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1.  The Effects of Reproductive Hormones on the Physical Properties of Cervicovaginal Fluid 
Objectives
The purpose of this study was to determine the impact of contraception, menopause and vaginal flora on the physical and biochemical properties of cervicovaginal fluid.
Study Design
Vaginal swabs, cervicovaginal fluid (CVF) and cervicovaginal lavage (CVL) were collected from a total of 165 healthy asymptomatic women including: post-menopausal women (n=29), women in the proliferative (n=26) or follicular (n=27) phase, and women using the levonogestrel intrauterine device (LNG-IUD) (n=28), depomedroxyprogesterone acetate (DMPA) (n=28) or combined oral contraceptives (OCPs) (n=27). Vaginal smears were evaluated using the Nugent score. The osmolality, viscosity, density and pH of CVL samples were measured.
Results
CVL from postmenopausal women and women with abnormal vaginal flora was less viscous and had higher pH than premenopausal women and women with normal flora, respectively. Women using hormonal contraceptives had more viscous CVL as compared to premenopausal women not using hormonal contraceptives, but this increase in viscosity was mitigated in the presence of bacterial vaginosis. Women using DMPA had less total protein in the CVL as compared to women using the LNG-IUD, and had similar protein content when compared to postmenopausal women.
Conclusion
The differences in CVL protein content between DMPA and LNG-IUD suggest that type of progesterone and route of delivery impact the vaginal environment. Contraceptive hormone users had more viscous CVL than women not using contraceptives. However, the presence of bacterial vaginosis impacted both the pH and viscosity (regardless of hormonal contraceptive use), demonstrating that vaginal flora has a greater impact on the physical properties of cervicovaginal fluid than reproductive hormones.
doi:10.1016/j.ajog.2014.03.041
PMCID: PMC4149850  PMID: 24662718
Contraceptives; cervicovaginal fluid; bacterial vaginosis
2.  Evaluation of PD 404,182 as an Anti-HIV and Anti-Herpes Simplex Virus Microbicide 
PD 404,182 (PD) is a synthetic compound that was found to compromise HIV integrity via interaction with a nonenvelope protein viral structural component (A. M. Chamoun et al., Antimicrob. Agents Chemother. 56:672–681, 2012). The present study evaluates the potential of PD as an anti-HIV microbicide and establishes PD's virucidal activity toward another pathogen, herpes simplex virus (HSV). We show that the anti-HIV-1 50% inhibitory concentration (IC50) of PD, when diluted in seminal plasma, is ∼1 μM, similar to the IC50 determined in cell culture growth medium, and that PD retains full anti-HIV-1 activity after incubation in cervical fluid at 37°C for at least 24 h. In addition, PD is nontoxic toward vaginal commensal Lactobacillus species (50% cytotoxic concentration [CC50], >300 μM), freshly activated human peripheral blood mononuclear cells (CC50, ∼200 μM), and primary CD4+ T cells, macrophages, and dendritic cells (CC50, >300 μM). PD also exhibited high stability in pH-adjusted Dulbecco's phosphate-buffered saline with little to no activity loss after 8 weeks at pH 4 and 42°C, indicating suitability for formulation for transportation and storage in developing countries. Finally, for the first time, we show that PD inactivates herpes simplex virus 1 (HSV-1) and HSV-2 at submicromolar concentrations. Due to the prevalence of HSV infection, the ability of PD to inactivate HSV may provide an additional incentive for use as a microbicide. The ability of PD to inactivate both HIV-1 and HSV, combined with its low toxicity and high stability, warrants additional studies for the evaluation of PD's microbicidal candidacy in animals and humans.
doi:10.1128/AAC.02000-13
PMCID: PMC3910842  PMID: 24217696
3.  Is Wetter Better? An Evaluation of Over-the-Counter Personal Lubricants for Safety and Anti-HIV-1 Activity 
PLoS ONE  2012;7(11):e48328.
Because lubricants may decrease trauma during coitus, it is hypothesized that they could aid in the prevention of HIV acquisition. Therefore, safety and anti-HIV-1 activity of over-the-counter (OTC) aqueous- (n = 10), lipid- (n = 2), and silicone-based (n = 2) products were tested. The rheological properties of the lipid-based lubricants precluded testing with the exception of explant safety testing. Six aqueous-based gels were hyperosmolar, two were nearly iso-osmolar, and two were hypo-osmolar. Evaluation of the panel of products showed Gynol II (a spermicidal gel containing 2% nonoxynol-9), KY Jelly, and Replens were toxic to Lactobacillus. Two nearly iso-osmolar aqueous- and both silicone-based gels were not toxic toward epithelial cell lines or ectocervical or colorectal explant tissues. Hyperosmolar lubricants demonstrated reduction of tissue viability and epithelial fracture/sloughing while the nearly iso-osmolar and silicon-based lubricants showed no significant changes in tissue viability or epithelial modifications. While most of the lubricants had no measurable anti-HIV-1 activity, three lubricants which retained cell viability did demonstrate modest anti-HIV-1 activity in vitro. To determine if this would result in protection of mucosal tissue or conversely determine if the epithelial damage associated with the hyperosmolar lubricants increased HIV-1 infection ex vivo, ectocervical tissue was exposed to selected lubricants and then challenged with HIV-1. None of the lubricants that had a moderate to high therapeutic index protected the mucosal tissue. These results show hyperosmolar lubricant gels were associated with cellular toxicity and epithelial damage while showing no anti-viral activity. The two iso-osmolar lubricants, Good Clean Love and PRÉ, and both silicone-based lubricants, Female Condom 2 lubricant and Wet Platinum, were the safest in our testing algorithm.
doi:10.1371/journal.pone.0048328
PMCID: PMC3492332  PMID: 23144863
4.  Development and Characterization of a Vaginal Film Containing Dapivirine, a Non- nucleoside Reverse Transcriptase Inhibitor (NNRTI), for prevention of HIV-1 sexual transmission 
Dapivirine, a non-nucleoside reverse transcriptase inhibitor, is a potent and promising anti-HIV molecule. It is currently being investigated for use as a vaginal microbicide in two dosage forms, a semi-solid gel and a silicone elastomer ring. Quick-dissolving films are promising and attractive dosage forms that may provide an alternative platform for the vaginal delivery of microbicide drug candidates. Vaginal films may provide advantages such as discreet use, no product leakage during use, lack of requirement for an applicator for insertion, rapid drug release and minimal packaging and reduced wastage. Within this study the in vitro bioactivity of dapivirine as compared to the NNRTI UC781 was further established and a quick dissolve film was developed for vaginal application of dapivirine for prevention of HIV infection. The developed film was characterized with respect to its physical and chemical attributes including water content, mechanical strength, drug release profile, permeability, compatibility with lactobacilli and bioactivity. The anti-HIV activity of the formulated dapivirine film was confirmed in in vitro and ex vivo models. Importantly the physical and chemical properties of the film as well as its bioactivity were maintained for a period of 18 months. In conclusion, a vaginal film containing dapivirine was developed and characterized. The film was shown to prevent HIV-1 infection in vitro and ex vivo and have acceptable characteristics which make this film a promising candidate for testing as vaginal microbicide.
doi:10.1007/s13346-011-0022-6
PMCID: PMC3375737  PMID: 22708075
Dapivirine; TMC120; UC781; Microbicides; Thin Films; HIV prevention; Vaginal Drug Delivery
5.  Degradation of naturally occurring and engineered antimicrobial peptides by proteases 
We hypothesized that current antimicrobial peptides should be susceptible to proteolytic digestion. The antimicrobial peptides: Griffithinsin, RC-101, LL-37, LSA-5, PSC-RANTES and DJ007 were degraded by commercially available proteases. Two different species of anaerobic vaginal flora, Prevotella bivia and Porphyromonas asaccharolytica also degraded the materials. Griffithsin was resistant to digestion by 8 of the 9 proteases and the bacteria while LL-37 was the most sensitive to protease digestion. These data suggests most of the molecules may not survive for very long in the proteolytic rich environments in which they are intended to function.
doi:10.4236/abb.2011.26059
PMCID: PMC3354962  PMID: 22611520
Microbicides; HIV; Anti-HIV; Antimicrobial Peptides; Proteases
6.  In Vitro and Ex Vivo Testing of Tenofovir Shows It Is Effective As an HIV-1 Microbicide 
PLoS ONE  2010;5(2):e9310.
Background
Tenofovir gel has entered into clinical trials for use as a topical microbicide to prevent HIV-1 infection but has no published data regarding pre-clinical testing using in vitro and ex vivo models. To validate our findings with on-going clinical trial results, we evaluated topical tenofovir gel for safety and efficacy. We also modeled systemic application of tenofovir for efficacy.
Methods and Findings
Formulation assessment of tenofovir gel included osmolality, viscosity, in vitro release, and permeability testing. Safety was evaluated by measuring the effect on the viability of vaginal flora, PBMCs, epithelial cells, and ectocervical and colorectal explant tissues. For efficacy testing, PBMCs were cultured with tenofovir or vehicle control gels and HIV-1 representing subtypes A, B, and C. Additionally, polarized ectocervical and colorectal explant cultures were treated apically with either gel. Tenofovir was added basolaterally to simulate systemic application. All tissues were challenged with HIV-1 applied apically. Infection was assessed by measuring p24 by ELISA on collected supernatants and immunohistochemistry for ectocervical explants. Formulation testing showed the tenofovir and vehicle control gels were >10 times isosmolar. Permeability through ectocervical tissue was variable but in all cases the receptor compartment drug concentration reached levels that inhibit HIV-1 infection in vitro. The gels were non-toxic toward vaginal flora, PBMCs, or epithelial cells. A transient reduction in epithelial monolayer integrity and epithelial fracture for ectocervical and colorectal explants was noted and likely due to the hyperosmolar nature of the formulation. Tenofovir gel prevented HIV-1 infection of PBMCs regardless of HIV-1 subtype. Topical and systemic tenofovir were effective at preventing HIV-1 infection of explant cultures.
Conclusions
These studies provide a mechanism for pre-clinical prediction of safety and efficacy of formulated microbicides. Tenofovir was effective against HIV-1 infection in our algorithm. These data support the use of tenofovir for pre-exposure prophylaxis.
doi:10.1371/journal.pone.0009310
PMCID: PMC2824823  PMID: 20174579
7.  Oleate lipase activity in Gardnerella vaginalis and reconsideration of existing biotype schemes 
BMC Microbiology  2009;9:78.
Background
Gardnerella vaginalis is a facultative gram positive organism that requires subculture every 1–2 days to maintain viability. It has been linked with bacterial vaginosis (BV), a syndrome that has been associated with increased risk for preterm delivery, pelvic inflammatory disease and HIV acquisition. About 10% of the G. vaginalis isolates have been reported to produce sialidase, but there have not been any studies relating sialidase production and biotype. Sialidase activity is dramatically increased in the vaginal fluid of women with BV and bacterial sialidases have been shown to increase the infectivity of HIV in vitro. There are 8 different biotypes of G. vaginalis. Biotypes 1–4 produce lipase and were reported to be associated with BV and the association of these biotypes with BV is under dispute. Other studies have demonstrated that G. vaginalis biotype 1 can stimulate HIV-1 production. Because of the discrepancies in the literature we compared the methods used to biotype G. vaginalis and investigated the relationship of biotype and sialidase production.
Results
A new medium for maintenance of Gardnerella vaginalis which allows survival for longer than one week is described. Some isolates only grew well under anaerobic conditions. Sialidase producing isolates were observed in 5 of the 6 biotypes tested. Using 4-methylumbelliferyl-oleate to determine lipase activity, instead of egg yolk agar, resulted in erroneous biotypes and does not provide reliable results.
Conclusion
Previous studies associating G. vaginalis biotype with bacterial vaginosis were methodologically flawed, suggesting there is not an association of G. vaginalis biotypes and bacterial vaginosis. Sialidase activity was observed in 5 of the 8 biotypes.
doi:10.1186/1471-2180-9-78
PMCID: PMC2680412  PMID: 19386125

Results 1-7 (7)