Neelima Chandra

Interleukin (IL)-1, IL-6, and IL-8 Predict Mucosal Toxicity of Vaginal Microbicidal Contraceptives

Abstract Inflammation of the female reproductive tract increases susceptibility to HIV-1 and other viral infections and, thus, it becomes a serious liability for vaginal products. Excessive release of proinflammatory cytokines may alter the mucosal balance between tissue destruction and repair and be linked to enhanced penetration and replication of viral pathogens upon chemical insult. The present study evaluates four surface-active microbicide candidates, nonoxynol-9 (N-9), benzalkonium chloride (BZK), sodium dodecyl sulfate, and sodium monolaurate for their activity against human sperm and HIV, and their capacity to induce an inflammatory response on human vaginal epithelial cells and by the rabbit vaginal mucosa. Spermicidal and virucidal evaluations ranked N-9 as the most potent compound but were unable to predict the impact of the compounds on vaginal cell viability. Interleukin (IL)-1 release in vitro reflected their cytotoxicity profiles more accurately. Furthermore, IL-1 concentrations in vaginal washings correlated with cumulative mucosal irritation scores after single and multiple applications (P < 0.01), showing BZK as the most damaging agent for the vaginal mucosa. BZK induced rapid cell death, IL-1 release, and IL-6 secretion. The other compounds required either more prolonged or repeated contact with the vaginal epithelium to induce a significant inflammatory reaction. Increased IL-8 levels after multiple applications in vivo identified compounds with the highest cumulative mucosal toxicity (P < 0.01). In conclusion, IL-1, IL-6, and IL-8 in the vaginal secretions are sensitive indicators of compound-induced mucosal toxicity. The described evaluation system is a valuable tool in identifying novel vaginal contraceptive microbicides, selecting out candidates that may enhance, rather than decrease, HIV transmission.

Depot Medroxyprogesterone Acetate Increases Immune Cell Numbers and Activation Markers in Human Vaginal Mucosal Tissues

The relationship between exogenous contraceptive hormones and permissiveness of the female genital tract to human immunodeficiency virus type 1 (HIV-1) is the subject of renewed debate. To better characterize the effect of depot medroxyprogesterone acetate (DMPA) on HIV-1 cellular targets and epithelial integrity in the vagina, we compared leukocyte populations, markers of activation and proliferation, and the density of intercellular junctional proteins in the vaginal epithelium of women during the follicular and luteal phases of the menstrual cycle and approximately 12 weeks after receiving a DMPA injection. This prospective cohort study involved 15 healthy women. Vaginal biopsies were obtained in the follicular and luteal phases of the menstrual cycle, and approximately 12 weeks following a 150-mg intramuscular injection of DMPA. Leukocyte populations, activation phenotype, and epithelial tight junction and adherens proteins were evaluated by immunohistochemistry. After receiving DMPA, the numbers of CD45, CD3, CD8, CD68, HLA-DR, and CCR5 bearing immune cells were significantly (p<0.05) increased in vaginal tissues, compared to the follicular and/or luteal phases of untreated cycles. There were no significant differences in immune cell populations between the follicular and luteal phases of the control cycle. There were also no statistically significant differences in epithelial thickness and density of epithelial tight junction and adherens proteins among the follicular, luteal, and post-DMPA treatment sampling points. In this pilot study, vaginal immune cell populations were significantly altered by exogenous progesterone, resulting in increased numbers of T cells, macrophages, and HLA-DR- and CCR5-positive cells.

A 90-Day Tenofovir Reservoir Intravaginal Ring for Mucosal HIV Prophylaxis

ABSTRACT A vaginal gel containing the antiretroviral tenofovir (TFV) recently demonstrated 39% protection against HIV infection in women. We designed and evaluated a novel reservoir TFV intravaginal ring (IVR) to potentially improve product effectiveness by providing a more controlled and sustained vaginal dose to maintain cervicovaginal concentrations. Polyurethane tubing of various hydrophilicities was filled with a high-density TFV/glycerol/water semisolid paste and then end-sealed to create IVRs. In vitro, TFV release increased with polyurethane hydrophilicity, with 35 weight percent water-swelling polyurethane IVRs achieving an approximately 10-mg/day release for 90 days with mechanical stiffness similar to that of the commercially available NuvaRing. This design was evaluated in two 90-day in vivo sheep studies for TFV pharmacokinetics and safety. Overall, TFV vaginal tissue, vaginal fluid, and plasma levels were relatively time independent over the 90-day duration at approximately 104 ng/g, 106 ng/g, and 101 ng/ml, respectively, near or exceeding the highest observed concentrations in a TFV 1% gel control group. TFV vaginal fluid concentrations were approximately 1,000-fold greater than levels shown to provide significant protection in women using the TFV 1% gel. There were no toxicological findings following placebo and TFV IVR treatment for 28 or 90 days, although slight to moderate increases in inflammatory infiltrates in the vaginal epithelia were observed in these animals compared to naïve animals. In summary, the controlled release of TFV from this reservoir IVR provided elevated sheep vaginal concentrations for 90 days to merit its further evaluation as an HIV prophylactic.

Preclinical assessment of the proinflammatory potential of microbicide candidates.

The efficacy of a microbicide depends on the balance between its specific activity and its safety. The experience with nonoxynol-9, the first microbicide to be tested in clinical trials, provides a good example of a compound with high in-vitro activity and poor clinical performance, possibly because of underestimated local safety issues. In order to identify compounds that may induce epithelial toxicity and inflammation early in drug development, we have established a preclinical evaluation system based on the assessment of cytotoxicity, cytokine secretion, and tissue inflammatory reaction using a human vaginal cell line (VK-2/E6E7) and a refined rabbit vaginal irritation model. Evaluated through this system, nonoxynol-9 displayed high cytotoxicity and proinflammatory activity. VK-2 cells incubated for 6 h with nonoxynol-9 released significant amounts of IL-1, IL-6 and IL-8. Gels containing 2 and 4% nonoxynol-9, administered to female rabbits intravaginally for 3 days, induced a potent inflammatory reaction evidenced by high levels of IL-1b and CD3+ T cells in cervicovaginal lavages and a significant influx of CD4 and nuclear factor kappa B cells into mucosal and submucosal tissues. Interestingly, cervicovaginal epithelium exposed to nonoxynol-9 also showed high levels of active nuclear factor kappa B immunoreactivity. This combined, sequential, preclinical evaluation system based on VK-2 cells in culture and a refined rabbit vaginal irritation model represents a valuable tool to assess the local safety profile of anti-HIV microbicide candidates.

Biomarkers of leukocyte traffic and activation in the vaginal mucosa

Abstract Development of novel vaginal spermicides and anti-human immunodeficiency virus (HIV) microbicides requires careful assessment of their potential to recruit and activate CD4+ HIV-1 host cells in the female genital tract mucosa, two events that facilitate HIV-1 infection. Leukocyte traffic and activation are mediated by proinflammatory cytokines and chemokines, e.g. interleukin (IL)-1, IL-6 and IL-8, which have been detected in vaginal secretions in association with epithelial damage and infections. These proinflammatory mediators, however, have bidirectional, destructive as well as beneficial, effects on the mucosal barrier, and may be counterbalanced by endogenous inhibitors. Here we propose additional biomarkers for the evaluation of compound-induced cervicovaginal mucosal inflammation. Displaying different temporal patterns of detection, the levels of soluble E-selectin, vascular adhesion molecule-1, CD14 and myeloperoxidase in vaginal secretions reflected the mucosal leukocyte reaction to proinflammatory compounds being evaluated for safety in an improved rabbit vaginal irritation model. These biomarkers, which were also detected in human vaginal secretions, may be used to enhance the characterization of mucosal safety of vaginally applied compounds, both in animal as well as clinical studies.

Six-day randomized safety trial of intravaginal lime juice

Objectives:Nigerian women reportedly apply lime juice intravaginally to protect themselves against HIV. In vitro data suggest that lime juice is virucidal, but only at cytotoxic concentrations. This is the first controlled, randomized safety trial of lime juice applied to the human vagina. Methods:Forty-seven women were randomized to apply water or lime juice (25%, 50%, or undiluted) intravaginally twice daily for two 6-day intervals, separated by a 3-week washout period. Product application also was randomized: during 1 interval, product was applied using a saturated tampon and in the other by douche. Vaginal pH, symptoms, signs of irritation observed via naked eye examination and colposcopy, microflora, and markers of inflammation in cervicovaginal lavages were evaluated after 1 hour and on days 3 and 7. Results:The largest reduction in pH was about one-half a pH unit, seen 1 hour after douching with 100% lime juice. We observed a dose-dependent pattern of symptoms and clinical and laboratory findings that were consistent with a compromised vaginal barrier function. Conclusions:The brief reduction in pH after vaginal lime juice application is unlikely to be virucidal in the presence of semen. Lime juice is unlikely to protect against HIV and may actually be harmful.

Bacterial Vaginosis and Subclinical Markers of Genital Tract Inflammation and Mucosal Immunity

Bacterial vaginosis (BV) has been linked to an increased risk of human immunodeficiency virus (HIV) acquisition and transmission in observational studies, but the underlying biological mechanisms are unknown. We measured biomarkers of subclinical vaginal inflammation, endogenous antimicrobial activity, and vaginal flora in women with BV and repeated sampling 1 week and 1 month after completion of metronidazole therapy. We also compared this cohort of women with BV to a healthy control cohort without BV. A longitudinal, open label study of 33 women with a Nugent score of 4 or higher was conducted. All women had genital swabs, cervicovaginal lavage (CVL) fluid, and cervicovaginal biopsies obtained at enrollment and received 7 days of metronidazole treatment. Repeat sampling was performed approximately 1 week and 1 month after completion of therapy. Participants baseline samples were compared to a healthy, racially matched control group (n=13) without BV. The CVL from women with resolved BV (Nugent 0-3) had significantly higher anti-HIV activity, secretory leukocyte protease inhibitor (SLPI), and growth-related oncogene alpha (GRO-α) levels and their ectocervical tissues had significantly more CD8 cells in the epithelium. Women with persistent BV after treatment had significantly higher levels of interleukin-1β, tumor necrosis factor alpha (TNF-α), and intercellular adhesion molecule 1 (ICAM-1) in the CVL. At study entry, participants had significantly greater numbers of CCR5(+) immune cells and a higher CD4/CD8 ratio in ectocervical tissues prior to metronidazole treatment, compared to a racially matched cohort of women with a Nugent score of 0-3. These data indicate that BV is associated with changes in select soluble immune mediators, an increase in HIV target cells, and a reduction in endogenous antimicrobial activity, which may contribute to the increased risk of HIV acquisition.

Seminal plasma induces prostaglandin-endoperoxide synthase (PTGS) 2 expression in immortalized human vaginal cells: involvement of semen prostaglandin E2 in PTGS2 upregulation.

ABSTRACT Inflammation of the cervicovaginal mucosa is considered a risk factor for HIV infection in heterosexual transmission. In this context, seminal plasma (SP) may play an important role that is not limited to being the main carrier for the virions. It is known that SP induces an inflammatory reaction in the cervix called postcoital leukocytic reaction, which has been associated with promotion of fertility. The mechanisms by which SP triggers this reaction, however, have not been clearly established. Previously we reported the expression of prostaglandin-endoperoxide synthase 2 (PTGS2), also known as cyclooxygenase 2 (COX-2), in human vaginal cells in response to toll-like receptor (TLR) ligands and other proinflammatory stimuli. In this study, we demonstrate that SP induces transcriptional and translational increase of COX-2 expression in human vaginal cells and cervicovaginal tissue explants. Furthermore, SP potentiates vaginal PTGS2 expression induced by other proinflammatory stimulants, such as TLR ligands and a vaginal mucosal irritant (nonoxynol-9) in a synergistic manner. SP-induced PTGS2 expression is mediated by intracellular signaling pathways involving MAPKs and NF-κB. Using fractionation and functional analysis, seminal prostaglandin (PG)-E2 was identified as a one of the major factors in PTGS2 induction. Given the critical role of this PG-producing enzyme in mucosal inflammatory processes, the finding that SP induces and potentiates the expression of PTGS2 in cervicovaginal cells and tissues has mechanistic implications for the role of SP in fertility-associated mucosal leukocytic reaction and its potential HIV infection-enhancing effect.

Induction of Cyclooxygenase (COX)-2 in Human Vaginal Epithelial Cells in Response to TLR ligands and TNF-α

Mucosal inflammation caused by infections of the female lower genital tract is considered to be an important cofactor for HIV transmission. We hypothesize that COX‐2, a key inflammation‐related enzyme, is involved in these responses and is upregulated by microbial ligands and pro‐inflammatory cytokines.

A quantitative multiplex nuclease protection assay reveals immunotoxicity gene expression profiles in the rabbit model for vaginal drug safety evaluation.

Any vaginal product that alters the mucosal environment and impairs the immune barrier increases the risk of sexually transmitted infections, especially HIV infection, which thrives on mucosal damage and inflammation. The FDA-recommended rabbit vaginal irritation (RVI) model serves as a first line selection tool for vaginal products; however, for decades it has been limited to histopathology scoring, insufficient to select safe anti-HIV microbicides. In this study we incorporate to the RVI model a novel quantitative nuclease protection assay (qNPA) to quantify mRNA levels of 25 genes representing leukocyte differentiation markers, toll-like receptors (TLR), cytokines, chemokines, epithelial repair, microbicidal and vascular markers, by designing two multiplex arrays. Tissue sections were obtained from 36 rabbits (6 per treatment arm) after 14 daily applications of a placebo gel, saline, 4% nonoxynol-9 (N-9), and three combinations of the anti-HIV microbicides tenofovir (TFV) and UC781 in escalating concentrations (highest: 10% TFV+2.5%UC781). Results showed that increased expression levels of toll-like receptor (TLR)-4, interleukin (IL)-1β, CXCL8, epithelial membrane protein (EMP)-1 (P<0.05), and decreased levels of TLR2 (P<0.05), TLR3 and bactericidal permeability increasing protein (BPI) (P<0.001) were associated with cervicovaginal mucosal alteration (histopathology). Seven markers showed a significant linear trend predicting epithelial damage (up with CD4, IL-1β, CXCL8, CCL2, CCL21, EMP1 and down with BPI). Despite the low tissue damage RVI scores, the high-dose microbicide combination gel caused activation of HIV host cells (SLC and CD4) while N-9 caused proinflammatory gene upregulation (IL-8 and TLR4) suggesting a potential for increasing risk of HIV via different mechanisms depending on the chemical nature of the test product.