Peggy A. Crowley-Nowick

Cytokine Profile in Genital Tract Secretions from Female Adolescents: Impact of Human Immunodeficiency Virus, Human Papillomavirus, and Other Sexually Transmitted Pathogens

Quantitative enzyme-linked immunosorbent assays were used to measure interleukin (IL)-2, IL-10, and IL-12 in cervical secretions from female adolescents with and without sexually transmitted infections. Compared with human immunodeficiency virus [HIV]-negative patients, HIV-positive patients had higher concentrations of IL-10 (118.2 pg/mL vs. 34.5 pg/mL; P=.002) and IL-12 (175.5 pg/mL vs. 85.1; P=.03). IL-2 concentrations were not statistically different. Furthermore, genital tract infections were predictors of IL-10 and IL-12 concentrations. Coinfection with HIV and human papillomavirus predicted the highest IL-10 concentrations; coinfection with HIV, human papillomavirus, and other sexually transmitted pathogens predicted the highest IL-12 concentrations. The data indicate that concomitant infection of the genital tract with HIV and other viral, bacterial, or protozoan pathogens influences the local concentrations of some immunoregulatory cytokines.

Risk of high-grade squamous intraepithelial lesion in HIV-infected adolescents.

BACKGROUND The risk of developing the human papillomavirus (HPV)-associated precancer high-grade squamous intraepithelial lesion (HSIL) in human immunodeficiency virus (HIV)-infected adolescents is unknown. We examined the risk of developing HSIL among adolescents with and without HIV infection. METHODS HIV-infected (n = 172) and -uninfected (n = 84) girls aged 13-18 years who were participating in a multicenter study of primarily horizontally acquired HIV infections in adolescents (Reaching for Excellence in Adolescent Health Care) and who did not have HSIL on cytologic examination at study entry or at the first follow-up visit were followed at 6-month intervals. HIV-uninfected girls were recruited for comparison in a 2:1 ratio (HIV infected:HIV uninfected). The primary outcome was cytologic diagnosis of HSIL confirmed by expert review. RESULTS Incidence of HSIL by the end of follow-up was higher for HIV-infected girls than for HIV-uninfected girls (21.5% vs. 4.8%, respectively). In multivariate analysis, use of hormonal (either estrogen/progesterone oral combination or medroxyprogesterone acetate intramuscular) contraceptives, high cervical mucous concentrations of interleukin (IL)-12, a positive HPV test, and persistent low-grade squamous intraepithelial lesion (LSIL) were significantly associated with the development of HSIL. CONCLUSIONS The incidence of HSIL was alarmingly high in HIV-infected adolescent girls. However, when other predictors were considered in multivariate analysis, HIV status was not retained in the model. The heightened risk for HSIL associated with persistent LSIL underscores the need to closely monitor HIV-infected adolescents with LSIL. The risk for HSIL associated with high concentrations of IL-12 may be suggestive of a local immune dysregulation. The role of hormonal contraception as a risk factor deserves further investigation.

Serologic response to hepatitis B vaccine in HIV infected and high-risk HIV uninfected adolescents in the REACH cohort

PURPOSE To evaluate hepatitis B (HBV) vaccine response rates in HIV infected and high-risk HIV uninfected youth and examine associations with responsiveness in the HIV infected group. METHODS Cohorts within the Reaching for Excellence in Adolescent Care and Health (REACH) study population were defined based on receipt of HBV vaccine both retrospectively and prospectively. Sero-responsiveness was determined by HBsAb measurements. Testing was done for HBsAg, HBsAb, and HBcAb. For HBsAb, a value of > 10 International Units per liter was considered a positive response, and the data were collected as either positive or negative from each of the reporting laboratories. Covariates of responsiveness were explored in univariate and multivariate models for each cohort. RESULTS Sixty-one subjects had received a three-dose vaccination course at the time of entry into REACH. HIV uninfected subjects had significantly higher rates of response by serology compared with HIV infected subjects (70% vs. 41.1%; chi(2) = .05; RR = .586, 95% CI: .36-.96). By the time of an annual visit 43 subjects had received three vaccinations with at least one occurring in the study period. The rates of response were similar for the HIV infected and uninfected groups (37.1% vs. 37.5%) in this cohort. Univariate and multivariate analysis in the prospective HIV infected group (N = 35) found an association between elevated CD8(+)/CD38(+)/HLA-DR(+) T cells and lack of HBV vaccine responsiveness (6.7% vs. 60%; chi(2) = .03; RR = .12, 95% CI: .02- .55). CONCLUSIONS The poor HBV vaccine response rate in the HIV uninfected high-risk adolescents was unexpected and suggests that HBV vaccination doses have not been optimized for older adolescents. This is the first report of decreased responsiveness in HIV infected subjects being associated with elevated CD8(+)/CD38(+)/HLA(-)DR(+) T cells and suggests that ongoing viral replication and concomitant immune system activation decreases the ability of the immune system in HIV infected subjects to respond to vaccination.

Cytokine and immunoglobulin concentrations in cervical secretions: reproducibility of the Weck-cel collection instrument and correlates of immune measures

Elucidation of local immune response at the cervix is important for understanding and evaluating STD vaccine approaches currently being proposed. However, no well-validated method exists for the collection of cervical secretions for evaluation of cervical immune response. The purpose of this study was to determine the reproducibility of the Weck-cel sponge used to collect cervical secretions for immunological assessment. Additionally, it was possible to examine correlates of immunity as part of our investigation. Two cervical secretion specimens were collected sequentially from each of 120 women using Weck-cel sponges. Cervical secretions were collected prior to Pap smear sampling to avoid blood contamination. At the laboratory, the duplicate specimens were weighed and tested in replicate wells to determine the concentration of two cytokines (IL-10 and IL-12) and two immunoglobulin isotypes (IgG and IgA). IL-12, total IgG, and total IgA showed a strong correlation between samples from the same woman ranging from 0.78 to 0.84. Kappa coefficients obtained after categorizing assay results ranged from 0.62 to 0.67. Variance components analysis suggested that 69% to 85% of the variance observed was accounted for by between-women variance, with the remaining variability attributed to variation between samples collected from the same woman. IL-10 results were less reproducible than those obtained from the other assays examined, suggesting problems with the assay used to measure this cytokine rather than with the Weck-cel sampling instrument. Various factors were found to significantly correlate with cytokine and immunoglobulin measures at the cervix. Age and reproductive status were associated with all four immune measures; women over 50 years of age and those who were postmenopausal had increased concentrations of IL-10, IL-12, IgG, and IgA. Hemoglobin concentrations were positively correlated with IgG and IL-10 concentrations, but not with IgA or IL-12 concentrations, suggesting local production of IgA and IL-12. The concentration of all immune measures decreased with increasing volume of collection. No significant association was observed between time from collection to freezing of specimens and concentrations of cytokines or immunoglobulins. Overall, our data suggest that measurement of immunological parameters in cervical secretions collected using Weck-cel sponges are reproducible. In addition, various correlates of cytokine and immunoglobulin concentrations were identified.

Mucosal Immunity of the Adolescent Female Genital Tract

This study sought to characterize mucosal immunity of the adolescent genital tract during the cycle and determine if adolescents have more suppressed immunoglobulin levels in the follicular phase than adults. Daily from cycle day 9 until ovulation, then every other day until menses, cervical secretions for IgA, IgG, and cytokines were collected via Weck-Cel sponge and serum for luteinizing hormone (LH), estradiol, and progesterone was obtained from three adolescent girls (mean age 16.8 years). Immunoglobulin and cytokine levels varied during the menstrual cycle, reaching their nadir around ovulation. Compared with 13 adults, adolescents had a greater drop in IgG in the follicular phase (mean beta-953 vs. -269 microg/mL/day, p = .045), but a similar rate of rise in IgG in the luteal phase (mean beta +118 vs. +100 microg/mL/day, p = .252). Rates of change in IgA did not differ between adolescents and adults for either phase. Although limited by the small sample size, these findings suggest that adolescents may be more sensitive to unopposed estrogen and warrant further investigation.

Douching practices among HIV infected and uninfected adolescents in the United States

PURPOSE To characterize sexual behaviors and sociodemographic factors that are associated with douching among geographically diverse adolescent women with and without HIV infection. METHODS HIV infected subjects recruited preferentially and behaviorally comparable high-risk HIV uninfected subjects were enrolled in a prospective HIV study from 15 sites in 13 U.S. cities. Baseline interview data from 1996 to 1999 for females aged 12 to 19 years were analyzed using one-way analysis of variance and multiple logistic regression. RESULTS Among the 342 females/young women, 74.9% were black (non-Hispanic), 11.1% Hispanic/Latina, and 14.0% white or other race/ethnicity; 63.5% were HIV infected. Young women who had dropped out of high school comprised 23.4% of subjects. In the 3 months before the interview, 179 (52.3%) adolescents had douched at least once. In a multivariable logistic regression model, recent douching was more common among sexually active females (OR = 2.2; 95% CI: 1.2-4.2), Blacks (OR = 2.2; 95% CI: 1.2-4.1 vs. Hispanics/Whites/others), females who dropped out of high school (OR = 2.1; 95% CI: 1.2-3.7), and HIV infected females (OR = 1.7; 95% CI: 1.04-2.7). CONCLUSIONS In this nationwide study, adolescents who are sexually active, African-American, dropped out of high school, and HIV infected were most likely to douche. Interventions to discourage douching should pay special attention to these populations.

T-lymphocyte profile and total and virus-specific immunoglobulin concentrations in the cervix of HIV-1-infected women

Background:The mucosal lymphocyte population is the largest in the body, and the gastrointestinal compartment has been well characterized in HIV infection. Much less is known about the effects of HIV on the genital tract. Objective:To examine the T-lymphocyte phenotype and receptor repertoire as well as total and virus-specific immunoglobulin concentrations in the endocervix of HIV-infected women at different stages of infection as compared with uninfected women. Patients and Methods:Participants were 12 seronegative women, 10 HIV-infected “slow progressors” not taking antiretroviral therapy, and 9 HIV-infected women whose antiretroviral therapy was failing. We used multiparameter flow cytometry to enumerate T-cell populations on cytobrush-obtained cervical specimens, the immunoscope technique to determine the T-cell receptor (TCR) repertoire, and quantitative enzyme-linked immunosorbent assays for antibody determinations on cervical secretions absorbed onto ophthalmic sponges. Nonparametric statistical analyses were performed. Results:We found marked depletion of leukocytes and CD4+ T lymphocytes in the endocervix of HIV-infected women as compared with uninfected women; this was significant at more advanced disease stages. Naive T cells were rare in the endocervix of all groups. Activation marker expression was higher in endocervical T lymphocytes than in peripheral blood among control and slow-progressing HIV-infected women but not in women failing therapy. Endocervical T lymphocytes showed highly restricted utilization of Vβ TCR families. Unlike other mucosal sites, the cervix contained IgG as the predominant immunoglobulin isotype. HIV-IgG was detected in the cervix of most HIV-infected women and in blood of all infected women. Conclusions:HIV infection induces substantial changes in the immune profile of the female genital tract. Further study of the implications of these findings for HIV acquisition and transmission is needed.

Immunology and the REACH Study: HIV immunology and preliminary findings

This review paper presents the immunology findings in human immunodeficiency virus (HIV) infected and uninfected youth in the Reaching for Excellence in Adolescent Care and Health (REACH) Project within the context of basic and HIV immunology concepts. Methods employed in the study for specimen collection, management, and laboratory analysis are presented. This paper reviews published analyses of cross-sectional data; longitudinal analyses are underway. These preliminary data extend the work of others in demonstrating the potential for substantial thymic reserve in youth. This finding in HIV infected adolescents has implications for a fuller response to antiretroviral or immune-based therapies compared to that seen in adults. Dysregulation in mucosal immunity may appear before systemic HIV effects are seen and requires attention particularly to screening and treatment of genital co-infections. REACH has demonstrated gender differences in immunologic measures irrespective of HIV infection status.

Immunogenetic Correlates of Neisseria gonorrhoeae Infection in Adolescents

Background: Understanding host factors modulating immunity to Neisseria gonorrhoeae infection may benefit work on vaccine development. Methods: We analyzed longitudinal data collected from 485 male and female adolescents to determine genetic correlates of genital gonorrhea. Cytokine data from 388 females were analyzed to assess immunologic markers of gonorrhea and their relationship to genetic correlates. Results: The T-G haplotype defining interleukin-2 (IL-2) gene promoter and intron 1 polymorphisms (−330T and −166G) was more frequently found in individuals who had gonorrhea (relative odds = 3.2, P = 0.01). Among 3 endocervical cytokines measured, IL-10 and IL-12 concentrations were higher and IL-2 lower when gonorrhea was detected. The decrease in endocervical IL-2 after gonorrhea acquisition was mostly restricted to subjects with the IL2 T-G haplotype, which may reflect involvement of a pathogen-specific and genetically mediated mechanism for differential IL-2 responses at genital mucosa. In addition, 2 human leukocyte antigen variants (Cw*04 and DQB1*05) were also independently associated with gonorrhea (adjusted relative odds = 1.9 and 0.5, respectively; P <0.05 for both). Conclusions: Confirmation of immunogenetic correlates of gonorrhea in larger cohorts may be useful in guiding further research on both innate and adaptive immune responses to N. gonorrhoeae.

Implications of blood contamination for assessment of local cellular immunity in the endocervix.

Cervical cytobrushes provide a tool to sample endocervical T cells for assessment of local immunity. However, most previous studies in HIV-seropositive women have excluded samples containing blood and hence have analyzed selected populations of patients. As determined by multiple-parameter flow cytometric analysis of T lymphocytes from two sequential cytobrushes and concurrently collected blood samples, this study found a minimal effect of blood contamination on cervical T cell phenotypic parameters in normal women. The consequences of blood in endocervical samples will ultimately depend on the design and objective of each study, but these data suggest studies could be more inclusive and should not automatically discard samples that contain red blood cells.