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Activation of Human Immunodeficiency Virus Type 1 Expression
by Gardnerella vaginalis
Farhad B. Hashemi, Mahmood Ghasse...
JID 1999;179 (April) Effect of G. vaginalis on HIV Replication 925
PMA or tumor necrosis factor (TNF)-a [18]. The HIV-1MN ...
926 Hashemi et al. JID 1999;179 (April)
Figure 1. Dose-dependent activation of HIV-1 expression in U1 cells
by 4 heated or...
JID 1999;179 (April) Effect of G. vaginalis on HIV Replication 927
Figure 3. Physical characterization of HIV-activating f...
928 Hashemi et al. JID 1999;179 (April)
Figure 5. NF-kB and AP-1 binding activity in U1 cells treated with G. vaginalis ly...
JID 1999;179 (April) Effect of G. vaginalis on HIV Replication 929
Table 1. The effect of G. vaginalis lysates on cytokine...
930 Hashemi et al. JID 1999;179 (April)
8. European Collaborative Study. Risk factors for mother-to-child transmission
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  1. 1. 924 Activation of Human Immunodeficiency Virus Type 1 Expression by Gardnerella vaginalis Farhad B. Hashemi, Mahmood Ghassemi, Kenneth A. Roebuck, and Gregory T. Spear Department of Immunology/Microbiology, Rush University, and Department of Medicine, Section of Infectious Diseases, University of Illinois at Chicago Bacterial vaginosis (BV) is associated with an increased rate of sexual transmission of human immunodeficiency virus (HIV) type 1, and Gardnerella vaginalis is frequently isolated from the genital tracts of women with BV. G. vaginalis lysates were found to significantly stimulate HIV expression in monocytoid cells. Stimulation was significantly higher when lysates were heated at 100ЊC for 5 min but was reduced by treatment with lysozyme or protease. G. vaginalis lysates also activated HIV expression in certain T cell lines. G. vaginalis lysates activated HIV long-terminal repeat transcription in HIV-infected cells and increased NF-kB binding activity, indicating an effect by G. vaginalis on HIV transcription. The activation of HIV production by G. vaginalis suggests that genital tract infection with G. vaginalis increases the risk of HIV transmission by increasing HIV expression in the genital tract. This may explain, at least in part, the increased rate of HIV transmission in women with BV. Sexual transmission of human immunodeficiency virus (HIV) continues to be the major route of the infection, accounting for 70%–80% of cases worldwide [1–3]. In the United States, women are a growing demographic group of newly diagnosed AIDS cases [4, 5]. Retrospective population studies [6–9] and a recent prospective investigation [10] show that vaginal infec- tions, such as bacterial vaginosis (BV) and sexually transmitted diseases, are associated with an increased risk of HIV trans- mission. BV is a common disorder characterized by changes in the vaginal flora in which the normally predominant lactobacilli are replaced by potential pathogens such as Gardnerella vagin- alis, genital Mycoplasma infections, and eventually anaerobic bacteria, accompanied by an increase in vaginal pH [11]. BV-associated microorganisms may increase the incidence of sexual transmission of HIV by several mechanisms. For ex- ample, cell-associated or secreted products of microorganisms may affect the differentiation or proliferation state of HIV tar- get cells in the genital tract, leading to increased susceptibility to HIV infection [12]. Alternatively, infected cells, such as mac- rophages, T cells, and dendritic cells in the genital tract may be stimulated by these organisms or their products, leading to Received 24 July 1998; revised 9 December 1998. The subjects in this study gave informed consent as a part of the Women’s Interagency HIV Study at Rush-Presbyterian St. Luke’s Medical Center. Human investigation guidelines of the US Department of Health and Hu- man Services and of Rush-Presbyterian St. Luke’s Medical Center were observed. Grant support: NIH (AI-34993, AI-31812). Reprints or correspondence: Dr. Farhad B. Hashemi, Dept. of Immu- nology/Microbiology, Rush University, 1653 W. Congress Pkwy., Chicago, IL 60612 ( The Journal of Infectious Diseases 1999;179:924–30 ᭧ 1999 by the Infectious Diseases Society of America. All rights reserved. 0022-1899/99/7904-0020$02.00 increased HIV expression. Focusing on the latter mechanism, we hypothesized that G. vaginalis can increase HIV expression. G. vaginalis, a gram-variable, rod-shaped bacteria, is com- monly isolated from the female genital tract [13]. Since G. va- ginalis is among the bacteria frequently (р95%) associated with BV [14], we studied whether G. vaginalis can modulate HIV expression in infected cells. Materials and Methods Organisms and cells. G. vaginalis cultures were grown on choc- olate agar plates (Remel Microbiology Products, Lenexa, KS) at 37ЊC for 48 h in a humidified atmosphere with 5% CO2. In this study, we used G. vaginalis ATCC strain 14018 (American Type Culture Collection, Rockville, MD), referred to as isolate B, and 3 genital isolates from patients. A genital tract yeast isolate (Can- dida albicans) and coagulase (Ϫ) staphylococci (referred to as Staphylococcus species—not aureus or SSNA) was isolated from 1 of the clinical samples and cultured as above. Lactobacillus aci- dophilus was isolated from a clinical specimen from the genital tract and grown in MRS broth (Remel) under anaerobic conditions. Microorganisms were identified by standard procedures recom- mended by the American Society for Microbiology [15]. We obtained the U1, U38, J1.1, and HeLa cervix epitheloid carcinoma cell lines from the AIDS Research and Reference Re- agent Program (Division of AIDS, National Institute of Allergy and Infectious Diseases, NIH, Rockville, MD; U1 and J1.1 cells contributed by T. Folks; U38 cells by B. Felber and G. Pavlaskis; and HeLa cells by R. Axel) [16–18]. Both U1 and U38 are trans- formed monocytoid cells derived from the U937 cell line. The for- mer contains a stable copy of the complete HIV-1IIIB genome and expresses very low amounts of CD4 [16]. The latter has the HIV-1 long-terminal repeat (LTR) promoter linked to the chloramphen- icol acetyl transferase (CAT) gene and is a very sensitive indicator of HIV-1 transactivating (Tat) protein [17]. The J1.1 cells are la- tently HIV-1–infected Jurkat T cells capable of being induced with atTehranUniversityofMedicalScienceonMarch9,2011jid.oxfordjournals.orgDownloadedfrom
  2. 2. JID 1999;179 (April) Effect of G. vaginalis on HIV Replication 925 PMA or tumor necrosis factor (TNF)-a [18]. The HIV-1MN and HIV-1RF preparations were also obtained from the AIDS Research and Reference Reagent Program [19, 20]. The human peripheral blood–acute lymphocytic leukemia (HPB-ALL) cell line is a trans- formed T lymphocyte cell line [21]. All cell lines were maintained in RPMI 1640 medium supple- mented with 10 mM HEPES, 2 mM glutamine, and 10% fetal bovine serum (FBS; BioWhittaker, Walkersville, MD). This culture medium is referred to as complete medium. The cells were fed twice weekly using complete medium. Prior to each experiment, cell vi- ability was routinely checked by trypan blue dye exclusion. All cells were negative for mycoplasma by polymerase chain reaction [22]. Preparation of lysates from G. vaginalis, L. acidophilus, coagulase (Ϫ) staphylococci, and yeast. Culture plates growing confluent lawns of G. vaginalis, SSNA, or C. albicans were harvested, washed with PBS, centrifuged (700 g) for 10 min at 24ЊC, and then resus- pended in 3 mL of PBS. For L. acidophilus, broth cultures were centrifuged for 10 min at 700 g after 24 h of incubation. The bacterial pellet was then resuspended in 5 mL of PBS. Lysates were prepared using bacterial pellets as previously described [23]. In brief, suspensions were sonicated on ice for 3 min using a flat-tip probe sonifier (model 250; Branson Ultrasonics, Danbury, CT) at setting 4. Prior to sonication, harvested cultures were tested for purity by culturing a sample on chocolate agar plates. The protein concentration of lysates was determined by BSA protein assay (Pierce, Rockford, IL). Lysates were stored at Ϫ20ЊC until use. Prior to bioassay, lysates were diluted in complete medium to a final concentration of 5000, 500, or 50 ng/mL lysate protein. Stimulation of U1, J1.1, HPB-ALL, and HeLa cells by G. va- ginalis lysates. To test the HIV-inducing activity, U1 or4 4 ϫ 10 J1.1 cells were incubated with lysates in microtiter plate wells at 37ЊC. After 3 days, culture supernatant fluids were collected, mixed with Triton X-100 (final concentration 0.1%), and p24 concentra- tions were measured by ELISA (National Cancer Institute, Rock- ville, MD). Similar procedures were used for the HPB-ALL cul- tures, except that before treatment with lysates, HPB-ALL cells were infected with HIV-1 as described previously [24]. In brief, HPB-ALL cells were incubated with HIV-1MN (6 4 2 ϫ 10 3 ϫ 10 TCID/mL) at 37ЊC for 2 h. Cells were then washed once and re- suspended in complete medium at cells/mL and treated with5 5 ϫ 10 lysates followed by culture for 3 days at 37ЊC. Where appropriate, complete medium, TNF-a (50 U; R&D Systems, Minneapolis), or PMA (100 ng/mL) were included as controls. HeLa cells ( total) were incubated with 5000 ng/mL pro-5 2 ϫ 10 tein of heated lysate in 24-well plates (Corning, New York, NY) at 37ЊC. After 3 days, culture supernatant fluids were collected, and cytokine concentrations were determined. Cytokine measurements by ELISA. Cytokine levels in super- natant fluids of Hela cell cultures were determined by ELISA. To test interleukin (IL)-8 concentrations, we used an ELISA (Cyto- Screen; Biosource International, Camarillo, CA). For TNF-a, IL- 1a, and IL-6 measurements, we used CYTELISA kits (CYT- immune, College Park, MD). All determinations were made ac- cording to manufacturers’ recommendations. Heat, lysozyme, and protease treatments of G. vaginalis lysates. For heat treatment, G. vaginalis lysates were heated at 100ЊC in a water bath for 5 min. For protease treatment, proteinase K (Sigma, St. Louis) was added to heated G. vaginalis lysates at 10 mg/mL final concentration. For lysozyme treatment, egg white lysozyme (Sigma) was added to heated lysates at 250 mg/mL final concen- tration. The mixture of enzyme plus lysate were incubated for 30 min at 37ЊC and then heated again at 100ЊC for 5 min. Samples were diluted in complete medium and added to culture wells. As controls, the G. vaginalis lysates and enzymes were incubated for 30 min at 37ЊC in separate tubes. Samples were then heated and added to the cells. Stimulation of HIV-LTR activity by G. vaginalis lysates. HIV LTR activity was measured as previously described [17]. Briefly, U38 cells were infected with HIV-1RF (∼100 pg of p246 3 ϫ 10 content) in the presence or absence of G. vaginalis–heated lysates (5000 ng/mL protein). After 48 h, cells were harvested and washed. Protein extracts were prepared by 4 freeze-thaw cycles of cell pellets. Pellets were resuspended in 100 mL of 0.25 M Tris (pH 7.8), frozen at Ϫ70ЊC for 10 min, and thawed at 37ЊC. After the fourth thaw, samples were centrifuged for 5 min, and protein concentrations of supernatants were determined by protein assay (Bio-Rad Labo- ratories, Hercules, CA). Equal amounts of protein extract were analyzed by CAT assay as described [25] with minor modifications. In brief, 50 mL of cell extract, adjusted to contain 100 mg of protein, was incubated for 5 h at 37ЊC with 50 mL of CAT assay mix (containing 20 mL of 0.01 Ci/mL [3 H]chloramphenicol [DuPont, Boston], 5 mL of 5 mg/ mL butyl CoA [Sigma], 5 mL of 2 M Tris-Cl, pH 8, and 20 mL of distilled water). The acetylated chloramphenicol was then extracted with the addition of 200 mL of 2:1 (vol/vol) tetramethylpentade- cane/xylenes (Sigma) by vigorous shaking. After the tubes were centrifuged, 85% of the organic phase was transferred to scintil- lation vials and counts per minute were determined. Electrophoretic mobility shift assays (EMSA). U1 cells were washed in serum-free RPMI 1640 and resuspended in RPMI sup- plemented with 0.5% FBS. After overnight incubation at 37ЊC, cells were adjusted to /mL and treated with heated G. vaginalis6 5 ϫ 10 lysate (5000 ng/mL protein) for 2 h at 37ЊC. Cells were then washed in PBS and centrifuged (500 g) for 5 min. Cell pellets were used to prepare nuclear protein extracts for the EMSA by the method of Osborn et al. [26] with the addition of proteinase inhibitors. Extracted proteins (5 mg) were incubated with AP-1 or NF-kB 32 P- labeled oligonucleotide (AP-1, 5-CGCTTGATGAGTCAGCCG- GAA-3; NFk-B, 5-AGTTGAGGGGACTTTCCCAGGC-3; Pro- mega, Madison, WI) for 30 min at 24ЊC. Positive controls included extracts from cells stimulated with PMA or TNF-a. Incubated mixtures were separated on a 5% nondenaturing polyacrylamide gel. Scanning and densitometry were done by personal scanner SI (Molecular Dynamics, Sunnyville, CA). To generate stimulation index ratios, the density of an empty lane on the scanned film was selected as the background value for band comparison. Results Stimulation of HIV production in U1 monocytic cells by G. vaginalis lysates. In order to determine the effect of G. va- ginalis on HIV expression, lysates from 4 G. vaginalis isolates were prepared by sonication. The resulting lysates were then added to cultures of the chronically HIV-infected U1 cell line. These cells have been used as a model to test the effects of atTehranUniversityofMedicalScienceonMarch9,2011jid.oxfordjournals.orgDownloadedfrom
  3. 3. 926 Hashemi et al. JID 1999;179 (April) Figure 1. Dose-dependent activation of HIV-1 expression in U1 cells by 4 heated or unheated G. vaginalis lysates. Isolates were incubated with U1 cells at 50, 500, or 5000 ng/mL G. vaginalis lysate protein; heated and unheated lysates of coagulase (Ϫ) staphylococci (SSNA) and Lactobacillus acidophilus at 50, 500, or 5000 ng/mL lysate protein were also used. Control media (dashed line). After 3 days, culture supernatant fluids were collected, and p24 concentrations were mea- sured by ELISA. Values are mean of triplicate culture wells. Data are representative of 2 separate experiments. Figure 2. Effects of G. vaginalis lysates on HIV expression in J1.1 T cells. Heated or unheated G. vaginalis lysates were incubated with J1.1 or HIV-infected HPB-ALL cells at 50, 500, or 5000 ng/mL lysate protein. After 3 days, p24 concentrations in culture supernatant fluids were measured by ELISA. Controls included TNF-a (50 U/well) and media (dashed line). Data are mean of triplicate culture wells from 1 experiment. various substances, including cytokines and bacterial products [27, 28] on HIV replication in monocytoid cells. Although untreated U1 cells produced low levels of HIV as measured by release of p24 into culture medium, addition of 5000 ng/mL G. vaginalis lysate protein significantly increased HIV replication compared with that in cells cultured in medium alone ( , one-group t test; figure 1). The increase wasP ϭ .003 13- to 77-fold higher than in untreated cells. For cells that received 500 ng/mL G. vaginalis lysate protein, the increase in p24 production was 6- to 11-fold above control cultures (figure 1). Lower amounts of lysate (50 ng/mL; figure 1) did not sig- nificantly increase HIV production. As shown in figure 1, in contrast to G. vaginalis lysates, lysates of genital tract–derived coagulase (Ϫ) staphylococci (SSNA), L. acidophilus, and C. albicans (not shown), at lysate protein levels of 50–5000 ng/ mL, did not increase HIV production in U1 cells. To assess the heat stability of the stimulatory factor in G. vaginalis, the lysates were heated at 100ЊC for 5 min. The heated lysates at 5000 and 500 ng/mL stimulated higher p24 produc- tion from U1 cells than unheated lysates ( and .04,P ϭ .002 respectively, paired t test; figure 1). Addition of heated lysates from isolates C and D at 50 ng/mL also increased p24 pro- duction 18- to 20-fold higher than control, whereas isolates A and B at 50 ng/mL isolates did not affect p24 production. These results show that the stimulatory activity from G. vaginalis is not only heat-stable, but that 100ЊC treatment for 5 min also increases its activity. Stimulation of HIV production in T lymphocyte lines by G. vaginalis lysates. The ability of G. vaginalis lysates to stim- ulate HIV replication in 2 T lymphocytic cell lines was also evaluated. Although the effect of G. vaginalis in J1.1 cells was less remarkable than in U1 cells, as a group, G. vaginalis lysates (5000 ng/mL lysate protein) significantly increased HIV ex- pression in J1.1 than in cells cultured in medium alone (P ϭ , one-group t test; figure 2). Isolate B did not stimulate HIV.017 expression at any concentration; however, for isolates A, C, and D, the increase in p24 production was 1.2- to 2.6-fold above control cultures. For cells that received 500 ng/mL G. vaginalis lysate protein of isolates C and D, the increase in p24 produc- tion was 1.7- and 2.0-fold over control cultures, respectively (figure 2). Isolates A and B did not stimulate p24 production in J1.1 cells at 500 ng/mL lysate protein (figure 2). Overall, the group of J1.1 cultures treated with 500 ng/mL lysates did not show any significant increase over control cultures ( ,P ϭ .13 one-group t test; figure 2). Similar to U1 cells, neither 50 ng/ mL lysate protein (figure 2) nor C. albicans lysates (not shown) affected p24 production by J1.1 cells. Heating significantly increased stimulatory activity of the lys- ates at 5000 ng/mL compared with unheated samples (P ϭ , paired t test; figure 2). Although less marked, heat also.006 enhanced the stimulatory activity of lysates at the 500 ng/mL protein lysate level ( , paired t test; figure 2). The J1.1P ϭ .09 cells responded to TNF-a (50 U) treatment by production of pg/mL p24 protein (not shown).5 4 ϫ 10 atTehranUniversityofMedicalScienceonMarch9,2011jid.oxfordjournals.orgDownloadedfrom
  4. 4. JID 1999;179 (April) Effect of G. vaginalis on HIV Replication 927 Figure 3. Physical characterization of HIV-activating factor(s) from G. vaginalis lysates. Heated lysates (5000 ng/mL lysate protein) from 4 G. vaginalis isolates were incubated with lysozyme (250 mg/mL) or proteinase K (10 mg/mL) and heated at 100ЊC for 5 min to inactivate enzymes then incubated with U1 cells. Culture supernatant fluids were collected after 3 days, and p24 levels were measured by ELISA. Data represent 2 separate experiments run in triplicate culture wells. Figure 4. Effect of G. vaginalis lysates on HIV long-terminal repeat activation in U38 cells. Cells were treated with heated G. vaginalis lysates (5000 ng/mL lysate protein) in presence or absence of exogenous HIVRF. After 48 h, cells were harvested, and chloramphenicol acetyl transferase (CAT) activity of cellular extract was determined. Data are representative of 3 independent experiments. Lysates from G. vaginalis isolates A and C were also tested for stimulatory activity on the HPB-ALL T cell line (not shown). In contrast to U1 and J1.1 cells, heated or unheated lysates from these isolates did not stimulate any increase in HIV production by HPB-ALL cells, whereas TNF-a (50 U) induced HIV production by these cells (not shown). Physical properties of the G. vaginalis stimulatory substance. Although the above experiments showed that the stimulatory activity in G. vaginalis lysates was heat-stable, further char- acteristics of the stimulatory activity were determined. Lysates from isolates were heated at 100ЊC and then treated with either lysozyme or proteinase K. Both lysozyme and proteinase K treatment significantly reduced the ability of the lysates to stim- ulate HIV production by U1 cells (figure 3), suggesting that the stimulatory substance(s) contained a protein, lipoprotein, or peptidoglycan-like component that was required for full ac- tivity. Control samples containing only enzymes had no effect on p24 production by U1 cells (not shown). In further experiments, G. vaginalis lysates were separated into soluble and particulate material by ultracentrifugation and then added separately to U1 cells. All of the recoverable stim- ulatory activity for all 4 G. vaginalis isolates was associated with the particulate fraction (not shown), suggesting that the stimulatory component is associated with the G. vaginalis cell wall or membrane. Taken together, these results provide evi- dence that the HIV activating factor(s) may be a protein de- rivative associated with the G. vaginalis cell wall or membrane. Effect of G. vaginalis on HIV transcription through activation of HIV-LTR. In order to determine the effect of G. vaginalis on HIV-1 LTR promoter activity, U38 cells were infected with HIV in the presence or absence of heated G. vaginalis lysates followed by measurement of HIV-LTR activity by CAT assay. Since the U38 cells have the HIV-1 LTR promoter linked to the CAT gene, they are a sensitive indicator of HIV-1 transac- tivating (Tat) protein [17]. As expected, HIV infection of U38 cells enhanced HIV-LTR CAT transcription. When cells were infected in the presence of lysates, the enhancement of HIV-LTR CAT expression was sig- nificantly higher than in untreated HIV-infected cells (P ϭ , one-group t test; figure 4). The increases over cells that.018 were HIV-infected but not treated with lysates ranged from 1.6- to 3.4-fold for isolates A and D, respectively. Incubation with G. vaginalis lysates did not increase CAT expression in unin- fected U38 cells (not shown). Effect of G. vaginalis lysates on transcription factors NF-kB and AP-1. The mechanism of stimulation of HIV production by G. vaginalis lysates was further investigated by assessing induction of transcription factors NF-kB and AP-1 in U1 cells. Lysates from G. vaginalis isolates A and B induced increases in NF-kB binding activity (figure 5A) that corresponded to about a 15-fold induction of NF-kB binding over untreated U1 cells. In contrast, G. vaginalis lysates did not affect AP-1 binding in these cells (figure 5B). Effect of G. vaginalis lysates on cytokine production by HeLa epithelial cells. Another possible mechanism of increasing HIV replication in the genital tract by G. vaginalis is to induce proinflammatory cytokine production by epithelial cells. To de- termine whether G. vaginalis stimulates these cells, Hela cells were incubated with heated G. vaginalis lysates (5000 ng/mL lysate protein) for 72 h, and proinflammatory cytokines levels in culture supernatant fluids were measured. Heated G. vaginalis lysates from isolates A and B did not increase secretion of IL- 6, IL-1a, TNF-a, or IL-8 by Hela cells (table 1). In contrast, the positive controls increased secretion of these cytokines (ta- ble 1). atTehranUniversityofMedicalScienceonMarch9,2011jid.oxfordjournals.orgDownloadedfrom
  5. 5. 928 Hashemi et al. JID 1999;179 (April) Figure 5. NF-kB and AP-1 binding activity in U1 cells treated with G. vaginalis lysates. U1 cells were treated with heated lysates of G. vaginalis (5000 ng/mL lysate protein) for 2 h at 37ЊC. Complete medium, tumor necrosis factor (TNF)-a, and PMA served as controls. Nuclear protein extracts were analyzed by electrophoretic mobility shift assay using consensus NFk-B (A) or AP-1 (B) probes. Autoradiographs were scanned and optical density was measured by densitometry. Density of empty lane on scanned film was selected as background (index ) forratio ϭ 1 comparison of bands. Bar graph compares stimulatory ratio of lysate-treated U1 cells with cells treated with PMA, TNF-a, and complete medium alone. Discussion BV is associated with a higher incidence of sexual transmis- sion of HIV. The process by which microbial or host-related factors in the genital tract leads to increased risk of HIV trans- mission is not well understood. We hypothesized that G. va- ginalis, which is frequently associated with BV, activates HIV expression in cells in the genital tract. This activation could then contribute to the higher risk of HIV sexual transmission by increasing the amount of virus in the genital tract. To test this hypothesis, we evaluated the effect of G. vaginalis on HIV replication. We found that G. vaginalis activated HIV expression in monocytoid and in certain T cells. The HIV-inducing effect was specific to G. vaginalis, since common nonpathogenic bacteria often isolated in the vaginal tract (e.g., coagulase [Ϫ] staphy- lococci and L. acidophilus) did not induce HIV expression. All 4 G. vaginalis isolates used in this study stimulated HIV pro- duction by U1 cells, although the stimulatory activity of some isolates was modest unless the lysates were heated. These results suggest that some strains of G. vaginalis may be more effective at increasing HIV expression in the genital tract than others. Although we have not studied the mechanism by which heat treatment releases the HIV-inducing activity by G. vaginalis lysates, it is possible that in the vaginal tract, factors such as degradative enzymes or the low pH of vaginal secretions release the activity of G. vaginalis similar to how heat acts on G. va- ginalis components in vitro. Thus, our data support the hy- pothesis that increased HIV production by the cells in the gen- atTehranUniversityofMedicalScienceonMarch9,2011jid.oxfordjournals.orgDownloadedfrom
  6. 6. JID 1999;179 (April) Effect of G. vaginalis on HIV Replication 929 Table 1. The effect of G. vaginalis lysates on cytokine production by HeLa cells. Cytokine concentration (pg/mL) IL-6 IL-1 TNF-a IL-8 G. vaginalis A !16 59 !16 96 G. vaginalis B 744 9 109 78 Culture medium 907 16 161 214 TNF-a (50 U) 11,844 55 NA 2301 NOTE. Cells ( ) were incubated with G. vaginalis (5000 ng/mL lysate5 2 ϫ 10 protein) at 37ЊC. Lysates were heated (100ЊC for 5 min) before incubation. After 3 days, culture supernatant fluids were collected and cytokine levels were deter- mined by ELISA. Values are mean of duplicate cultures; data are representative of 3 separate experiments. IL, interleukin; TNF, tumor necrosis factor; NA, not applicable. ital tract may affect the risk of HIV transmission by increasing virus load in the genital tract. It will be of interest to investigate whether other organisms associated with BV also activate HIV expression independent or in combination with G. vaginalis. We hypothesized that G. vaginalis could also activate HIV replication in the genital tract indirectly by inducing secretion of proinflammatory cytokines by epithelial cells. In fact, other bacteria from the genital tract, such as Chlamydia species, have increased inflammatory cytokine production [29], and some studies have shown increased IL-1a, IL-8, IL-1b, IL-6, and TNF-a in vaginal secretions of pregnant women with BV [30, 31]. However, G. vaginalis did not stimulate production of IL- 1a, IL-6, TNF-a, or IL-8 in epithelial cells. Perhaps the lack of inflammatory cytokine secretion by epithelial cells in re- sponse to G. vaginalis is one way this organism evades the immune response. For instance, Darveau et al. [32] recently reported that Porphromonas gingivalis not only does not induce IL-8 secretion by epithelial cells but that it also degrades IL-8 and inhibits its production. Although we have not identified the HIV-inducing factor(s) in G. vaginalis lysates, the evidence of its heat stability and its sensitivity to protease suggest that the active component is at least in part a heat-stable lipoprotein or proteoglycan. The HIV activation seems to be specific to G. vaginalis components, since other vaginal gram-positive bacteria with generally similar cell wall structure, such as Staphylococcus species (not aureus) and L. acidophilus, did not induce HIV expression. The reduction of activity after lysozyme treatment combined with the recovery of HIV-inducing activity from only the particulate part of the lysate indicate that the activating factor(s) is a cell wall or membrane-associated component of G. vaginalis, such as the proteogylcans. In fact, ultrastructure studies reveal that G. va- ginalis has no outer membrane and the thin peptidoglycan layer of cell wall is exposed on the surface [33]. That the enzymes had various degrees of effect on the G. vaginalis lysates from different isolates suggests a qualitative or quantitativedifference in isolates. For example, the side chains on the active protein or proteoglycan could vary or the concentration of the active factor(s) may vary in different isolates. The up-regulation of HIV by G. vaginalis was probably not due to lipopolysaccharide (LPS) for the following reasons. First, G. vaginalis does not possess LPS [33]. Second, flow cy- tometric analysis of U1 cells by our laboratory confirmed re- ports by other investigators [34] that U1 cells do not express the LPS receptor (CD14 molecule) unless they are stimulated with granulocyte-macrophage colony-stimulating factor. In ad- dition, in our hands, Escherichia coli LPS at 1 mg/mL (Sigma) had no effect on HIV replication in U1 cells (unpublished data). Thus, the activation of HIV by G. vaginalis was probably not due to LPS. The induction of HIV expression by G. vaginalis in chroni- cally infected U1 cells indicates an effect on the expression of HIV after integration of the virus rather than infection by HIV. This is consistent with the observation that the G. vaginalis lysate also activated HIV replication in chronically infected J1.1 cells. Increased HIV-driven CAT activity induced by G. vagin- alis indicates that the mechanism by which G. vaginalis lysates stimulated increased production of virus is due to up-regulation of HIV transcription. Since the presence of exogenous HIV was required for the HIV-LTR activation by the G. vaginalis lysates in U38 cells, we conclude that the G. vaginalis–mediated en- hancement of HIV expression involves a pathway that requires HIV infection. We believe this to be the first report to demonstrate that G. vaginalis activates HIV expression. These findings warrant fur- ther studies to evaluate the in vivo significance of G. vaginalis activation of HIV-1. 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