Human Ectocervical Tissue Research Articles

Pre-clinical evaluation of antiproteases as potential candidates for HIV-1 pre-exposure prophylaxis.

Previous studies on highly HIV-1-exposed, yet persistently seronegative women from the Punwami Sex Worker cohort in Kenya, have shed light on putative protective mechanisms, suggesting that mucosal immunological factors, such as antiproteases, could be mediating resistance to HIV-1 transmission in the female reproductive tract. Nine protease inhibitors were selected for this study: serpin B4, serpin A1, serpin A3, serpin C1, cystatin A, cystatin B, serpin B13, serpin B1 and α-2-macroglobulin-like-protein 1. We assessed in a pilot study, the activity of these antiproteases with cellular assays and an ex vivo HIV-1 challenge model of human ecto-cervical tissue explants. Preliminary findings with both models, cellular and tissue explants, established an order of inhibitory potency for the mucosal proteins as candidates for pre-exposure prophylaxis when mimicking pre-coital use. Combination of all antiproteases considered in this study was more active than any of the individual mucosal proteins. Furthermore, the migration of cells out of ecto-cervical explants was blocked indicating potential prevention of viral dissemination following amplification of the founder population. These findings constitute the base for further development of these mucosal protease inhibitors for prevention strategies.

Preformulation Characterization of Griffithsin, a Biopharmaceutical Candidate for HIV Prevention.

Griffithsin (GRFT) has shown potent anti-HIV activity, and it is being developed as a drug candidate for HIV prevention. Successful implementation requires thorough understanding of its preformulation characterization. In this work, preformulation assessments were conducted to characterize GRFT and identify its degradation pathways under selected conditions of temperature, light, pH, shear, ionic strength, and oxidation. Compatibility with vaginal fluid simulant, vaginal enzymes, Lactobacillus spp., and human cervicovaginal secretions was assessed. The purity, melting temperature, and HIV gp120-binding affinity of GRFT stored at 4°C and 25°C in phosphate-buffered saline (PBS) were assessed for 2 years. Chemical modifications were evaluated by intact mass analysis and peptide sequencing. Excised human ectocervical tissue permeability and localization of GRFT were evaluated. Our results demonstrated GRFT to be safe and stable under all the preformulation assessment conditions studied except oxidative stress. When GRFT was exposed to hydrogen peroxide or human cervicovaginal secretion, methionine 78 in the protein sequence underwent oxidation. GRFT did not permeate through human cervical tissue but adhered to the superficial epithelial tissue. The 2-year stability study revealed no significant change in GRFT’s aggregation, degradation, melting temperature, or gp120-binding affinity despite a slow increase in oxidation over time. These studies elucidated desirable safety and bioactivity profile for GRFT, showing promise as a potential drug candidate for HIV prevention. However, susceptibility to oxidative degradation was identified. Effective protection of GRFT from oxidation is required for further development.

Development of human ectocervical tissue models with physiologic endocrine and paracrine signaling†.

There is a shortage of research models that adequately represent the unique mucosal environment of human ectocervix, limiting development of new therapies for treating infertility, infection, or cancer. We developed three microphysiologic human ectocervix models to study hormone action during homeostasis. First, we reconstructed ectocervix using decellularized extracellular matrix scaffolds, which supported cell integration and could be clinically useful. Secondly, we generated organotypic systems consisting of ectocervical explants co-cultured with murine ovaries or cycling exogenous hormones, which mimicked human menstrual cycles. Finally, we engineered ectocervix tissue consisting of tissue-specific stromal-equivalents and fully-differentiated epithelium that mimicked in vivo physiology, including squamous maturation, hormone response, and mucin production, and remained viable for 28days in vitro. The localization of differentiation-dependent mucins in native and engineered tissue was identified for the first time, which will allow increased efficiency in mucin targeting for drug delivery. In summary, we developed and characterized three microphysiologic human ectocervical tissue models that will be useful for a variety of research applications, including preventative and therapeutic treatments, drug and toxicology studies, and fundamental research on hormone action in a historically understudied tissue that is critical for women's health.

Distinct follicular and luteal transcriptional profiles in engineered human ectocervical tissue dependent on menstrual cycle phase.

To investigate genomic pathways that may influence physiology and infectivity during the menstrual cycle, RNA sequence analysis was performed on patient-matched engineered ectocervical tissue after follicular and luteal phase (LP) hormone treatments. We developed distinct cellular, molecular, and biological profiles in ectocervical epithelium dependent on the menstrual cycle phase. Follicular phase hormones were associated with proliferation, transcription, and cell adhesion, while LP samples expressed genes involved in immune cell recruitment, inflammation, and protein modifications. Additionally, our analysis revealed mucins not previously reported in ectocervical tissue, which could play an important role in fertility and disease prevention. This study provides insight into the phenomenon of increased LP vulnerability to infection and identifies potential targets for future research.

Epidermal Growth Factor Receptor Signaling Enhances the Proinflammatory Effects of Staphylococcus aureus Gamma-Toxin on the Mucosa.

Staphylococcus aureus (S. aureus) produces many different exotoxins including the gamma-toxins, HlgAB and HlgCB. Gamma-toxins form pores in both leukocyte and erythrocyte membranes, resulting in cell lysis. The genes encoding gamma-toxins are present in most strains of S. aureus, and are commonly expressed in clinical isolates recovered from menstrual Toxic Shock Syndrome (mTSS) patients. This study set out to investigate the cytotoxic and proinflammatory effects of gamma-toxins on vaginal epithelial surfaces. We found that both HlgAB and HlgCB were cytotoxic to cultured human vaginal epithelial cells (HVECs) and induced cytokine production at sub-cytotoxic doses. Cytokine production induced by gamma-toxin treatment of HVECs was found to involve epidermal growth factor receptor (EGFR) signaling and mediated by shedding of EGFR ligands from the cell surface. The gamma-toxin subunits displayed differential binding to HVECs (HlgA 93%, HlgB 97% and HlgC 28%) with both components (HlgAB or HlgCB) required for maximum detectable binding and significant stimulation of cytokine production. In studies using full thickness ex vivo porcine vaginal mucosa, HlgAB or HlgCB stimulated a dose-dependent cytokine response, which was reduced significantly by inhibition of EGFR signaling. The effects of gamma-toxins on porcine vaginal tissue and cultured HVECs were validated using ex vivo human ectocervical tissue. Collectively, these studies have identified the EGFR-signaling pathway as a key component in gamma-toxin-induced proinflammatory changes at epithelial surfaces and highlight a potential therapeutic target to diminish toxigenic effects of S. aureus infections.

The Effect of Commonly Used Excipients on the Epithelial Integrity of Human Cervicovaginal Tissue.

Pharmaceutical excipients are widely used in vaginal drug products. The epithelial integrity of the cervicovaginal tissue is important for HIV-1 prevention. However, the effects of excipients on cervicovaginal epithelium remain unknown. This study aims at assessing the effects of vaginal product excipients on the integrity of human cervicovaginal epithelium and on a lead HIV prevention antiretroviral drug, tenofovir (TFV). In the current study, nine excipients commonly used in vaginal formulations were incubated for 6 h with excised human ectocervical tissue. The effects of the excipients were examined by measuring the transepithelial electrical resistance (TEER), epithelial morphology, paracellular/transcellular permeability, and cell viability. The efficacy of TFV for preventing HIV-1 infection in the ex vivo cultured ectocervix was also tested. We found that disodium ethyl-enediaminetetraacetate (EDTA), sorbic acid, and benzoic acid had no effect on the tissue TEER. Butylated hydroxyanisole, glycerin, propylene glycol, methylparaben, and propylparaben slightly to moderately decreased tissue TEER, whereas citric acid significantly decreased the TEER in a time-dependent manner. Tissue morphology observed post-exposure strongly correlated with TEER data; however, a less strong correlation was observed between paracellular permeability and TEER data after exposure to different excipients. In addition, treatment with EDTA, methylparaben, and propylene glycol at tested levels had no effect on the efficacy of TFV in preventing tissue HIV-1 infection. In conclusion, the combined measurements of TEER, morphology, permeability, and viability using human cervicovaginal tissue represent a clinically relevant platform for safety evaluation of excipients and formulated products for HIV-1 prevention.

Increased Dapivirine Tissue Accumulation through Vaginal Film Codelivery of Dapivirine and Tenofovir

The HIV-1 replication inhibitor dapivirine (DPV) is one of the most promising drug candidates being used in topical microbicide products for prevention of HIV-1 sexual transmission. To be able to block HIV-1 replication, DPV must have access to the viral reverse transcriptase enzyme. The window for DPV to access the enzyme happens during the HIV-1 cellular infection cycle. Thus, in order for DPV to exert its anti-HIV activity, it must be present in the mucosal tissue or cells where HIV-1 infection occurs. A dosage form containing DPV must be able to deliver the drug to the tissue site of action. Polymeric films are solid dosage forms that dissolve and release their payload upon contact with fluids. Films have been used as vaginal delivery systems of topical microbicide drug candidates including DPV. For use in topical microbicide products containing DPV, polymeric films must prove their ability to deliver DPV to the target tissue site of action. Ex vivo exposure studies of human ectocervical tissue to DPV film revealed that DPV was released from the film and did diffuse into the tissue in a concentration dependent manner indicating a process of passive diffusion. Analysis of drug distribution in the tissue revealed that DPV accumulated mostly at the basal layer of the epithelium infiltrating the upper part of the stroma. Furthermore, as a combination microbicide product, codelivery of DPV and TFV from a polymeric film resulted in a significant increase in DPV tissue concentration [14.21 (single entity film) and 31.03 μg/g (combination film)], whereas no impact on TFV tissue concentration was found. In vitro release experiments showed that this observation was due to a more rapid DPV release from the combination film as compared to the single entity film. In conclusion, the findings of this study confirm the ability of polymeric films to deliver DPV and TFV to human ectocervical tissue and show that codelivery of the two agents has a significant impact on DPV tissue accumulation. These findings support the use of polymeric films for topical microbicide products containing DPV and/or TFV.

Herpes simplex virus type-2 stimulates HIV-1 replication in cervical tissues: implications for HIV-1 transmission and efficacy of anti-HIV-1 microbicides.

Herpes Simplex virus Type-2 (HSV-2) increases the risk of HIV-1 acquisition, yet the mechanism for this viral pathogen to regulate the susceptibility of the cervicovaginal mucosa to HIV-1 is virtually unknown. Using ex vivo human ectocervical tissue models, we report greater levels of HIV-1 reverse transcription, DNA integration, RNA expression, and virions release in HIV-1/HSV-2 co-infected tissues compared with HIV-1 only infected tissues (P<0.05). Enhanced HIV-1 replication was associated with increased CD4, CCR5, and CD38 transcription (P<0.05) and increased number of CD4+/CCR5+/CD38+ T cells in HIV-1/HSV-2 co-infected tissues compared with tissues infected with HIV-1 alone. Tenofovir (TFV) 1% gel, the leading microbicide candidate, demonstrated only partial protection against HIV-1, when applied vaginally before and after sexual intercourse. It is possible that mucosal inflammation, in particular that induced by HSV-2 infection, may have decreased TFV efficacy. HSV-2 upregulated the number of HIV-1-infected cells and elevated the concentration of TFV needed to decrease HIV-1 infection. Similarly, only high concentrations of TFV inhibited HSV-2 replication in HIV-1/HSV-2-infected tissues. Thus, HSV-2 co-infection and mucosal immune cell activation should be taken into consideration when designing preventative strategies for sexual transmission of HIV-1.

Development of a liposome microbicide formulation for vaginal delivery of octylglycerol for HIV prevention

The feasibility of using a liposome drug delivery system to formulate octylglycerol (OG) as a vaginal microbicide product was explored. A liposome formulation was developed containing 1% OG and phosphatidyl choline in a ratio that demonstrated in vitro activity against Neisseria gonorrhoeae, HSV-1, HSV-2 and HIV-1 while sparing the innate vaginal flora, Lactobacillus. Two conventional gel formulations were prepared for comparison. The OG liposome formulation with the appropriate OG/lipid ratio and dosing level had greater efficacy than either conventional gel formulation and maintained this efficacy for at least 2 months. No toxicity was observed for the liposome formulation in ex vivo testing in a human ectocervical tissue model or in vivo testing in the macaque safety model. Furthermore, minimal toxicity was observed to lactobacilli in vitro or in vivo safety testing. The OG liposome formulation offers a promising microbicide product with efficacy against HSV, HIV and N. gonorrhoeae.

Formulation Development of Retrocyclin 1 Analog RC-101 as an Anti-HIV Vaginal Microbicide Product

RC-101 is a synthetic microbicide analog of retrocyclin, which has shown in vitro activity against X4 and R5 HIV-1. In an effort to develop a safe and effective RC-101 vaginal microbicide product, we assessed safety in ex vivo macaque and human models and efficacy using in vitro and ex vivo models. A polyvinyl-alcohol vaginal film containing RC-101 (100 μg/film) was developed. Formulation assessment was conducted by evaluating disintegration, drug content, mechanical properties, and stability. Efficacy was evaluated by in vitro peripheral blood mononuclear cells (PBMC) assay and ex vivo human ectocervical tissue explant model. Ex vivo safety studies were conducted by exposing RC-101 to an excised monkey reproductive tract and excised human ectocervical tissue. RC-101 100 μg films were shown to be safe to human and monkey tissue and effective against HIV-1 in vitro and ex vivo in human ectocervical tissue. The 90% inhibitory concentration (IC90) for RC-101 films at 2,000 μg (IC90=57.5 μM) using an ex vivo model was 10-fold higher than the IC90 observed using an in vitro model (IC90=5.0 μM). RC-101 films were stable for 1 month at 25°C, with in vitro bioactivity maintained for up to 6 months. RC-101 was developed in a quick-dissolve film formulation that was shown to be safe in an ex vivo model and effective in in vitro and ex vivo models. RC-101 film formulations were shown to maintain bioactivity for a period of 6 months. Findings from the present study contribute to the development of a safe and effective topical microbicide product.

The Glycogen Content of Human Ectocervical Tissue of Females Receiving Steroid Contraceptive Therapy

Cervical biopsies were obtained at random from women of reproductive age attending either a family planning or gynecologic clinic to determine the effect of oral contraceptives on glycogen content of ectocervical tissue. Glycogen was precipitated with 95% ethyl alcohol reacted with an acidic anthrone solution and the resulting color determined at a wave length of 620 nm. Selected cervical biopsies were obtained for histochemical localization of periodic acid-Schiff positive material. Observations were divided into 3 groups: Group 1 encompassed Days 1-10 of the menstrual cycle; Group 2 Days 11-16; Group 3 Days 17-32. Glycogen content of the ectocervical tissue remains fairly constant and is not under the influence of either endogenous or exogenous steroids. The glycogen content was shown to be limited to the cervical squamous layer.