Alvarez-Olmos, Martha I. MD*†; Barousse, Melissa M. BS†; Rajan, Latta MD‡; Van Der Pol, Barbara J. MS§; Fortenberry, Dennis MD§; Orr, Donald MD§; Fidel, Paul L. Jr. PhD†
THE VAGINA AND ITS MICROFLORA form a balanced ecosystem that is an important health-maintaining biologic feature providing host defense against infection. The human adult vaginal ecosystem is composed of both anaerobic and aerobic bacterial species predominated by lactobacilli.1 Lactobacillus acidophilus and Lactobacillus fermentum have been identified as the primary species in the vagina along with Lactobacillus crispatus, Lactobacillus gasseri, and Lactobacillus jensenii.2,3 The production of several antimicrobial compounds, including lactic acid, hydrogen peroxidase, lactacin, and acidolicin is attributed to lactobacilli. Of these, H2O2 is considered the most important, although its production is not restricted to lactobacilli species. H202 activity could be bacteriostatic or bactericidal depending on the bacterial species.4
Bacterial vaginosis (BV) is considered the leading cause of vaginal discharge and malodor in adult women caused by replacement of the vaginal lactobacilli with high concentrations of characteristic sets of aerobic and anaerobic bacteria.5 BV has been associated with adverse pregnancy outcomes6 (ie, preterm delivery, preterm labor, premature rupture of membranes), pelvic inflammatory diseases,7 postoperative pelvic infections,8 and higher prevalence of HIV among certain high-risk populations.9,10
It is now recognized that the mucosal and systemic compartments of the immune system display a significant degree of independence. The current knowledge of the human mucosal response to the normal flora and sexually transmitted disease (STD) pathogens is limited. Cytokines and chemokines are critical mediators of the immune response. Th1-type cytokines characterized by IL-2, IFN-γ, and IL-12 are involved in the phagocyte-mediated defense against infections, especially with intracellular microorganisms, whereas Th2-type cytokines characterized by IL-4, IL-5, and IL-10 are involved in the resistance to extracellular pathogens.11 Proinflammatory cytokines are critical for innate resistance and links to adaptive immunity. Chemokines are important for migration of leukocytes to sites of infection.12 We and others have shown that cytokines and chemokines can be detected in vaginal lavage fluid of normal healthy women that could be indicative of a normal protective response maintaining a state of immunologic homeostasis.13,14
It is well known that adolescents have the highest age-specific risk for acquiring STDs, including HIV-1 infection. Of more than 12 million annual cases of STDs in the United States, 25% occur among female adolescents between 13 and 18 years of age.15 Almost half of polled adolescents report being sexually active. Adolescence is a critical period during which lifelong health behaviors are established, providing great opportunity for reducing risks through health promotion and preventive strategies. Although information on vaginal ecology, STDs, BV, and genital tract immunity has been studied frequently in adult women,16,17 there is a paucity of information for these parameters in adolescent females.18 Accordingly, the purpose of this study was to characterize the vaginal presence of lactobacilli in adolescents, systemic immune sensitization to lactobacilli, and relationships of lactobacilli and BV to local immunomodulators to better understand potential factors in the susceptibility of adolescents to STDs.
Materials and Methods
Participants and Specimens
A cross-sectional sampling of 89 adolescent females ranging from 14 to 18 years of age enrolled as part of a longitudinal study of the Mid-America Adolescent Sexually Transmitted Diseases Clinical Research Center (MASTI-CRC from the University of Indiana) was conducted between January and October 2001. Enrollment was based on attendance at an adolescent health clinic for a number of different reasons (ie, oral contraception, pregnancy testing, gynecologic symptoms, attendance with a friend, and so on). Neither having a STD nor being sexually active was a requirement for enrollment in the parent study. However, this cross-sectional study excluded any adolescent with Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis, or a combination of these conditions. After obtaining written informed consent from adolescents and their parents, all adolescents answered a standardized questionnaire addressing demographic, behavioral, and health information. An adolescent specialist performed a complete physical and pelvic examination and specimens were collected. All procedures were followed in the conduct of clinical research in accordance with the Institutional Review Boards at the Louisiana State University Health Sciences Center, New Orleans, Louisiana, and Indiana University Medical Center, Indianapolis, Indiana. Participants were enrolled and specimens were collected at Indiana University Medical Center.
Specimens collected from enrolled subjects included a lateral vaginal swab, cervicovaginal lavage, and venous blood at scheduled visits by a provider. The vaginal lavage was collected after a 30 to 40 seconds continuous aspiration with 5 mL of nonpyrogenic sterile saline. A portion of each sample was processed at Indiana University Medical Center (IUMC), Indianapolis, Indiana, and the remainder shipped overnight to Louisiana State University Health Sciences Center (LSUHSC) in New Orleans where they were further processed. The vaginal swab was used for the identification of lactobacilli (culture) and BV (Gram stain). Cervicovaginal lavages were used for detection of cytokines, whereas blood (30 mL) obtained by venipuncture into Vacutainer CPT cell preparation tubes (Becton Dickinson, Sparks, MD) was taken in a subgroup of this population for antigen-stimulated lymphocyte responses.
The vaginal swab was immediately placed on Amies transport medium-free self-contained anaerobic-atmosphere containers (Becton Dickinson Vacutainer Systems, Rutherford, NJ) at Indiana and inoculated within 24 hours onto nonselective media (Columbia Blood Agar plates; Difco, Detroit, MI) microaerophilically at 37°C and onto lactobacilli selective media (Rogosa Agar in Tomato Juice, CLBS media; BD Microbiology Systems, Cockeysville, MD) at LSU Health Sciences Center under anaerobic conditions for 48 to 72 hours.10 Lactobacilli were identified phenotypically at the genus level by Gram stain, colony morphologies, and standard biochemical reactions using API-CH50 (Pasteur Merieux, Paris, France).
H2O2-Producing Lactobacilli Screening
At least 3 lactobacillus colonies recovered from each plate were stored in Rogosa SL broth (BD-Biosciences, USA) with 20% glycerol at −70°C. Subsequently, the colonies were subcultured (0.1 mL of a suspension of lactobacilli) on 0.25 mg of tetramethylbenzidine (TMB) per milliliter of Rogosa agar containing 0.01 mg of horseradish peroxidase (type IV from Sigma-Aldrich, USA).19 After 2 days of incubation in an anaerobic glove box at 37°C, agar plates were exposed to environmental air. Blue colonies represented H2O2-producing lactobacilli. This reaction was qualitatively scored as positive or negative according to the presence or absence of color.
Conventional Gram stain was performed using the same vaginal swab. The flora was scored according to Nugent’s criteria (0–3: normal; 4–6: intermediate or disturbed flora, and 7–10: BV).20
Peripheral Lymphocyte Responses
L. crispatus (BCRC 2,116), provided by Dr. Sharon Hillier (University of Pittsburgh, PA), was inoculated and diluted 1/100 before use in tissue culture assays. The bacteria were killed by exposure to 70°C for 15 minutes and confirmed by negative cultures on agar plates. Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation (Ficoll-Paque) as previously described.21 The cells collected from the interface were washed in Hank’s balanced salt solution (HBSS) and resuspended in serum-free lymphocyte tissue culture medium (AIM V; GIBCO, Grand Island, NY) supplemented with 100 mmol/L glutamine. Subsequently, 1.5 × 106 PBMC/mL were cultured in triplicate in sterile 96-well tissue culture plates (Costar Corp., Cambridge, MA) in a total volume of 200 μL and either 1 × 107 colonies of live or heat-killed L. crispatus, 5 × 106 Candida albicans heat-killed blastospores (HKB)/mL, 125 μg/mL of C. albicans soluble cytoplasmic substances (SCS) (provided by Dr. Judith Domer, Appalachian State University, Boone, NC), or 20 μg/mL Phytohemagglutinin (PHA) (Sigma-Aldrich, St. Louis, MO).
To establish a standard bacterial concentration and time for culture, we performed preliminary experiments with PBMCs from 5 participants cultured with different bacterial concentrations (5 × 104, 5 × 106, and 1 × 107 bacteria/mL) in AIM V medium or with AIM V medium alone. Cells were incubated at 37°C, 5% CO2 for 4, 5, and 6 days. During the final 6 hours of culture, 1.0 μCi of 3H-thymidine (ICN Radiochemical, Costa Mesa, CA) was added to each well. The contents of the wells were then harvested onto glass fiber filters, and the radioactivity incorporated into the cells was counted in a liquid scintillation counter (Beckman Instruments, Irving, CA). Data were expressed as proliferation indices (mean counts per minute [cpm] for stimulated cultures divided by mean cpm in unstimulated cultures).
Cytokine Production and Evaluation
The production of cytokines by PBMC was similar to that for proliferation except that the culture was done in 24-well plates and in a 2-mL volume. All cultures were stimulated with 1 x 107 live L. crispatus. The supernatants were collected at 48 hours postculture, aliquoted, and frozen at −70°C until use. Th1-type (IFN-γ, IL-12) and Th2-type/immunoregulatory (IL-4, IL-10, and TGF-β) cytokines were quantified by enzyme-linked immunosorbent assay (ELISA) using commercial capture and biotinylated antibodies (BD Pharmingen, San Diego, CA) and the respective recombinant human cytokine as the standard, as previously described.22 The assays were performed in EIA/A2 96-well plates (Costar, Corning, NY) according to the manufacturer’s instructions. Absorbance was read at 450 nm using an automated plate reader (Bio-Tek Instruments, Winooski, VT).
Protein quantification in CVL was measured using a commercial BCA protein assay kit (Pierce Chemical, Rockford, IL) and bovine serum albumin (BSA) as the standard (Sigma-Aldrich). CVL was tested for Th2-type/immunoregulatory cytokines (IL-4, IL-10, and TGF-β), Th1-type cytokines (IL-12, IFN-γ), proinflammatory cytokines (IL-1, IL-6, and TNF-α), and the chemokine IL-8 by capture ELISA as previously described.22 Concentrations for each sample were extrapolated from the standard curve and expressed as pg/mL. Cytokines in CVL were ultimately normalized to total protein in the sample and expressed as pg/mg protein.
Detection of Total Antibodies
IgA and IgG antibodies were quantified by ELISA, using commercial capture and detection antibodies (Sigma) and the respective human purified antibody (Sigma) as the standard. For each assay, optimal coating concentrations, detection dilutions, and standard concentrations were predetermined as previously described.24 The coating antibody concentrations were as follows: IgA, 20 μg/mL, and IgG, 5 μg/mL. The highest concentrations used for purified IgA and IgG standards were 40 and 625 μg/mL, respectively. The detection antibody (biotinylated) dilutions were 1:30,000 for both anti-IgA and anti-IgG antibodies. O-phenylene diamine dihydrochloride (OPD) was used as the substrate. Total antibody concentrations were determined by extrapolation from the standard curve and then normalized to total protein in CVL and expressed as ng/mg protein.
Clinical, epidemiologic, and laboratory data were entered into the SPSS for Windows database (version 10.0 software; SPSS Inc., Chicago, IL). Descriptive statistics were used for data analysis. We compared categorical variables by chi-squared test and continuous variables by Student ttest or Mann-Whitney Utest. A P value of 0.05 (2-tailed) was defined as statistically significant.
Demographics and Distribution by Vaginal Culture
At the time of the visit and specimen collection, the mean age of subjects enrolled in the cohort (n = 89) was 15 ± 0.93 years. The majority were blacks (82%) followed by whites (16%) and Hispanics (2%). Sexual activity (vaginal intercourse) and vaginal douching were reported in 81% (n = 72) and 65% (n = 57) of subjects, respectively. Oral and progesterone contraception was reported in 22.4% (n = 19) and 37% (n = 31) of 85 subjects with available information.
Vaginal Lactobacilli Colonization and Speciation
Vaginal lactobacilli were identified in 65 of 89 subjects (73%). Of those with lactobacilli, 85% were found to be H2O2-producing strains. The most frequent lactobacilli species detected were L. acidophilus (49%), followed by L. rhamnosus (10.2%), L. crispatus and L. lactis (8.2% each), L. plantarum (6.1%), L. raffinolactis and L. mesenchemoris (4.1% each), and others (10.1%). There was no difference in the distribution by age, ethnicity, contraceptive use, sexual activity, numbers of partners, and use of vaginal douches when subjects were stratified by the presence or absence of vaginal lactobacilli, either H2O2-producing or non-H2O2-producing (data not shown).
Systemic Lactobacilli Sensitization.
A subset of 28 participants was selected for evaluation of systemic sensitization of peripheral blood lymphocytes to vaginal lactobacilli. Results showed positive responses (defined as proliferation index [PI] >3.0) for 50% of subjects in response to heat-killed L. crispatus (Fig. 1A). Similar results were observed for live lactobacilli (data not shown). This was compared with 93% positive responses to killed Candida, 77% positive responses to soluble Candida antigens, and 50% positive responses to streptokinase. Positive responses to PHA were observed in 100% of subjects. The degree of positive responses to L. crispatus did not appreciably change if a PI >2.0 was used as a definition of positive responses (data not shown). The magnitude of responses was highest in response to PHA followed by heat-killed Candida, soluble Candida antigen, streptokinase, and lactobacilli (Fig. 1A). The cytokine pattern in response to L. crispatus was evaluated in the supernatants from similar cell cultures. Immunoregulatory cytokines, including TGF-β and IL-10, were detected in the highest concentrations with Th1-type (IFN-γ and IL-12) and Th2-type (IL-4) cytokines detected at considerably lower concentrations (Fig. 1B).
Bacterial Vaginosis and Vaginal Lactobacilli
BV was detected in 32 of 86 processed specimens (36%), whereas 48% (n = 43) and 12% (n = 11) were defined as normal and intermediate, respectively. There was a significant inverse relationship between BV and the presence of vaginal lactobacilli (P = 0.034, Fig. 2). However, in those with and without BV in which lactobacilli was present, H2O2-producing lactobacilli was present more often than H2O2-nonproducing lactobacilli (85 vs. 15%, respectively).
Local Immunity and Vaginal Lactobacilli
When vaginal cytokine levels were compared between those positive or negative for lactobacilli, a similar pattern was detected for each (Fig. 3A). Among those positive or negative for H2O2-producing lactobacilli, a similar pattern was also observed (Fig. 3B). Total vaginal IgG antibodies (Fig. 4A) were in lower concentrations than IgA antibodies (Fig. 4B). Although a similar pattern of IgG and IgA antibodies were observed in those with or without lactobacilli, significant reductions in both IgG and IgA antibody concentrations were observed in those with H2O2-nonproducing isolates (Fig. 4).
Local Immunity and Bacterial Vaginosis
When vaginal cytokine levels were compared between those positive or negative for BV (overt and/or intermediate), a similar pattern was detected except for IL-1, which was in higher concentrations in those with overt and intermediate BV compared with those without BV (P = 0.022, Fig. 5), but not in those with overt BV only (P = 0.06) (data not shown). Local concentrations of IgA and IgG antibodies were similar between those with or without BV (data not shown).
The vaginal microflora and ecosystem are important elements in the resistance and susceptibility to STDs. There is considerable knowledge of the vaginal microflora and microenvironmental conditions in adult populations. However, there is a paucity of information known about adolescents who are extremely susceptible to STDs. Our data show that an urban population of adolescents, seemingly representative of a general adolescent population about sexual behaviors, contraception, and so on, has considerable vaginal lactobacilli colonization, although somewhat reduced compared with adults (73% vs. 90%).23 Most, but not all, strains of lactobacilli produce hydrogen peroxide. H2O2 production has been proposed as a mechanism by which lactobacilli inhibit the growth of other genital microorganisms.23 The prevalence of H2O2-producing lactobacilli ranges from 56% to 96% of sexually active women with a healthy vaginal ecosystem,24,25 whereas it is approximately 38% among postmenopausal women.26 Premenarchal girls from 1 to 6 years of age are unlikely to be colonized with H2O2-producing lactobacilli.27 We observed that 85% of lactobacilli from this middle adolescent cohort were H2O2-producing strains. Thus, adolescents appear similar to adults relative to H2O2-producing lactobacilli. Interestingly, sexual activity that was associated with yeast colonization in this population28 was not associated with lactobacilli colonization. Other parameters such as contraceptive use, age, ethnicity, sexual behaviors, and STDs had no effect on lactobacilli or yeast colonization.
L. acidophilus and L. fermentum are the primary species colonizing the vagina of adult women using phenotypic identification methods such as biochemical assays, whereas L. crispatus and L. jensenii are recognized as the predominant vaginal lactobacillus species based on DNA homology methods.1,3 In this group of adolescents, L. acidophilus was identified in the highest percentage by phenotypic methods, similar to adults. It is unclear whether the phenotypic results would parallel that by DNA technology,29 but based on the adult studies, they could be expected to be different. Accordingly, we recognize that molecular techniques could identify additional lactobacillus species or existing species in different percentages.
Host defenses protect the reproductive tract from pathogenic organisms. However, there must also be considerable immune tolerance to antigens of the local flora. Host defenses can be divided into 2 functionally independent compartments: systemic and mucosal. This compartmentalization is essential in the female genital tract.30 Immune sensitization against lactobacilli is largely unknown. It would be predicted that some level of detectable immunity would be present as a result of antigen exposure, but that local toleration would be present so as to avoid inflammatory responses at sites of colonization. Indeed, our results showed considerable responsiveness to live and killed lactobacilli (50% positive), suggesting at least some level of lactobacilli-specific systemic immune sensitization. To our knowledge, this is the first attempt to measure lactobacilli-specific immunity in adolescents. Compared with other antigens, responses to lactobacilli were similar to streptokinase but inferior to Candida antigens. This could be the result of the species used in the culture that, although common, was not detected in a majority of adolescents, at least by phenotypic methods. However, without knowledge of any cross-reactivity between antigens for different species, the moderate responsiveness could represent the natural maturation process of these adolescents in the acquisition of adult normal flora. Alternatively, the results could reflect mechanisms of tolerance in some individuals but not others. A formal proliferation study in adults as well as comparative species analysis in adolescents to address antigen cross-reactivity will be required to gain a better understanding of this important issue.
In other evidence of systemic sensitization, results of lactobacilli-specific cytokine production showed a predominant pattern of immunoregulatory cytokines, mainly IL-10 (Th2-type) and TGF-β. The presence of IL-10 is consistent with the response expected to extracellular bacteria as well as with the low levels of Th1-type cytokines. It is unclear why IL-4, another Th2-type cytokine, was low. Perhaps this would be reflective in low levels of lactobacilli-specific antibodies. TGF-β is an important pleiotropic cytokine that together with IL-10 counteracts the effects of proinflammatory cytokines by inhibiting the proliferation of T cells and activation of macrophages,31 thereby creating a condition of tolerance.
Bacterial vaginosis was present in 36% of enrolled adolescents. This is similar to that reported in adult women attending clinics for STDs32 but much higher than the 5% diagnosis rate in women visiting a college health clinic for routine examination.33 Interestingly, the enrolled adolescents attending the health clinic with symptoms of STDs were in a minority in the parent study and were excluded in our cross-sectional analysis. Thus, the adolescent population was more similar to the adult college cohort. Accordingly, our results suggest that BV in adolescents is relatively common and can be acquired as early as 14 years of age. We recognize, however, that the population being studied is from an urban setting and could reflect a more significant susceptibility to BV. It is also possible that the reduced presence of lactobacilli in adolescents compared with adults (73% vs. 90%) contributed to the relatively high rate of BV. Several studies have demonstrated an inverse relationship between the presence of H2O2-producing lactobacilli and BV for both pregnant and nonpregnant adult women.20,24,25,34 We demonstrated a similar relationship, suggesting a protective effect of H2O2-producing lactobacilli in adolescents. Interestingly, in those with BV who had lactobacilli present, a high distribution of H2O2-producing lactobacilli was evident similar to those without BV. Although this could suggest a lack of protection by H2O2-producing lactobacilli in those with BV, numbers of adolescents with BV and lactobacilli was relatively small (n = 18) compared with the number of adolescents without BV but with lactobacilli (n = 43).
Few studies have evaluated the vaginal cytokine milieu in adolescents.18 The complete local cytokine profile in this cohort is the subject of a separate report.28 We could not identify any differences between the concentration of local cytokines or cytokine profile when stratified by the presence or absence of vaginal lactobacilli or H2O2-producing species in the adolescent population. The lack of any correlates to the presence of lactobacilli is consistent with it being part of the normal flora and as such, provoking little in the way of inflammatory responses. There were also few differences of local cytokines when comparing between adolescents with and without BV. The exception was IL-1 that was significantly elevated in adolescents collectively with any level of BV, but not in those with overt BV alone. Elevated IL-1 has been reported previously for pregnant and nonpregnant adults with BV.35,36 Although BV is considered a noninflammatory syndrome, some data indicate a potential link between BV and inflammation.35 We do not, however, rule out the alternative possibility that IL-1 was elevated in response to other vaginal conditions in several of the adolescents explaining the lack of significance in those with overt BV.
The distribution of the immunoglobulins in vaginal secretions of adolescents showing higher levels of total IgA than IgG differs from the distribution in adults. This too was the subject of the previous report.28 No differences were detected in vaginal antibodies between adolescents with or without vaginal lactobacilli, although reduced levels of both IgG and IgA were found in those with H2O2-nonproducing lactobacilli. No differences were detected between those with or without BV. However, not until organism-specific antibodies are quantified will the significance of the antibodies relative to type of lactobacilli or BV be realized.
In summary, we found that the vaginal lactobacilli microflora in an urban population of adolescents, whereas more similar to adults than children, have several unique distinctions. The rates of colonization and species distribution were somewhat different to adults and they have high rates of BV. Local immunity in adolescents showed few differences in cytokines or antibodies as a function of lactobacilli or BV despite evidence of lactobacilli-specific systemic immune sensitization. Although separately none of the differences observed would be expected to increase susceptibility to STDs, together they could contribute to increased risk. Although this analysis did not include those with STDs so that baseline levels of vaginal flora, BV, immunity, and so on, could adequately be established, future studies can include a formal analysis of all these parameters in a cohort of adolescents with and without STDs to identify critical risk factors. In any event, the differences to adults revealed in this study, together with the differences reported in vaginal yeast and vaginal immunity,28 serves to emphasize the distinct nature of the adolescent vaginal ecosystem that is part of the developmental changes that occur during the course to adulthood.
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