, Chieko Kurono2, Masanori Baba3, Claude Saliou1,4, Tsuyoshi Soji2, Lester Packer4 and Takashi Okamoto1

1 Departments of Molecular Genetics and
2 Anatomy, Nagoya City University Medical School,Nagoya 467-8601, Japan
3 Division of Human Retroviruses, Center for Chronic Viral Diseases, Faculty of Medicine, Kagoshima University, Kagoshima 890-0075, Japan
4 Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA

Correspondence to: T. Okamoto


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
NF-{kappa}B is a potent cellular activator of HIV-1 gene expression. Down-regulation of NF-{kappa}B activation is known to inhibit HIV replication from the latently infected cells. Gold compounds have been effectively used for many decades in the treatment of rheumatoid arthritis. We previously reported that gold compounds, especially aurothioglucose (AuTG) containing monovalent gold ion, inhibited the DNA-binding of NF-{kappa}B in vitro. In this report we have examined the efficacy of the gold compound AuTG as an inhibitor of HIV replication in latently infected OM10.1 and Ach2 cells. Tumor necrosis factor (TNF)-{alpha}-induced HIV-1 replication in OM10.1 or Ach2 cells was significantly inhibited by non-cytotoxic doses of AuTG (>10 µM in OM10.1 cells and >25 µM in Ach2 cells), while 25 µM of the counter-anion thioglucose (TG) or gold compound containing divalent gold ion, HAuCl3, had no effect. The effect of AuTG on NF-{kappa}B-dependent gene expression was confirmed by a transient CAT assay. Specific staining as well as electron microscopic examinations revealed the accumulation of metal gold in the cells, supporting our previous hypothesis that gold ions could block NF-{kappa}B–DNA binding by a redox mechanism. These observations indicate that the monovalent gold compound AuTG is a potentially useful drug for the treatment of patients infected with HIV.

Keywords: AIDS, antiviral, NF-{kappa}B, rheumatoid arthritis, viral latency


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
NF-{kappa}B is a cellular transcription factor found in a large number of cell types, and is able to regulate a wide variety of cellular genes including cytokines such as IL-6, IL-8 and tumor necrosis factor (TNF)-{alpha} (for review see 1–4). In addition to cellular gene regulation, NF-{kappa}B has also been shown to be a potent activator of HIV-1 gene expression (46). NF-{kappa}B usually exists in the cytoplasm in a complex with an inhibitory molecule, I{kappa}B (7). Upon stimulation of the cells, I{kappa}B dissociates and NF-{kappa}B translocates to the nucleus where it site-specifically binds to DNA to activate the expression of target genes (7,8). In cells latently infected with HIV-1, activation of NF-{kappa}B could trigger the transcription of viral genes including the trans-activator Tat which would result in an explosive increase in HIV-1 replication (9,10). Thus, down-regulation of NF-{kappa}B activity has long been sought in order to inhibit the latently infected HIV and prevent clinical development of AIDS in the HIV-infected individuals (11,12).

Gold compounds such as aurothioglucose (AuTG), auranofin and aurothiomalate have been effectively used for many years in the treatment of rheumatoid arthritis (RA) (called `chrysotherapy'). Chrysotherapy has been shown to reduce the production of cytokines IL-6 and IL-8 in serum (13), monocytes (14), macrophages (15) and in synovial cells (1618) of RA patients. Since these cytokine productions are known to be under the control of NF-{kappa}B, NF-{kappa}B was considered to be one of the targets of chrysotherapy (19). In vitro, monovalent gold compounds such as AuTG were shown to inhibit the DNA binding (20) of NF-{kappa}B, which was consistent with an experiment using cell lines (19). We demonstrated that the monovalent gold ion Au(I), not the divalent gold ion Au(II), could efficiently block NF-{kappa}B–DNA binding and proposed a possible mechanism of its action: Au(I) may oxidize the thiolate anions, associated with Zn2+, on the NF-{kappa}B molecule into disulfides and thus abrogate the DNA-binding activity due to the higher oxidation potential of Au(I) over Zn2+ (20). It has yet to be proven, however, that cells treated with AuTG should contain an accumulation of metal gold as the result of reduction by thiolate anions.

In this study we investigated the efficacy of AuTG as a potential anti-HIV agent using the cell lines OM10.1 (21) and Ach2 (22), which are latently infected with HIV-1, as a model system. The effects of AuTG on HIV-1 gene expression was examined by a transient CAT assay. We also examined accumulation of metal gold by a specific staining procedure and transmission electron microscopy.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Reagents
AuTG, thioglucose (TG), HAuCl3, {alpha}-lipoic acid, reduced glutathione (GSH), ß-nicotinamide adenine dinucleotide phosphate, reduced form (NADPH), glutathione reductase, dithiobis-2-nitrobenzoic acid (DTNB) and 5-sulfosalicylic acid (5-SSA) were supplied by Sigma (St Louis, MO). Stock solutions of AuTG, TG and HAuCl3 were made in deionized water and stored at –80°C.

Cell culture
The human monocytic cell line latently infected with HIV-1, OM10.1 (21), and the human T cell line latently infected with HIV-1, Ach2 (22), were maintained at 0.2–1.0x106 cells/ml in RPMI 1640 supplemented with 10% (v/v) FCS. To maintain the latency of the HIV-1 in these cells, 20 µM AZT was added in culture media and was excluded at least 2 weeks before experiments. Cultures were incubated with AuTG, TG or HAuCl3 for either 3, 6 or 12 days and the cell media were changed every 3 days.

p24 antigen assay
Viral p24 antigen levels in the cell supernatant of OM10.1 and Ach2 cells were determined using the Retro-tek HIV-1 p24 antigen ELISA kit (Cellular Products, Buffalo, NY). Samples were harvested from cells which had been stimulated with 5 ng/ml TNF-{alpha} for 24 h. The concentration of p24 was determined based on a standard curve constructed from authentic p24.

CAT assay
Jurkat cells were transfected with 2 µg of pHIVSCAT by LipofectAMINE (Gibco/BRL, Rockville, MD). Cells were harvested 24 h after the stimulation with TNF-{alpha} and lysed in Triton lysis buffer. CAT assays were performed as previously described using 3H-labeled acetyl coenzyme A (23,24). Experiments were performed 3 times and SEM were indicated as bars.

GSH assay
Total cellular GSH concentration was measured essentially as described previously (25,26). Briefly, samples (~107 cells) were centrifuged for 5 min at 2000 g and the cell pellet was re-suspended in 200 µl of 2.5% 5-SSA to precipitate the proteins. Samples were then centrifuged for 10 min at 10,000 g and the supernatant was isolated. Then 10 µl of sample was combined with 200 µl of a premix solution of 1 mM DTNB (Ellman's reagent) and 0.35 mM NADPH, incubated for 5 min, then 40 µl of 8.5 IU/ml of GSSG reductase was added. The resulting chromophore, TNB, was monitored spectrophotometrically at 405 nm. Quantitation was achieved by comparison with the absorption of known GSH concentrations, carried out in parallel.

Staining of metal gold in the cells
OM10.1 cells were incubated with various concentrations of AuTG for 12 days. Cells were fixed with 2.5 % glutaraldehyde in PBS and stained with p-dimethylaminobenzylidene rhodamine according to the method previously described (27).

Electron microscopy
Cell suspensions were centrifuged for 10 min at 200 g and the cell pellet was immersed for 30 min in a fixative containing 2.5% glutaraldehyde and 2% sucrose in 0.05 M cacodylate buffer (pH 7.4). Fixed samples were washed for 30 min and post-fixed for 2 h with 1% osmium tetroxide buffered by 2% sucrose and 0.05 M sodium cacodylate (pH 7.4). After post-fixation, the samples were subsequently dehydrated in a graded series of ethanol and then embedded in epoxy resin (28) without propylene oxide penetration procedure. Thin sections were stained with uranyl acetate and lead citrate, and then observed by using a transmission electron microscope (H-7000; Hitachi, Tokyo, Japan).


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
AuTG, but not TG or HAuCl3, inhibits HIV-1 replication in vitro
To evaluate if AuTG inhibited TNF-{alpha}-stimulated HIV-1 replication, we incubated Ach2 or OM10.1 cells for 6 days with either AuTG, TG (the counter-anion of AuTG) or HAuCl3, a compound containing Au(II). Cell cultures were then stimulated with 5 ng/ml TNF-{alpha} for 24 h to induce NF-{kappa}B and activate HIV-1 replication. Then the HIV-1 p24 concentrations in cell-free supernatants was analyzed. As shown in Fig. 1Go(A), in OM10.1 cells, at concentrations >=10 µM AuTG, p24 levels in the cell supernatant were significantly decreased. In contrast, 25 µM TG had no effect and 25 µM HAuCl3 significantly increased p24 concentrations. In the Ach2 cells (Fig. 2AGo), a similar effect was observed; 25 µM AuTG significantly inhibited p24 levels. Moreover, we did not see any significant cytotoxicity (Figs 1B and 2BGoGo). The concentrations of gold compounds used in the previous experiments were similar to those found in the synovial joint tissue of RA patients under chrysotherapy (29). In a shorter incubation with AuTG for 3 days, there was no significant effect on the TNF-{alpha}-induced HIV-1 replication. When cells were further incubated for 12 days, a more profound effect was observed, although significant cytotoxicity was observed (Fig. 3Go). After 12 days of incubation with 100 µM AuTG, the p24 antigen levels were below control levels. However, there was also a high level of toxicity. After 9 days of incubation, there was a significant decrease in the viability of cells treated with 100 µM AuTG and levels of cell replication had decreased in cells treated with 25 and 100 µM AuTG (data not shown). After 12 days of incubation, the viability of cells treated with 10, 25 and 100 µM AuTG had decreased to 60% of the control (Fig 3BGo).



View larger version (16K):
[in this window]
[in a new window]
 
Fig. 1. Effects of gold compounds on HIV-1 replication in latently infected OM10.1 cells. (A) p24 antigen expression in OM10.1 cells. Cells were incubated with 0, 4, 10 and 25 µM AuTG, 25 µM TG, and 25 µM HAuCl3 for 6 days. Cultures were then stimulated with TNF-{alpha} for 24 h. (B) Cell viability in OM10.1 cells treated with 0, 4, 10 and 25 µM AuTG for 6 days. Each value indicates the mean of three replicate cultures. Error bars show SD values.

 


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 2. Effects of gold compounds on HIV-1 replication in latently infected Ach2 cells. (A) p24 antigen expression in Ach2 cells. Cells were incubated with 0, 4, 10 and 25 µM AuTG, 25 µM TG, and 25 µM HAuCl3 for 6 days. Cultures were then stimulated with TNF-{alpha} (5 ng/ml) for 24 h. (B) Cell viability in Ach2 cells treated with 0, 4, 10 and 25 µM AuTG for 6 days. Each value indicates the mean of three replicate cultures. Error bars show SD values.

 


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 3. Effects of long-term treatment of OM10.1 cells by AuTG. (A) OM10.1 cells were incubated with the indicated concentrations of AuTG for 12 days and the amount of HIV p24 antigen was measured 24 h after stimulation with 5 ng/ml. Each value indicates the mean of three replicate cultures. Error bars show SD values. The asterisk indicates that nearly half of the cells in this column were dead, probably due to the cytotoxicity of 100 µM AuTG. (B) Cell viability in OM10.1 cells treated with 0, 10, 25 and 100 µM AuTG (•) or TG ({circ}) for 12 days.

 
Inhibition of the TNF-{alpha}-induced activation of HIV-1-LTR CAT by AuTG
To study the effect of AuTG on TNF-{alpha}-stimulated transcription of HIV-1-LTR, we performed CAT assays with Jurkat cells treated with AuTG or TG. Jurkat cells were incubated for 6 days with various concentrations of AuTG or TG following transfection with HIV-1-LTR CAT reporter plasmid (pHIVSCAT). After 24 h, these cells were stimulated with 5 ng/ml TNF-{alpha} for 24 h to induce NF-{kappa}B activation and stimulate transcription from HIV-1-LTR. Then the CAT enzymatic activity was measured to analyze the transcriptional activation from HIV-1-LTR. As shown in Fig. 4Go, AuTG inhibited trans-activation by TNF-{alpha} in a dose-dependent manner, whereas 25 µM TG had little inhibitory effect on the trans-activation. There was no effect of AuTG on the basal transcription level using a reporter plasmid containing a mutated {kappa}B sequence (data not shown). These results indicate that AuTG inhibited trans-activation of HIV-1-LTR by inhibiting TNF-{alpha}-induced NF-{kappa}B activation.



View larger version (33K):
[in this window]
[in a new window]
 
Fig. 4. Effects of AuTG on HIV-1 gene expression. Jurkat cells were incubated for 6 days with various concentration of AuTG or TG following the transfection with HIV-1-LTR CAT reporter plasmid (pHIVSCAT). These cells were then stimulated with 5 ng/ml TNF-{alpha} for 24 h to induce NF-{kappa}B and activate gene expression from HIV-1-LTR. The CAT enzymatic activity was measured to analyze the transcriptional activation of HIV-1-LTR. Experiments were performed 3 times and SEM are indicated.

 
AuTG does not increase cellular GSH
We have previously shown in vitro that AuTG might block the DNA binding of NF-{kappa}B by catalyzing oxidation of its redox-sensitive cysteins (5,20). However, it is also possible that AuTG may inhibit NF-{kappa}B activation by changing the redox status of the cell, thereby blocking the NF-{kappa}B activation cascade. To address this question, we measured the total GSH level, as an indicator of redox status, in the OM10.1 cells. As shown in Fig. 5Go(A), AuTG did not significantly increase total GSH levels. This was compared with the effect of {alpha}-lipoic acid, an antioxidant which was previously shown to inhibit HIV-1 replication and NF-{kappa}B activation through a redox pathway (4,30) (Fig. 5BGo). In contrast to the action of AuTG, treatment of cells with {alpha}-lipoic acid resulted in a dose-dependent increase of GSH which correlated with a decrease in p24 antigen.



View larger version (16K):
[in this window]
[in a new window]
 
Fig. 5. GSH concentration and p24 expression in OM10.1 cells (A) Cells were incubated with 0, 4, 10 and 25 µM AuTG for 6 days. GSH was measured after 6 days from an aliquot of culture. Cells were then stimulated with TNF-{alpha} for 24 h and assayed for p24 antigen. (B) Cells were incubated with 0, 50, 100, 500 and 1000 µM {alpha}-lipoic acid for 24 h. GSH was measured after 24 h from an aliquot of culture. Cells were then stimulated with TNF-{alpha} for 24 h and assayed for p24 antigen. GSH (•) and p24 antigen ({blacksquare}). Each value indicates the mean of three replicate cultures. Error bars show SD values. Asterisks indicate the statistical significance (P < 0.01).

 
Accumulation of metal gold in the AuTG-treated cells
Experiments described in Figs 1–3GoGoGo indicated a cumulative nature of the effect of AuTG over time during incubation with the cell. In addition, our previous findings with AuTG (20) indicated conversion of gold monovalent ions into metal gold. Therefore, in order to examine the possible accumulation of metal gold in the OM10.1 cells treated with AuTG, cells were stained with p-dimethylaminobenzylidene rhodamine (27). As shown in Fig. 6Go, accumulation of metal gold was demonstrated in a dose-dependent manner for the AuTG concentration.



View larger version (56K):
[in this window]
[in a new window]
 
Fig. 6. Metal staining of OM10.1 cells treated with AuTG. Cells were treated with the indicated concentrations of AuTG for 12 days, fixed by 2.5% paraformaldehyde and stained by p-dimethylaminobenzylidene rhodamine. Since the only heavy metal ion contained in the culture media was gold, the dense stained material in the cells was considered metal gold. Original magnification: x400.

 
We then examined the intracellular accumulation of metal gold by transmission electron microscopy. Figure 7Go demonstrates the electron microscopic morphology of the OM10.1 cells treated for 6 days with either 25 µM AuTG, 25 µM HAuCl3 or 25 µM TG. Although the cells treated with 25 µM TG (Fig. 7AGo) or 25 µM HAuCl3 (Fig. 7CGo) showed no cytological changes as compared with control (Fig. 7AGo), the cells treated with AuTG exhibited characteristic changes in mitochondria. As shown in Fig. 7Go(D), after treatment with 25 µM AuTG most of the mitochondria in OM10.1 cells were filled with electron-dense material, most likely due to gold metal, and cristae were irregularly arranged and distorted. In the cells treated with 4 µM AuTG only a few mitochondria contained electron-dense material (data not shown). It was also noted that in the cells treated with AuTG, no HIV virus production was observed, whereas in control cells HIV virion production was demonstrated in the cytoplasmic vesicles (e.g. see the inset of Fig. 7AGo).



View larger version (208K):
[in this window]
[in a new window]
 
Fig. 7. Electron microscopic examination of OM10.1 cells treated with gold compounds. (A) Control cells (x8000), (B) treated with 25 µM TG (x8000), (C) treated with 25 µM HAuCl3 (x8000) and (D) treated with 25 µM AuTG (x30,000). The cell organella in the cells treated with TG (B), HAuCl3 (C) or control (A) appeared to be normal. Inset of (A) shows HIV virions in the cytoplasmic vesicle (indicated by arrows) as well as normal morphology of mitochondria. In the cells treated with AuTG (D), a number of degenerating mitochondria were observed in the cytoplasm. In such degenerating mitochondria, small dense granules were observed (inset of D). It seemed that the dense inclusions which were scattered in cytoplasm might be transferred and accumulated into the mitochondria matrix (numbers in the inset indicate successive changes of mitochondria). Scale bars are indicated at 1 µM except for the insets (0.3 µm).

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
We have demonstrated that the gold compound AuTG significantly inhibited TNF-{alpha}-induced HIV-1 replication in latently infected OM10.1 and Ach2 cells. Inhibition occurs at the concentrations of AuTG which are clinically relevant and similar to those found in patients receiving chrysotherapy (29). These observations are reinforced by the recent report by Shapiro et al. (31) that chrysotherapy of a patient with psoriatic arthritis who was infected with HIV resulted in a significant and sustained increase in CD4 cells. Although gold compounds have been used for decades to treat patients with RA, their mechanism of action is still not entirely known. In the present study, it is most likely that AuTG leads to a decrease in HIV replication by inhibiting NF-{kappa}B action.

In chrysotherapy, the active component of AuTG seems to be the monovalent gold ion, Au(I), and not the counter-anion, TG (1820). This is supported by our present data which shows that Au(I) alone had an effect on HIV-1 replication since the counter-anion TG had no inhibitory effect (Figs 1–3GoGoGo). The inhibitory effect of AuTG was more profound in OM10.1 cells than in Ach2 cells, most likely due to the fact that monocyte/macrophage cells reacted more strongly to TNF-{alpha} in the induction of HIV-1 replication, as previously reported (32,33).

There are a few possibilities as to how gold compounds inhibit NF-{kappa}B action. First, gold compounds may be able to change the redox status of the cell and therefore prevent the activation of NF-{kappa}B by inhibiting the redox-controlled signal transduction pathway (34). However, we have shown, using total cellular GSH levels as a measure of the redox status of the cell, that AuTG did not change the redox status of the cell. Incubation of cells with AuTG did not result in a significant increase in total GSH concentration (Fig. 4AGo) unlike other compounds known to block NF-{kappa}B activation (4,30,35,36). These observations indicate that AuTG inhibits NF-{kappa}B in a manner other than through the modulation of the redox status of the cell.

A second possibility is that the monovalent gold ion can block the DNA-binding activity of NF-{kappa}B as suggested by the in vitro study (18,20). Gold compounds like AuTG as well as another monovalent gold compound, aurothiomalate, inhibited the DNA binding of NF-{kappa}B, but not nuclear translocation (18). We have also demonstrated that the NF-{kappa}B, upon treatment with oxidative reagents such as diamide, lost the DNA-binding activity even before the dissociation of I{kappa}B in vitro (5,24,26). In addition, we and others (20,37) have reported that in order to bind to DNA, NF-{kappa}B requires Zn2+. It was shown in Yang et al. (20) that Au(I) could inhibit the NF-{kappa}B–DNA binding by oxidizing the protein thiols associated with Zn and not by replacing them, at least in vitro (20). We thus proposed a possibility that Au(I) oxidizes the redox-sensitive thiolate anions (cysteine residues) on the NF-{kappa}B molecule into the disulfide form to limit DNA-binding activity. This oxidation should result concomitantly in the accumulation of free gold metal in the cell. We then examined the AuTG-treated cells by specific staining for heavy metals and found accumulation of metal gold (Fig. 6Go). Electron microscopic examinations have also revealed accumulation of electron dense materials, most likely gold metal, in the cytoplasm (Fig. 7Go). It was noted that this electron-dense material appeared to be located in the mitochondria, which is presumably due to the presence of the high concentration of free electrons in mitochondria. However, it is also possible for gold to interact with proteins other than NF-{kappa}B. So, the mechanism by which gold inhibits NF-{kappa}B binding in vivo needs to be further investigated.

In conclusion, we have established the efficacy of the monovalent gold compound AuTG as an anti-HIV replication agent. Since it is a compound that has been safely and effectively used in humans for years, it should be considered as a potential choice of therapy for the prevention of the clinical development of AIDS among those who are infected with HIV.


    Acknowledgments
 
This work was supported by grants-in-aid from the Ministry of Health and Welfare, the Ministry of Education, Science and Culture of Japan and from the Human Science Foundation.


    Abbreviations
 
5-SSA5-sulfosalicylic acid
AuTGaurothioglucose
DTNBdithiobis-2-nitrobenzoic acid
GSHglutathione
RArheumatoid arthritis
TGthioglucose
TNFtumor necrosis factor

    Notes
 
Transmitting editor: K. Okumura

Received 27 August 1998, accepted 13 October 1998.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Baeuerle, P. A. and Baichwal, V. R. 1997. NF-{kappa}B as a frequent target for immunosuppressive and anti-inflammatory molecules. Adv. Immunol. 65:111.[ISI][Medline]
  2. Baldwin, A. S. 1996. The NF-{kappa}B and I{kappa}B proteins: new discoveries and insights. Annu. Rev. Immunol. 14:649.[ISI][Medline]
  3. Thanos, D. and Maniatis, T. 1995. NF-{kappa}B: a lesson in family values. Cell 80:529.[ISI][Medline]
  4. Okamoto, T., Sakurada, S., Yang, J.-P. and Merin, J. P. 1997. Regulation of NF-{kappa}B and disease control: identification of a novel serine kinase and thioredoxin as effectors for signal transduction pathway for NF-{kappa}B activation. Curr. Topics Cell. Reg. 35:149.[ISI][Medline]
  5. Hayashi, T., Ueno, Y. and Okamoto, T. 1993. Oxidoreductive regulation of nuclear factor kappa B. J. Biol. Chem. 268:11380.[Abstract/Free Full Text]
  6. Okamoto, T., Josephs, S. F., Sadaie, M. R. and Wong-Staal, F. 1990. Transcriptional activation from the long-terminal repeat of human immunodeficiency virus in vitro. Virology 177:606.[ISI][Medline]
  7. Baeuerle, P. A. and Baltimore, D. 1988. I-kappa B: a specific inhibitor of the NF-kappa B transcription factor. Science 242:540.[ISI][Medline]
  8. Ghosh, S. and Baltimore, D. 1990. Activation in vitro of NF-kappa B by phosphorylation of its inhibitor I-kappa B. Nature 344:678.[ISI][Medline]
  9. Arya, S. R., Guo, C., Josephs, S. F. and Wong-Staal, F. 1985. Trans-activator gene of human lymphotropic virus type III (HTLVIII). Science 229:74.[ISI][Medline]
  10. Okamoto, T. and Wong-Staal, F. 1986. Demonstration of virus-specific transcriptional activator(s) in cells infected with HTLV-III by an in vitro cell-free system. Cell 47:29.[ISI][Medline]
  11. Okamoto, T., Matsuyama, T., Mori, S., Manamoto, Y., Kobayashi, N., Yamamoto, N., Josephs, S. F., Wong-Staal, F. and Shimotohno, K. 1989. Augmentation of human immunodeficiency virus type 1 gene expression by tumor necrosis factor alpha. AIDS Res. Hum Retroviruses 5:131.[ISI][Medline]
  12. Bohnlein, E., Lowenthal, J. W., Siekevitz, M., Ballard, D. W., Franza, B. R. and Greene, W. C. 1988. The same inducible nuclear proteins regulates mitogen activation of both the interleukin-2 receptor-alpha gene and type 1 HIV. Cell 53:827.[ISI][Medline]
  13. Madhok, R., Crilly, A., Murphy, E., Smith, J., Watson, J. and Capell, H. A, 1993. Gold therapy lowers serum interleukin levels in rheumatoid arthritis. J. Rheumatol. 20:630.[ISI][Medline]
  14. Crilly, A., Madhok, R., Watson, J., Capell, H. A. and Sturrock, R. D. 1994. Production of interleukin-6 by monocytes isolated from rheumatoid arthritis patients receiving second-line drug therapy. Br. J. Rheumatol. 33:821.[ISI][Medline]
  15. Yanni, G., Nabil, M., Farahat, M. R., Poston, R. N. and Panayi, G. S. 1994. Intramuscular gold decreases cytokine expression and macrophage numbers in the rheumatoid synovial membrane. Ann. Rheum. Dis. 53:315.[Abstract]
  16. Loetscher, P., Dewald, B., Baggiolini, M. and Seitz, M. 1994. Monocyte chemoattractant protein 1 and interleukin 8 production by rheumatoid synoviocytes. Effects of anti-rheumatic drugs. Cytokine 6:162.[ISI][Medline]
  17. Seitz, M, Dewald, B., Ceska, M., Gerber, N. and Baggiolini, M. 1992. Interleukin-8 in inflammatory rheumatic diseases: synovial fluid levels, relation to rheumatoid factors, production by mononuclear cells, and effects of gold sodium thiomalate and methotrexate. Rheumatol. Int. 12:159.[ISI][Medline]
  18. Yoshida, S., Kato, T., Sakurada, S., Kurono, C., Yang, J.-P., Matsui, N., Soji, T. and Okamoto, T. 1999. Inhibition of IL-6 and IL-8 induction from primary rheumatoid synovial fibroblasts by treatment with aurothioglucose. Int. Immunol. 11:151.[Abstract/Free Full Text]
  19. Williams, D. H., Jeffery, L. J. and Murray, E. J. 1992. Aurothioglucose inhibits induced NF-{kappa}B and AP-1 activity by acting as an IL-1 functional antagonist. Biochim. Biophys. Acta 1180:9.[ISI][Medline]
  20. Yang, J.-P., Merin, J. P., Nakano, T., Kato, T., Kitade, S. and Okamoto, T. 1995. Inhibition of the DNA-binding activity of NF-{kappa}B by gold compounds in vitro. FEBS Lett. 361:89.[ISI][Medline]
  21. Butera, S. T., Rerez, V. L., Wu, B.-Y., Nabel, G. J. and Folks, T. M. 1991. Oscillation of the human immunodeficiency virus surface receptor is regulated by the state of activation in a CD4+ cell model of chronic infection. J. Virol. 65:4645.[ISI][Medline]
  22. Folks, T. M., Clouse, K. A., Justement, J., Rabson, A., Duh, E., Kehrl, J. H. and Fauci, A. S. 1986. Tumor necrosis factor alpha induces expression of human immunodeficiency virus in a chronically infected T-cell clone. Proc. Natl Acad. Sci. USA 86:2365.
  23. Luo, Y., Madore, S. J., Parslow, T. G., Cullen, B. R. and Peterlin, B. M. 1993. Functional analysis of interactions between Tat and the trans-activation response element of human immunodeficiency virus 1 in cells. J. Virol. 67:5617.[Abstract]
  24. Okamoto, T., Ogiwara, H., Hayashi, T., Mitsui, A., Kawabe, T. and Yodoi, J. 1992. Human thioredoxin/adult T cell leukemia-derived factor activates the enhancer binding protein of human immunodeficiency virus type 1 by thiol redox control mechanism. Int. Immunol. 4:811.[Abstract]
  25. Vandeputte, C., Guizon, I., Genestie-Denis, I., Vannier, B. and Lorenzon, G. 1994. A microtiter plate assay for total glutathione and glutathione disulfide contents in cultures/isolated cells: performance study of a new miniaturized protocol. Cell Biol. Toxicol. 10:415.[ISI][Medline]
  26. Gallagher, E., Kavanagh, T. and Eaton, D. 1994. Glutathione, oxidized glutathione and mixed disulfides in biological samples. Methods Toxicol. 1B:349.
  27. Okamoto, K. and Utamura, M. 1938. Biologische untersuchungen des Goldes. 1 Mitteilung uber die histochemische Gold nachweis-methode. Acta Scholae. Med. Kioto. 22:373.
  28. Luft, J. 1961. Improvement in epoxy resin embedding methods. J. Biophys. Biochem. Cytol. 9:409.[Abstract/Free Full Text]
  29. Mascarenhas, B., Granda, J. and Freyberg, R. 1972. Gold metabolism in patients with rheumatoid arthritis treated with gold compounds—reinvestigated. Arthritis Rheum. 15:391.[ISI][Medline]
  30. Merin, J. P., Matsuyama, M., Kira, T., Baba, M. and Okamoto, T. 1996. Alpha-lipoic acid blocks HIV-1 LTR-dependent expression of hygromycin resistance in THP-1 stable transformants. FEBS Lett. 394:9.[ISI][Medline]
  31. Shapiro, D. L. and Masci, J. R. 1996. Treatment of HIV associated psoriatic arthritis with oral gold. J. Rheumatol. 23:1818.[ISI][Medline]
  32. Jacque, J. M., Fernandez, B., Arenzana-Seisdedos, F., Thomas, D,. Baleux, F., Virelizier, J. L. and Bachelerie, F. 1996. Permanent occupancy of the human immunodeficiency virus type 1 enhancer by NF-kB is needed for persistent viral replication in monocytes. J. Virol. 70:2930.[Abstract]
  33. Sato, T., Asamitsu, K., Yang, J.-P., Takahashi, N., Tetsuka, T., Yoneyama, A., Kanagawa, A. and Okamoto, T. 1998. Inhibition of HIV-1 replication by fasudil hydrochloride. AIDS Res. Hum. Retroviruses 14:293.[ISI][Medline]
  34. Vint, I. A., Foreman, J. C. and Chain, B. M. 1994. The gold anti-rheumatic drug auranofin governs T cell activation by enhancing oxygen free radical production. Eur. J. Immunol. 24:1961.[ISI][Medline]
  35. Tozawa, K., Sakurada, S., Kohri, K. and Okamoto, T. 1995. Effects of anti-nuclear factor {kappa}B reagents in blocking adhesion of human cancer cells to vascular endothelial cells. Cancer Res. 55:4162.[Abstract]
  36. Sakurada, S., Kato, T. and Okamoto,T. 1996. Induction of cytokines and ICAM-1 by proinflammatory cytokines in primary rheumatoid synovial fibroblasts and inhibition by N-acetyl-L-cysteine and aspirin. Int. Immunol. 8:1483.[Abstract]
  37. Zabel, U., Schreck, R. and Baeuerle, P. A. 1991. DNA binding of purified transcription factor NF-kappa B: Affinity, specificity, Zn2+ dependence, and differential half-site recognition. J. Biol. Chem. 266:252.[Abstract/Free Full Text]