Defined medium simulating genital tract secretions for growth of vaginal microflora.

A chemically defined medium that simulates female genital tract secretions was developed for the growth of the vaginal microflora. Qualitative and quantitative studies of the growth of predominant components of the vaginal microflora indicated that all vaginal isolates tested were able to grow in this defined medium.     

Effect of uterine immunization and oestradiol on specific IgA and IgG antibodies in uterine, vaginal and salivary secretions.

Levels of IgA and IgG antibodies were measured in uterine and vaginal secretions to examine the effect of uterine immunization on the genital tract humoral immune system. When ovariectomized animals were immunized on Day 0 and boosted 13 days later by placing sheep erythrocytes (SRBC) directly in the uterine lumen (UT/UT) immunization), a pronounced IgA and IgG antibody response was detected in uterine secretions measured on Day 26. This response was 20-30-fold greater than that measured following Peyer's patch immunization and boosting (PP/PP) and Peyer's patch immunization followed by uterine boosting (PP/UT). In contrast to uterine antibody responses that were oestradiol-dependent following PP/PP and PP/UT immunization, UT/UT immunization resulted in IgA and IgG antibody responses that were hormonally independent. To determine whether immunological information is distributed beyond the immediate site of immunization, ovariectomized rats were immunized and boosted by injection of SRBC into one uterine horn. When uterine secretions from the contralateral (non-immune) horns were analysed, IgA and IgG antibodies were found in uterine secretions after oestradiol stimulation. IgA and IgG antibodies were also present in vaginal secretions following UT/UT immunization and ligation of uteri at the utero-cervical junction. This response was hormonally dependent in that vaginal antibody levels were lowered by oestradiol treatment. IgG but not IgA antibodies were also found in saliva of UT/UT immunized animals. Oestradiol had no effect on salivary IgG levels in contrast to those of the genital tract. In summary, these experiments indicate that immunization of uteri can elicit pronounced IgA and IgG antibody responses in uterine secretions and this response is not altered by oestradiol. Moreover, immunization at one site in the genital tract results in the appearance of antibodies at other uterine sites (the contralateral-non-immunized horn), in vaginal secretions, in serum and at other mucosal sites, such as the salivary glands.     

Induction of antibody response to Chlamydia trachomatis in the genital tract by oral immunization.

Groups of BALB/c mice were orally immunized with Chlamydia trachomatis serovar L2/434/Bu in order to characterize the nature and kinetics of the chlamydial antibody response in the cervix and other mucosal sites. These animals were subsequently challenged intravaginally to determine whether oral immunization offers protection against chlamydial antigen shedding in the genital tract. Following oral immunization, immunoglobulin A antibody activity was detected in the genital tract as well as other mucosal sites. Subsequent intravaginal challenges exhibited booster effects on preexisting antibody activity in the genital tract. Significant protection against challenge infection in the genital tract was observed by oral immunization. This was indicated by the absence of any chlamydial antigen shedding in cervical secretions. On the other hand, passively administered chlamydial-specific serum immunoglobulin G antibody did not significantly influence the course of cervical shedding of the organism and did not confer any protection against a subsequent intravaginal challenge. It is concluded that prior oral immunization can induce a secretory antibody response in the genital tract and provide protection against subsequent infection.     

HIV-1 RNA in plasma and genital tract secretions in women infected with HIV-1

To assess antiretroviral therapy, all compartments, including the genital tract, need to be evaluated. HIV-1 RNA was quantified in whole cervicovaginal lavage fluid (CVL) and plasma of 56 women and in the cellular and supernatant fractions of 27 of these women. Overall, we detected HIV-1 RNA in 59% of whole CVL samples and in 61% and 44% of cellular and supernatant fractions of the subset of women, respectively. Detectability of HIV-1 RNA in CVL increased with increasing level of plasma RNA in both unfractionated and cell-associated CVL components (p = .0004 and .002, respectively), but not in the cell-free fraction (p = .29). Mean HIV-1 RNA levels in CVL increased with decreasing CD4 counts (p = .002,) and with increasing plasma HIV-1 RNA (p < .001). Adjusted odds ratios (OR) for detectable CVL RNA were highest for women with CD4 counts <200 cells/mm3 (OR, 10.1; 95% confidence interval [CI]: 1.6-82.7; p = .02) and >50,000 copies/ml of plasma RNA (OR, 25.2; 95% CI, 3.2-554; p = .01). Treatment did not seem to affect RNA detection in CVL after adjusting for plasma RNA and CD4. In conclusion, we found that detectability and level of CVL RNA were closely associated with the cellular fraction of genital secretions in women and strongly correlated with the level of plasma RNA and CD4. Genital tract secretions may need to be tested in the assessment of treatment efficacy and this can easily be accomplished with this rapid and easy procedure using whole CVL.     

Comparison of IgA versus IgG monoclonal antibodies for passive immunization of the murine respiratory tract.

The protective efficacy of anti-Sendai virus IgA was compared to that of IgG after topical application of monoclonal antibodies (MAb) to the respiratory tract of mice. BALB/c mice were passively intranasally immunized with 50 microliters ascites containing equivalent ELISA titers of MAb 1 h before and 4 and 24 h after intranasal challenge with Sendai virus. Lung viral titers were determined by plaque assay 3 days following challenge. In most instances IgA MAb afforded equivalent protection to IgG MAb in that there was no significant difference in virus recovery from the lungs of animals treated with either IgA or IgG MAb, including subclasses of IgG. When IgA MAb was fractionated into monomers and oligomers, there was no inherent advantage to the oligomeric form with respect to passive protection against viral challenge. The data indicate that IgA and IgG antibodies are equally efficacious in protecting the airways from viral infection. The experiments suggest that the advantage of IgA for protecting mucosal surfaces, such as the respiratory tract, relates to the presence of a specialized mechanism for transporting oligomeric IgA across epithelial surfaces. The results also support the rationale for active mucosal immunization protocols designed to generate an IgA response.     

Immunoglobulin G antibodies in human vaginal secretions after parenteral vaccination.

The induction of antibodies in vaginal secretions by systemic (intramuscular) immunization in humans was investigated by using the tetanus toxoid vaccine. Five women, 30 to 40 years old, were injected with a currently used dose of toxoid (40 IU), and serum, saliva, and vaginal secretion samples were collected on day 0 and on day 6 or day 10. All of these subjects had been previously vaccinated at least 5 years before; four were in good health, whereas one suffered from AIDS in clinical category B3. In most cases, analysis of specific antibodies in the vaginal wash showed a dramatic rise after boosting. These antibodies were primarily of the immunoglobulin G (IgG) isotype. The specific activity (ratio of antibody titer to IgG concentration) was shown to increase after the booster injection, irrespective of variations in the IgG level during the menstrual cycle. Comparison between serum and genital antibodies showed no difference in terms of both specific activity and level of avidity. These results demonstrate that parenteral injections can induce a systemic-derived antibody release in the vaginal fluid. Hence, systemic vaccinations can be efficient at the genital level and thus could reinforce or even replace a local vaccine.     

Specific antibody in the equine genital tract following systemic and local immunization.

An enzyme-linked immunosorbent assay (ELISA) was adapted for the horse to investigate the production of antibody in response to subcutaneous, intrauterine or intravaginal immunization with dinitro-phenylated human serum albumin. An IgM response was not detected, but antibodies of the IgGab, IgGc, IgGT and IgA isotypes were measured in serum, uterine and vaginal secretions. Local immunization produced antibody titres in serum and secretions, with evidence of significant local production.     

Methods for evaluation of humoral immune responses in human genital tract secretions

The compilation of epidemiological, virological, and immunological data clearly indicates that HIV-1 infection must be considered primarily as a disease of the mucosal immune system. The earliest and most dramatic alterations of the immune system occur in the mucosal compartment. However, the mucosal immune systems of the genital and intestinal tracts display remarkable immunological differences that must be considered in the evaluation of humoral immune responses in HIV-1-infected individuals or in volunteers immunized with experimental HIV vaccines. In this regard, marked differences in the dominant Ig isotypes, molecular forms of HIV-1-specific antibodies, and their distinct effector functions in the genital versus intestinal tracts must be carefully evaluated and considered in the measurement and interpretation of humoral immune responses. Appropriate controls and alternative immunochemical assays should be used to complement and confirm results generated by ELISA, which are prone to false positivity. Special precautions and rigorous controls must be used in the evaluation of antibody-mediated virus neutralization in external secretions of the genital and intestinal tracts.     

Comparison of different medium bases for the semiquantitative isolation of anaerobes from vaginal secretions.

Two studies were performed to determine the best medium for the isolation of anaerobes from vaginal secretions. In the first, three different medium bases (brucella, Centers for Disease Control [CDC], and Schaedler) were compared semiquantitatively for ability to support the growth of gram-negative anaerobes from vaginal fluid. Media were supplemented with laked sheep blood, kanamycin, and vancomycin. The brucella base agar formulation supported the growth of anaerobic gram-negative bacilli better than either the CDC or Schaedler base agar formulation. In a second study, nonselective brucella and CDC base sheep blood agar were compared for ability to support the growth of anaerobic gram-positive cocci. Anaerobic gram-positive cocci grew in higher concentrations on CDC base agar than on brucella base agar. On the basis of these observations, we recommend that a CDC base sheep blood agar be used for the nonselective plate and a brucella base plate supplemented with laked sheep blood, kanamycin, and vancomycin be used for isolation of gram-negative bacilli in studies of the anaerobic flora of the female genital tract.     

Determination of sperm antibodies in human genital secretions by an ELISA technique

Sera and genital secretions of 178 infertile men and 40 infertilewomen were evaluated for antibodies against spermatozoa by anenzymelinked immunosorbent assay (ELISA). It was shown thatcirculating sperm antibodies are not usually transudated intogenital secretions and, on the other hand, that local antibodiesfound in cervical mucus or seminal plasma are not detectablein serum. Furthermore it was demonstrated that the solubilizationof cervical mucus by bromelain almost completely removes antibodyactivities detectable by ELISA, whereas sonkation of mucus doesnot affect the immunoglobulins. Sonication should thus be appliedfor liquefaction of cervical mucus in order to assess its antibodyproperties.     

Microbial flora of the lower genital tract of women in labour in Zaria, Nigeria.

Nine genera of microbes isolated from the lower genital tract of 187 women in labour in Zaria have been identified. The work was undertaken to establish the nature of microorganisms in the lower genital tract of women in labour as a basis for further study. The isolates in order of prevalence were: Candida albicans (20.9%), Klebsiella sp (15.0%), Escherichia coli (9.1%), Streptococcus faecalis (6.4%), haemolytic streptococci (other than Streptococcus pyogenes (2.7%), Streptococcus viridans (2.1%), Staphylococcus aureus (2.1%), Aeromonas hydrophila (2.1%), Proteus mirabilis (1.1%), Peptostreptococcus putridus (1.1%), Streptococcus pyogenes (0.5%), and Streptococcus pneumoniae (0.5%). Neisseria gonorrhoeae, Haemophilus sp, Lactobacillus sp, and Clostridium sp were sought but not found. Chlamydia, viruses, and T-strains of mycoplasma and trichomonas were not sought. It appears from this study that the lower genital tract of most women in Zaria at the time of labour is heavily colonised by pathogens. For this reason alone prolonged labour and trauma to the genital tract at the time of delivery should be avoided.     

Preoperative treatment with oestradiol in women scheduled for vaginal operation for genital prolapse. A randomised, double-blind trial

Objective — To disclose a clinical and histopathological effect of local low-dose oestradiol treatment on the vagina. Design — A randomised, double-blind trial. Setting — Two gynaecological departments at University Hospitals. Subjects — Forty-eight postmenopausal women scheduled for surgery because of genital prolapse. Intervention — 25μg oestradiol or placebo, administered as vaginal pessaries daily, 3 weeks prior to surgery. Main outcome measures — Cytological, histological and clinical changes of the vaginal mucosa. Results — The thickness of the vaginal wall increased as did the oestrogenic index. No clinical effect was seen apart from decreased incidence of recurrent cystitis postoperatively. Conclusions — Preoperative oestrogen treatment has been shown to reduce the incidence of recurrent cystitis and may be needed for stimulation of vaginal mucosa; the short-term clinical effect is not convincing, however.     

Systemic and mucosal immune responses in mice after rectal and vaginal immunization with HIV-DNA vaccine.

We examined the feasibility of inducing local and systemic human immunodeficiency virus (HIV)-specific immune responses by rectal and vaginal application of an HIV-DNA vaccine. Mice were immunized with an HIV-DNA vaccine preparation via a rectal or vaginal route. After several applications, HIV-specific antibodies were detected in sera, fecal extract solutions, and vaginal washes, and these antibodies were potent in inhibiting the syncytium formation of a CD4-positive human T cell line by a cell line capable of inducing HIV-1 infection. Spleen cells from rectally and vaginally immunized mice showed antigen-mediated IFN-gamma-inducing activity. In addition, with rectal immunization, mononuclear cells from both the spleen and the regional lymph nodes of the rectal region were found to be potent at inducing a cytotoxic T lymphocyte response. These humoral and cell-mediated immune responses were enhanced by augmenting the vaccine with granulocyte-macrophage colony-stimulating factor-expressing plasmids or IL-12-expressing plasmid. Our results demonstrated that both rectal and vaginal immunization could induce systemic and mucosal immunity and that these responses were enhanced by the addition of the above cytokine-expressing plasmids.     

Utility of urine, vaginal, cervical, and rectal specimens for detection of Mycoplasma genitalium in women

This study assessed the utility of urine, vaginal, cervical, and rectal specimens for the detection of Mycoplasma genitalium in women by using our laboratory-developed PCR assay. The relative sensitivity was 85.7% for the vaginal swab specimen, 74.3% for the endocervical swab specimen, 61.4% for the urine specimen, and 24.3% for the rectal swab specimen.     

Tumor necrosis factor alpha activity in genital tract secretions of guinea pigs infected with chlamydiae.

Previous studies using the guinea pig model of chlamydial genital infection demonstrated that primary infection is associated with a marked acute inflammatory response early on, while chronic inflammation appears later, at a time when the level of infection is reduced. Challenge infections result primarily in a chronic inflammatory response. The stimuli that initiate inflammation and lead to tissue damage have not been defined. We investigated the possibility that tumor necrosis factors (TNFs) play a role in the inflammatory response to chlamydial genital tract infection. Cytotoxicity assays for TNF were performed on genital tract secretions collected from female guinea pigs during infection with the Chlamydia psittaci agent of guinea pig inclusion conjunctivitis. During the early days of primary infection, high levels of TNF-alpha were detected in genital tract secretions from inbred S2 strain and outbred Hartley strain guinea pigs. Significantly lower levels of TNF-alpha were detected in secretions from both strains during challenge infection. In general, the intensity of the TNF-alpha response was proportional to the intensity of infection. High TNF-alpha levels were present during primary infection at a time of marked neutrophil influx. Thus, TNF-alpha may play an important role in the response to primary chlamydial genital tract infection.     

Evaluation of a quantitative competitive PCR assay for measuring herpes simplex virus DNA content in genital tract secretions.

Previous studies have shown an association between the approximate titer of herpes simplex virus (HSV) DNA in clinical specimens and the ability to isolate HSV from genital secretions. To control for variance in amplification conditions, we developed a competitive quantitative PCR (QC PCR) for the detection of HSV DNA. The assay accurately measured from 10 to 10(6) copies of HSV DNA. We compared the QC PCR with our previous semiquantitative detection method and found concordance for 61 of 63 positive specimens. We also evaluated the HSV DNA content from individual swabs of genital secretions obtained from individual sites of the genital tract (cervix, vulva, and rectum) with that from one swab with secretions from all three sites. The concordance for detecting HSV DNA was 91%; for only 4 of 143 collection days was there a > 1 log difference between the two collection methods. A single swab with secretions from all three genital sites and evaluated in a QC PCR format can accurately measure the frequency of subclinical and clinical shedding of HSV and the titer of HSV shed from the genital region. Such an approach should be very useful in the evaluation of antiviral chemotherapy for HSV.     

Immune response of the female rat genital tract after oral and local immunization with keyhole limpet hemocyanin conjugated to the cholera toxin B subunit.

The immune response of the female rat genital tract was evaluated with Lewis rats given primary and secondary immunizations with keyhole limpet hemocyanin (KLH) alone or coupled to the cholera toxin (CT) B subunit (CTB) by the oral or intravaginal-uterine route or a combination of routes. CT (2 to 5 micrograms) was administered as an adjuvant with the KLH-CTB conjugate. While a significant mucosal immunoglobulin A (IgA) response was induced by KLH, there were no significant differences among the immunized groups in the levels of IgA antibodies in salivary gland, gut, vaginal, and uterine secretions, with the exception that rats immunized only orally with the KLH-CTB conjugate lacked a detectable vaginal response. Levels of IgA antibodies to CT, however, were significantly increased in genital tract secretions of rats immunized locally versus orally with the KLH-CTB conjugate. Antibody activity of the IgG isotype against both KLH and CT was significantly elevated in genital tract secretions of rats immunized with KLH-CTB by the oral or intravaginal-uterine route and given genital tract boosters, in comparison with the results for the other groups. IgM antibody titers were generally negligible in the different secretions. An enzyme-linked spot-forming assay revealed IgA and IgG antibody-secreting cells in salivary gland and uterine tissues. A highly significant correlation between the numbers of antibody-secreting cells and antibody titers existed for uterine IgG but not IgA responses to KLH among the different groups of rats. In conclusion, a vigorous local immune response was induced after immunization of the female rat reproductive tract alone or in combination with peroral challenge with the KLH-CTB conjugate.     

Bovine venereal vibriosis: variations in immunoglobulin class of antibodies in genital secretions and serum

The immunoglobulin classes of antibodies to Campylobacter (Vibrio) fetus in cervicovaginal mucus (CVM) were determined by the indirect fluorescent antibody test at sequential periods, since the order of class appearance has not been established for specific secretory immune responses. In the local immune response to C. fetus immunoglobulin M (IgM) antibodies appeared first, immunoglobulin A (IgA) antibodies next, and immunoglobulin G (IgG) last. IgM antibodies were quite transient, but IgG antibodies remained longer, and those of the IgA class persisted until the end of the experimental period (up to 10 months). Since differences appear to exist between immune mechanisms at cervicovaginal and uterine sites, as well as between immune responses induced by local and systemic immunizations, the immunoglobulin classes of antibodies in uterine secretions were compared with the classes in CVM and serum. Uterine antibodies arose coincidently with uterine lesions in heifers slaughtered after short periods of infection. In convalescent animals only IgA antibodies were found in CVM, whereas the predominant class of antibodies in the uterine secretions was IgG(1) in three of four animals studied. Only IgG antibodies were detected in CVM and uterine secretions of systemically immunized animals. These findings could account for faster clearance of C. fetus from the uterus than from the cervicovaginal area in locally infected animals and for failure of colonization in systemically immunized animals, because IgG antibodies are good opsonins and IgA antibodies are not. IgA antibodies do immobilize C. fetus, however, so they could prevent recolonization of the uterus in cervicovaginal carriers.