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Department of Immunology and Microbiology and Section of Infectious Diseases, Rush University
Section of Infectious Diseases, University of Illinois at Chicago, Chicago
Background.
Bacterial vaginosis (BV) is associated with complications of pregnancy and increased susceptibility to human immunodeficiency virus (HIV) sexual transmission.
Methods.
The ability of genital mucosal fluids from women with BV and of microbial flora associated with BV to induce tumor necrosis factor (TNF) secretion and Toll-like receptor (TLR) 2 and TLR4 mRNA expression was assessed.
Results.
Primary peripheral-blood mononuclear cells and THP-1 monocytic cells secreted TNF- in response to cervicovaginal lavage (CVL) samples from women with BV. Mycoplasma hominis and Gardnerella vaginalis also stimulated TNF- secretion. Strikingly, CVL samples from women with BV induced up to 60-fold increases in TLR4 mRNA expression, compared with CVL samples from women without BV and with bacteria not associated with BV. AntiTNF- antibody blocked increases in TLR4 mRNA expression induced by CVL samples from women with BV, indicating that TNF- plays a critical role in induction of TLR4. Both TLR2 and TLR4 mRNA expression were 60-fold higher in cells isolated from the lumen of the genital tract than in cervical mucosal tissue, but lumen TLR mRNA levels did not change significantly after BV treatment.
Conclusions.
These experiments show that genital mucosal fluids and certain bacteria from women with BV stimulate TNF- secretion and TLR4 mRNA expression, suggesting mechanisms whereby BV affects pregnancy and HIV transmission.
Bacterial vaginosis (BV) is a common alteration of the female genital-tract microbial flora and is a frequent cause of vaginal discharge [1]. Although Lactobacillus species are the predominant bacteria that normally colonize the female genital tract [2], during BV, lactobacilli are replaced by a complex mixture of microorganisms. The mixtures of bacteria that constitute BV are variable but usually consist of Gardnerella vaginalis and gram-negative anaerobes, such as Prevotella species, Bacteroides species, Mobiluncus species, and Peptostreptococcus species [3]. Mycoplasma hominis and Ureaplasma species are also frequently isolated during BV.
BV is associated with severe medical complications during pregnancy, including fetal loss, chorioamnionitis, preterm delivery, and low birth weight [4, 5]. It is thought that these problems arise when organisms associated with BV ascend from the lower reproductive tract, an occurrence that results in fetal stress due to induction of cytokines and other inflammatory products [6]. In both cross-sectional and prospective studies, BV has also been associated with an increase in the incidence of HIV sexual transmission [711]. Although BV may affect HIV sexual transmission in multiple ways, a possible explanation is that microbial flora associated with BV or the host factors produced in response to the microbial flora increase HIV replication in the genital tract by stimulating and/or recruiting HIV-infected cells [12, 13]. Evidence indicating that BV increases HIV expression by cells in the genital tract is provided by studies of HIV-infected women, in which BV was significantly associated with levels of HIV RNA in the genital tract [1416].
Expression of HIV by cells is enhanced in vitro and in vivo by such microorganisms as mycobacteria and Pneumocystis carinii [1720]. Stimulation of cells through Toll-like receptor (TLR) 2 and TLR4 with microbial products stimulates HIV expression through stimulation of tumor necrosis factor (TNF) secretion or direct activation of NF-B and subsequent binding of NF-B to the HIV promoter [2124]. Products from several genital-tract pathogens, such as Neisseria gonorrhoeae and Chlamydia trachomatis, have been shown to stimulate cells through either TLR2 or TLR4. [2527]. Epithelial cells in the vagina, ectocervix, and endocervix do not express TLR4 or myeloid differentiation2 but do express other TLRs, including TLR2 [28]. Leukocytesincluding neutrophils, monocytes, macrophages, and lymphocytesare normally found in vaginal and cervical tissues [29], and monocytes, macrophages, and neutrophils, but not lymphocytes, express both TLR2 and TLR4 [30].
In the present study, we tested the hypothesis that genital mucosal fluids from women with BV and common microbial flora associated with BV induce proinflammatory changes in cells, including TNF- secretion and TLR2 and TLR4 mRNA expression. Stimulation of cells that results in the release of proinflammatory cytokines or alteration of responses through TLRs could play a role in mediating the adverse effects of BV. Additionally, we compared TLR mRNA expression by cells in the lumen of the genital tract from women with and without BV and in cervical mucosal tissue, to determine whether BV affected these cells.
SUBJECTS, MATERIALS, AND METHODS
Genital-tract samples.
Cervicovaginal lavage (CVL) samples were collected from subjects enrolled at either the University of Illinois (U of I) or the Rush University site of the Chicago Consortium of the Women's Interagency HIV Study (WIHS), in accordance with the requirements of the institutional review boards. WIHS is a prospective epidemiological and natural-history study of HIV-infected and high-risk HIV-uninfected women. The U of I subjects were recruited from an existing Centers for Disease Control and Prevention cohort of women (in the VISION study) who were HIV seronegative but at high risk for HIV infection through heterosexual transmission. Informed consent was obtained from all subjects during their regularly scheduled study visit.
For all subjects, a pelvic exam was performed and vaginal secretions were collected from the posterior fornix by use of a swab, for preparation of a wet-mount slide. CVL samples were collected after the swab by directing a 10-mL stream of sterile normal saline at the cervical os and the endocervix. Fluid was aspirated and centrifuged, to pellet cells. Cells (total cell pellet) were frozen for later RNA extraction. All samples used in the present study were negative for chlamydia, trichomonas, and gonococci, as determined by wet-mount and probe-based tests [31]. Samples were determined to be positive for BV by use of the Amsel criteria [32]. Women whose CVL samples were positive for BV were treated with a single dose of 2 g of metronidazole. All posttreatment CVL samples used for experiments were negative for BV by the Amsel criteria. The median time between initial collection and follow-up was 21 days. All subjects were black; their median age was 41 years (range, 3249 years). CVL samples were not collected during menses. The present study used 2 nonoverlapping sets of CVL samples: a cross-sectional set consisting of 5 BV-positive and 5 BV-negative samples from subjects in the WIHS, and a longitudinal set of samples from subjects in the VISION study. These latter subjects were 9 women with BV who had been treated with antibiotics and returned after treatment to provide a second CVL sample. For studies of TLR mRNA expression in cervical mucosal tissue, RNA was extracted from portions of ectocervical tissue obtained from premenopausal women undergoing hysterectomies for benign disease.
Stimulation of cells for TNF- secretion.
THP-1 (monocytic), Hec 1A, and Hec 1B (endometrial epithelial) transformed cell lines were obtained from the American Type Culture Collection and were used in the present study as potential models for responses of primary cells. M. hominis, G. vaginalis, and Lactobacillus crispatus were obtained and grown in cultures as described elsewhere [33]. Peripheral-blood mononuclear cells (PBMCs) were obtained from healthy donors and were isolated on ficoll-hypaque (BioWhittakker). Cells (1 × 105) were cultured in RPMI 1640 medium supplemented with 2 mmol/L glutamine, 10% fetal bovine serum, and 0.5% gentamicin (all from BioWhittakker) in microtiter plates (Costar). Cells were stimulated with medium alone (0.2 mL), lipopolysaccharide (LPS; 1 g/mL; Sigma), bacteria (300 organisms/cell; 10% culture medium conditioned by the growth of bacteria or 10% unconditioned control medium), or CVL samples (10%). Culture supernatants were collected at 48 h, and TNF- levels were measured by ELISA (Biosource International).
Real-time polymerase chain reaction (PCR) quantification of TLR and 2-defensin mRNA.
PBMCs and THP-1 cells (3 × 106) were cultured in 24-well plates for 48 h with stimuli in 1 mL of medium and were collected by centrifugation. RNA was extracted by use of the RNeasy Mini Extraction Kit (Qiagen). RNA was obtained in the same way from CVL cell pellets and homogenized cervical mucosal tissues. cDNA was made from 1 g of RNA by use of the RT-PCR Kit (Clontech). The primers for TLR2, TLR9, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) have been described elsewhere [30]; the primers for TLR4 (5-CAGCTCTTGGTGGAAGTTGA-3 and 5-AGCATAAGGCCTGACATGTG-3 ) and 2-defensin (5-GATGCCTCTTCCAGGTGAGATG-3 and 5-AACTTCTACGCCATTCTTCCA-3 ) were designed by use of Primer Express (Applied Biosystems). SYBR Green PCR Core Reagents (PE Biosystems) were used for amplification. For all PCRs, reagents were used as follows: cDNA corresponding to 62 ng of RNA, 2.5 mmol/L MgCl2, 0.25 mmol/L dNTPs, 0.05 mol/L primers, 0.25 U/L ung, and 0.02 U/L Taq gold polymerase. Amplification was performed in an ABI Prism 5700 Thermocycler (Applied Biosystems), with the following thermocycle profile: stage 1, 50°C (2 min); stage 2, 95°C (10 min); and stage 3, 50 cycles of 95°C (45 s), 62°C (45 s), and 72°C (1 min) for TLR4. The annealing temperatures for GAPDH, 2-defensin, TLR2, and TLR9 were 61°C, 63°C, 61°C, and 62°C, respectively. During each thermocycler run, 6 standards consisting of doubling dilutions of 2 × 105 ng of cDNA from LPS-stimulated PBMCs were included, to generate a standard curve. The standard curve was used to calculate the relative amount of mRNA in samples after adjustment for GAPDH, a housekeeping gene that has been used in previous studies to normalize mRNA expression [30].
RESULTS
Induction of TNF- secretion by CVL samples from women with BV.
To determine whether substances in secretions from the genital tracts of women with BV can induce TNF- secretion, CVL samples collected from a cross-section of women were cultured with either primary PBMCs, the monocytic THP-1 cell line, or genital-tract epithelial cell lines. CVL samples from women with BV induced higher levels of TNF- secretion by PBMCs (mean ± SD, 898 ± 436 pg/mL) than did CVL samples from women without BV (mean ± SD, 194 ± 212 pg/mL) (P = .05) (figure 1A). To further explore the relationship between BV and induction of TNF- secretion, we tested CVL samples from 9 additional women with BV that were obtained both before and after antibiotic treatment. Although the CVL samples obtained before treatment induced significant TNF- secretion by PBMCs (mean ± SD, 1225 ± 492 pg/mL; P = .01, vs. control), the CVL samples obtained after treatment did not (mean ± SD, 148 ± 239 pg/mL; P > .05, vs. control; P < .05, for before treatment vs. after treatment [paired t test]) (figure 1A). THP-1 cells (figure 1B) also secreted significantly higher levels of TNF- when cultured with CVL samples from women with BV (mean ± SD, 186 ± 82 pg/mL; P = .02, t test) than when cultured with CVL samples from women without BV (mean ± SD, 21 ± 13 pg/mL; P > .05, vs. control). CVL samples from both women with BV and women without BV did not induce any detectable TNF- secretion by either the HEC-1A or the HEC-1B epithelial cell line, whereas stimulation with phorbol myristate acetate did induce TNF- secretion (data not shown). The responses of PBMCs were dose dependent, with higher concentrations of CVL samples inducing more TNF- secretion; CVL samples from women without BV induced little TNF- secretion, even when added at 20% of the culture volume (figure 1C and 1D).
Induction of TNF- secretion by bacteria associated with BV.
Because the above experiments showed that genital mucosal fluids from women with BV stimulate TNF- secretion, we determined whether whole bacteria associated with BV or soluble products released from such bacteria induce TNF- secretion. G. vaginalis was chosen for testing because it is found in essentially all cases of BV and may contribute to BV pathogenesis [34, 35]. M. hominis and L. crispatus were also tested, because we recently observed that counts of these organisms are significantly associated with genital-tract HIV load [16] and because L. crispatus is frequently isolated from women [3]. In multiple experiments, none of these bacteria induced any detectable TNF- secretion by either the HEC-1A or the HEC-1B epithelial cell line (data not shown). THP-1 cells secreted a significant amount of TNF- when cultured with M. hominis (mean ± SD, 1460 ± 180 pg/mL; P = .01, t test) and with G. vaginalis (mean ± SD, 2942 ± 311 pg/mL; P = .01, t test) (figure 2A). In contrast, L. crispatus did not induce significant TNF- secretion by THP-1 cells (mean ± SD, 297 ± 78 pg/mL; P = .29). Culture medium conditioned by the growth of bacteria or unconditioned control media from the 3 organisms did not significantly induce TNF- secretion by THP-1 cells (figure 2A). PBMCs (figure 2B) also produced a significant amount of TNF- when cultured with M. hominis (mean ± SD, 3198 ± 200 pg/mL; P = .04) and with G. vaginalis (mean ± SD, 9028 ± 560 pg/mL; P = .04), although PBMCs responded significantly to L. crispatus (mean ± SD, 6060 ± 350 pg/mL, P = .03) and culture medium conditioned by the growth of bacteria from M. hominis and G. vaginalis (P < .05). Dose-response experiments showed that as few as 60 M. hominis or G. vaginalis organisms/cell induced detectable TNF- secretion by THP-1 cells and PBMCs (data not shown).
Induction of TLR mRNA expression by genital-tract secretions and bacteria.
We also assessed the effect that genital mucosal fluids from 2 women with BV and 2 women without BV (selected from the cross-sectional cohort) had on TLR4 and TLR2 mRNA expression. Although the 2 CVL samples from the women without BV did not significantly increase TLR4 mRNA expression by PBMCs, the 2 CVL samples from the women with BV induced 47- and 62-fold increases in TLR4 mRNA expression by PBMCs (figure 3A). LPS also induced substantial increases in TLR4 mRNA expression (figure 3A). The fold increases in TLR4 mRNA expression induced by the CVL samples from the 2 women with BV ranged from 38 to 65 in 3 independent experiments, and the fold increases in TLR4 mRNA expression induced by LPS ranged from 40 to 70 in 5 independent experiments. The same CVL samples from the women with BV also significantly increased TLR4 mRNA expression by THP-1 cells, whereas the same CVL samples from the women without BV did not (data not shown). Blocking antibody to TNF- was added to cultures, to determine whether TNF- plays a role in the induction of TLR4 mRNA expression by genital mucosal secretions. AntiTNF- antibody significantly inhibited TLR4 mRNA expression by PBMCs (figure 3B) and THP-1 cells (data not shown), whereas an isotype control antibody did not. AntiTNF- antibody also blocked the increase in TLR4 mRNA expression induced by LPS (data not shown). The addition of M. hominis and G. vaginalis organisms to cultures induced only 4- and 8-fold increases in TLR4 mRNA expression by PBMCs, respectively, whereas LPS induced a significant increase in TLR4 mRNA expression by PBMCs (figure 3C). Thus, these experiments show that genital mucosal fluids from women with BV are highly effective in inducing TLR4 mRNA expression by cells and that TNF- is necessary for this response.
The 2 CVL samples from the women with BV that induced TLR4 mRNA expression also induced 30- and 37-fold increases in TLR2 mRNA expression by PBMCs (figure 4A) and induced smaller increases in TLR2 mRNA expression by THP-1 cells (data not shown). However, the 2 CVL samples from the women without BV also induced TLR2 mRNA expression (15- and 23-fold) that was significantly higher than that in controls (P < .05). The addition of M. hominis, G. vaginalis, or L. crispatus to cultures did not significantly increase TLR2 mRNA expression by PBMCs (figure 4B) or THP-1 cells (data not shown).
TLR mRNA expression by genital-tract cells.
Because the above experiments showed that genital mucosal secretions from women with BV increased TLR4 mRNA expression, we determined the level of TLR4 mRNA expression by cells isolated from the lumen of the genital tract of women with BV and compared it with the level of TLR4 mRNA expression by cells isolated from the same women after receipt of antibiotic treatment. TLR4 mRNA was detected in all 9 pairs of pre- and posttreatment CVL samples, although there was a 50-fold range in the total amount of TLR4 mRNA detected among different women (when adjusted for GAPDH mRNA levels). Although (1) 6 of the 9 women had significantly higher levels of TLR4 mRNA before treatment than after and (2) TLR4 mRNA expression by cells obtained from women with BV was twice that by cells obtained from women treated for BV, the difference in TLR4 mRNA expression before and after treatment was not significant for the 9 women as a group (P > .1, paired t test) (figure 5). Although TLR2 mRNA was also detected in all 9 sets of samples, as with TLR4 mRNA, there was no significant difference between pre- and posttreatment levels (figure 5). TLR9 mRNA levels were also assessed, because recent studies have shown that TLR9 is expressed in the genital tract [36]. Additionally, we measured levels of 2-defensin mRNA as a non-TLR control, because it is expressed by genital-tract cells and because we previously reported high levels of 2-defensin in genital mucosal secretions from women with Trichomonas infection [31]. Although TLR9 and 2-defensin mRNA was detected in all 9 sets of samples, there was no significant difference between pretreatment and posttreatment levels (figure 5).
DISCUSSION
Although it has been well established that BV adversely affects the outcome of pregnancy and increases acquisition of HIV, the mechanisms responsible for these effects have not been well defined. Overall, our experiments show that genital mucosal fluids from women with BV are potent stimulators of leukocytes, eliciting secretion of TNF- and expression of mRNA for both TLR4 and TLR2. Thus, our findings suggest that TNF- levels in genital mucosal fluids from women with BV would be higher than those in women with normal flora. It has been reported [37] that TNF- levels in undiluted genital mucosal fluid from women with BV were higher than those for women with normal flora, with median levels of 37 and 20 pg/mL, respectively. These levels are slightly higher than those in the CVL samples used in the present studywe found mean levels of 0.2 and 0.5 pg/mL in CVL samples from women without BV and with BV, respectively, and these correspond to 6 and 15 pg/mL, respectively, in undiluted mucosal fluid [38]. However, the use of undiluted genital fluid in the previous study probably contributed to their higher overall readings [37], because several of our CVL samples had TNF- levels that were below the limit of detection.
Studies suggest that induction of TNF- expression in the genital tract is likely to be involved in the adverse effects associated with BV; TNF- induces HIV expression via stimulation of the HIV promoter [24]. BV is associated with increased detection of HIV in the genital tract [14, 15], and a recent study has shown that levels of HIV are correlated positively with M. hominis infection but negatively with lactobacilli infection [16]. TNF- levels have also been associated with some of the adverse effects that BV has on the fetus; intrauterine inoculation of rabbits with BV-associated bacteria increased preterm births and was associated with increased TNF- secretion [39]. In a rat model of BV, prenatal exposure to LPS induced TNF- secretion and associated loss of fetal neurons [40]. TNF- levels in genital mucosal fluid are also associated with cervical inflammation, which, in turn, is associated with adverse outcomes of pregnancy [37].
The identities of the cell types in the genital tract that produce TNF- have not been identified. However, in light of Ficherova et al.'s demonstration that genital epithelial cells are unresponsive to LPS due to a lack of TLR4 [28], they may be leukocytes rather than epithelial cells. The experiments here have confirmed the unresponsiveness by genital epithelial cell lines to LPS and have shown that genital mucosal fluids from women with BV do not induce either TNF- secretion or TLR expression by epithelial cell lines. In this regard, our recent experiments suggest that the stimulatory activity in genital mucosal fluid from women with BV is mediated through TLR4 (M.R.Z., unpublished data). Most women with BV have increased leukocyte esterase and inflammatory cells in genital mucosal fluids [41]. Higher levels of both leukocytes and substances that induce TNF- secretion could help to explain the higher TNF- levels found during BV, although one study did not find an increased number of leukocytes in the genital tract during BV [42].
In the present experiments, we also found that genital mucosal fluids from women with BV strongly induced TLR4 mRNA expression by PBMCs and THP-1 cells in vitro. LPS also induced increased TLR4 mRNA expression, suggesting that the induction of TLR4 expression may be mediated through TLR4. When cells from the genital tract were assessed, 6 of 9 women had significantly higher TLR4 mRNA expression before antibiotic treatment than after, when BV had cleared. Differences in the severity of BV or the types of bacteria present during BV could have affected these results [43]. Also, the proportions of epithelial cells and leukocytes in CVL samples may have differed. We also observed that antiTNF- antibody blocked the increased expression of TLR4 mRNA in cells. Previous studies have shown that TNF- is important in the induction of TLR4 [44, 45].
Other studies have shown that infections with such pathogens as gonococci, chlamydia, trichomonads, and candida can induce TNF- secretion both in vitro and in vivo [31, 4648]; samples were screened for these organisms by use of molecular and microscopic assays in the present study, and samples found to be positive for them were excluded from our experiments. The stimulation that was seen with the CVL samples from women with BV, therefore, suggests that certain of the microorganisms associated with BV are the source of the stimulatory activity in the genital secretions, although the presence of a host factor cannot be ruled out. In fact, addition of G. vaginalis and M. hominis to cells potently induced TNF- secretion. In rabbits, genital inoculation with G. vaginalis causes increased TNF- expression in amniotic fluid [34]. Also, G. vaginalis activates HIV-1 expression in monocytic cell lines [35]. Few studies have assessed the ability of M. hominis to induce TNF- secretion, but this organism does not stimulate TNF- secretion by epithelial cells [49].
Induction of TLR expression during BV could have important effects on HIV. For example, stimulation through TLR4 and TLR2 has been shown to increase HIV expression by infected cells [21, 50]. Coinfections, such as with mycobacteria, significantly augment HIV replication in vivo through TLR [22, 51]. Thus, our experiments suggest that HIV-infected cells present in the genital tracts of women with BV may be directly stimulated through TLR to express increased levels of HIV, and this could have significant effects on HIV sexual transmission.
References
1. Holst E, Wathne B, Hovelius B, Mardh PA. Bacterial vaginosis: microbiological and clinical findings. Eur J Clin Microbiol 1987; 6:53641. First citation in article
2. Antonio MA, Hawes SE, Hillier SL. The identification of vaginal Lactobacillus species and the demographic and microbiologic characteristics of women colonized by these species. J Infect Dis 1999; 180:19506. First citation in article
3. Hillier SL, Krohn MA, Rabe LK, Klebanoff SJ, Eschenbach DA. The normal vaginal flora, H2O2-producing lactobacilli, and bacterial vaginosis in pregnant women. Clin Infect Dis 1993; 16(Suppl 4):S27381. First citation in article
4. Hillier SL, Nugent RP, Eschenbach DA, et al. Association between bacterial vaginosis and preterm delivery of a low-birth-weight infant. The Vaginal Infections and Prematurity Study Group [see comments]. N Engl J Med 1995; 333:173742. First citation in article
5. Ugwumadu AH. Bacterial vaginosis in pregnancy. Curr Opin Obstet Gynecol 2002; 14:1158. First citation in article
6. Locksmith G, Duff P. Infection, antibiotics, and preterm delivery. Semin Perinatol 2001; 25:295309. First citation in article
7. Sewankambo N, Gray RH, Wawer MJ, et al. HIV-1 infection associated with abnormal vaginal flora morphology and bacterial vaginosis [see comments]. Lancet 1997; 350:54650 (erratum: Lancet 1997; 350:1036). First citation in article
8. Taha TE, Gray RH, Kumwenda NI, et al. HIV infection and disturbances of vaginal flora during pregnancy. J Acquir Immune Defic Syndr Hum Retrovirol 1999; 20:529. First citation in article
9. Cohen CR, Duerr A, Pruithithada N, et al. Bacterial vaginosis and HIV seroprevalence among female commercial sex workers in Chiang Mai, Thailand. AIDS 1995; 9:10937. First citation in article
10. Martin HL, Richardson BA, Nyange PM, et al. Vaginal lactobacilli, microbial flora, and risk of human immunodeficiency virus type 1 and sexually transmitted disease acquisition. J Infect Dis 1999; 180:18638. First citation in article
11. Taha TE, Hoover DR, Dallabetta GA, et al. Bacterial vaginosis and disturbances of vaginal flora: association with increased acquisition of HIV. AIDS 1998; 12:1699706. First citation in article
12. Hillier SL. The vaginal microbial ecosystem and resistance to HIV. AIDS Res Hum Retroviruses 1998; 14(Suppl 1):S1721. First citation in article
13. Spear GT, al-Harthi L, Sha B, et al. A potent activator of HIV-1 replication is present in the genital tract of a subset of HIV-1-infected and uninfected women. AIDS 1997; 11:131926. First citation in article
14. Cu-Uvin S, Hogan JW, Caliendo AM, Harwell J, Mayer KH, Carpenter CC. Association between bacterial vaginosis and expression of human immunodeficiency virus type 1 RNA in the female genital tract. Clin Infect Dis 2001; 33:8946. First citation in article
15. Spinillo A, Debiaggi M, Zara F, Maserati R, Polatti F, De Santolo A. Factors associated with nucleic acids related to human immunodeficiency virus type 1 in cervico-vaginal secretions. BJOG 2001; 108:63441. First citation in article
16. Sha BE, Zariffard MR, Wang QJ, et al. Female genital tract HIV load correlates inversely with Lactobacillus species but positively with bacterial vaginosis and Mycoplasma hominis. J Infect Dis 2005; 191:2532. First citation in article
17. Orenstein JM. The macrophage in HIV infection. Immunobiology 2001; 204:598602. First citation in article
18. Wahl SM, Greenwell-Wild T, Peng G, Hale-Donze H, Orenstein JM. Co-infection with opportunistic pathogens promotes human immunodeficiency virus type 1 infection in macrophages. J Infect Dis 1999; 179(Suppl 3):S45760. First citation in article
19. Wahl SM, Greenwell-Wild T, Peng G, et al. Mycobacterium avium complex augments macrophage HIV-1 production and increases CCR5 expression. Proc Natl Acad Sci USA 1998; 95:125749. First citation in article
20. Ghassemi M, Novak RM, Khalili MF, Zhou J. Viable Mycobacterium avium is required for the majority of human immunodeficiency virus-induced upregulation in monocytoid cells. J Med Microbiol 2003; 52:87782. First citation in article
21. Equils O, Faure E, Thomas L, Bulut Y, Trushin S, Arditi M. Bacterial lipopolysaccharide activates HIV long terminal repeat through Toll-like receptor 4. J Immunol 2001; 166:23427. First citation in article
22. Equils O, Schito ML, Karahashi H, et al. Toll-like receptor 2 (TLR2) and TLR9 signaling results in HIV-long terminal repeat trans-activation and HIV replication in HIV-1 transgenic mouse spleen cells: implications of simultaneous activation of TLRs on HIV replication. J Immunol 2003; 170:515964. First citation in article
23. Pomerantz RJ, Feinberg MB, Trono D, Baltimore D. Lipopolysaccharide is a potent monocyte/macrophage-specific stimulator of human immunodeficiency virus type 1 expression. J Exp Med 1990; 172:25361. First citation in article
24. Osborn L, Kunkel S, Nabel GJ. Tumor necrosis factor alpha and interleukin 1 stimulate the human immunodeficiency virus enhancer by activation of the nuclear factor kappa B. Proc Natl Acad Sci USA 1989; 86:233640. First citation in article
25. Lien E, Sellati TJ, Yoshimura A, et al. Toll-like receptor 2 functions as a pattern recognition receptor for diverse bacterial products. J Biol Chem 1999; 274:3341925. First citation in article
26. Massari P, Ram S, Macleod H, Wetzler LM. The role of porins in neisserial pathogenesis and immunity. Trends Microbiol 2003; 11:8793. First citation in article
27. Prebeck S, Brade H, Kirschning CJ, et al. The Gram-negative bacterium Chlamydia trachomatis L(2) stimulates tumor necrosis factor secretion by innate immune cells independently of its endotoxin. Microbes Infect 2003; 5:46370. First citation in article
28. Fichorova RN, Cronin AO, Lien E, Anderson DJ, Ingalls RR. Response to Neisseria gonorrhoeae by cervicovaginal epithelial cells occurs in the absence of toll-like receptor 4-mediated signaling. J Immunol 2002; 168:242432. First citation in article
29. Givan AL, White HD, Stern JE, et al. Flow cytometric analysis of leukocytes in the human female reproductive tract: comparison of fallopian tube, uterus, cervix, and vagina. Am J Reprod Immunol 1997; 38:3509. First citation in article
30. Zarember KA, Godowski PJ. Tissue expression of human Toll-like receptors and differential regulation of Toll-like receptor mRNAs in leukocytes in response to microbes, their products, and cytokines. J Immunol 2002; 168:55461. First citation in article
31. Zariffard MR, Harwani S, Novak RM, Graham PJ, Ji X, Spear GT. Trichomonas vaginalis infection activates cells through toll-like receptor 4. Clin Immunol 2004; 111:1037. First citation in article
32. Amsel R, Totten PA, Spiegel CA, Chen KC, Eschenbach D, Holmes KK. Nonspecific vaginitis: diagnostic criteria and microbial and epidemiologic associations. Am J Med 1983; 74:1422. First citation in article
33. Zariffard MR, Saifuddin M, Sha BE, Spear GT. Detection of bacterial vaginosis-related organisms by real-time PCR for Lactobacilli, Gardnerella vaginalis and Mycoplasma hominis. FEMS Immunol Med Microbiol 2002; 34:27781. First citation in article
34. McDuffie RS Jr, Kunze M, Barr J, et al. Chronic intrauterine and fetal infection with Gardnerella vaginalis. Am J Obstet Gynecol 2002; 187:12636. First citation in article
35. Hashemi FB, Ghassemi M, Roebuck KA, Spear GT. Activation of human immunodeficiency virus type 1 expression by Gardnerella vaginalis. J Infect Dis 1999; 179:92430. First citation in article
36. Ashkar AA, Bauer S, Mitchell WJ, Vieira J, Rosenthal KL. Local delivery of CpG oligodeoxynucleotides induces rapid changes in the genital mucosa and inhibits replication, but not entry, of herpes simplex virus type 2. J Virol 2003; 77:894856. First citation in article
37. Sturm-Ramirez K, Gaye-Diallo A, Eisen G, Mboup S, Kanki PJ. High levels of tumor necrosis factor- and interleukin-1 in bacterial vaginosis may increase susceptibility to human immunodeficiency virus. J Infect Dis 2000; 182:46773. First citation in article
38. Belec L, Meillet D, Levy M, Georges A, Tevi-Benissan C, Pillot J. Dilution assessment of cervicovaginal secretions obtained by vaginal washing for immunological assays. Clin Diagn Lab Immunol 1995; 2:5761. First citation in article
39. Gibbs RS, McDuffie RS Jr, Kunze M, et al. Experimental intrauterine infection with Prevotella bivia in New Zealand White rabbits. Am J Obstet Gynecol 2004; 190:10826. First citation in article
40. Carvey PM, Chang Q, Lipton JW, Ling Z. Prenatal exposure to the bacteriotoxin lipopolysaccharide leads to long-term losses of dopamine neurons in offspring: a potential, new model of Parkinson's disease. Front Biosci 2003; 8:s82637. First citation in article
41. Mardh PA, Novikova N, Niklasson O, Bekassy Z, Skude L. Leukocyte esterase activity in vaginal fluid of pregnant and non-pregnant women with vaginitis/vaginosis and in controls. Infect Dis Obstet Gynecol 2003; 11:1926. First citation in article
42. Cauci S, Guaschino S, De Aloysio D, et al. Interrelationships of interleukin-8 with interleukin-1beta and neutrophils in vaginal fluid of healthy and bacterial vaginosis positive women. Mol Hum Reprod 2003; 9:538. First citation in article
43. Krohn MA, Hillier SL, Nugent RP, et al. The genital flora of women with intraamniotic infection. Vaginal Infection and Prematurity Study Group. J Infect Dis 1995; 171:147580. First citation in article
44. Wolfs TG, Buurman WA, van Schadewijk A, et al. In vivo expression of Toll-like receptor 2 and 4 by renal epithelial cells: IFN-gamma and TNF-alpha mediated up-regulation during inflammation. J Immunol 2002; 168:128693. First citation in article
45. Faure E, Thomas L, Xu H, Medvedev A, Equils O, Arditi M. Bacterial lipopolysaccharide and IFN-gamma induce Toll-like receptor 2 and Toll-like receptor 4 expression in human endothelial cells: role of NF-kappa B activation. J Immunol 2001; 166:201824. First citation in article
46. Netea MG, Selzman CH, Kullberg BJ, et al. Acellular components of Chlamydia pneumoniae stimulate cytokine production in human blood mononuclear cells. Eur J Immunol 2000; 30:5419. First citation in article
47. Lilic D, Gravenor I, Robson N, et al. Deregulated production of protective cytokines in response to Candida albicans infection in patients with chronic mucocutaneous candidiasis. Infect Immun 2003; 71:56909. First citation in article
48. Netea MG, Stuyt RJ, Kim SH, Van der Meer JW, Kullberg BJ, Dinarello CA. The role of endogenous interleukin (IL)18, IL-12, IL-1, and tumor necrosis factor in the production of interferon- induced by Candida albicans in human whole-blood cultures. J Infect Dis 2002; 185:96370. First citation in article
49. Baier J, Kruger TE. Induction of monocyte chemoattractant protein-1 by Mycoplasma hominis in respiratory epithelial cells. J Investig Med 2000; 48:45764. First citation in article
50. Bafica A, Scanga CA, Equils O, Sher A. The induction of Toll-like receptor tolerance enhances rather than suppresses HIV-1 gene expression in transgenic mice. J Leukoc Biol 2004; 75:4606. First citation in article
51. Bafica A, Scanga CA, Schito ML, Hieny S, Sher A. Cutting edge: in vivo induction of integrated HIV-1 expression by mycobacteria is critically dependent on Toll-like receptor 2. J Immunol 2003; 171:11237. First citation in article