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In vitro effects of intravaginal insertion products (IVIPs) on biomarkers of inflammation and immune cellular activation in the era of HIV.

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The use of vaginal products is associated with increased HIV acquisition risk, but the mechanism is not fully understood. Vaginal practices entail the use of a wide variety of products which can alter the vaginal environment to achieve a desired state. Strong motivations for vaginal practices include women’s desire to maintain stable relationships, manage health, hygiene and sexuality. This adjustment of the vaginal microenvironment may increase HIV acquisition risk. High levels of inflammation and immune activation in the female genital tract are associated with a threefold increase in HIV acquisition risk. We hypothesized that intravaginal insertion products (IVIPs) may be linked to high levels of inflammation and immune activation in the female genital tract which may subsequently lead to an increased risk of HIV acquisition Objective The pH of the IVIPs (Kuber, Snuff, Alum, Savlon and Rose water) was measured and the cytotoxicity of the IVIPs was evaluated by determining their effect on cell viability at different dilutions (Neat/stock, 1/5, 1/10, 1/100 and 1/1000). The mechanisms by which potassium aluminium sulfate (“Alum”) and smokeless tobacco (“snuff”) impact cellular activation and inflammation were investigated using peripheral blood mononuclear cells (PBMCs) in vitro. Methods The pH of alum, snuff, kuber, savlon and rose water was measured at different dilutions (Neat/Stock, 1/5, 1/10, 1/100 and 1/1000). The effect of the IVIPs on cell viability was determined by exposing PBMCs to the different dilutions of IVIPs mentioned above. PBMCs from 26 HIV-negative healthy donors were unstimulated (negative control) or stimulated for 3 hours at 37°C with 1/1000 dilutions of 450 mg/ml of alum or snuff and 10μg/ml of PHA (positive control). The PBMC supernatants were collected following PBMC stimulation, and eleven cytokines were measured from 12 of the 26 PBMC supernatants. Pro-inflammatory (IL-1β, TNF-α, IL-6), chemokines (IL-8, IP-10, MIP-1α, MIP-1β, MCP-1), hematopoietic (IL-7, GM-CSF) and regulatory (IL-10) cytokines were measured using Bio-Plex multiplex assay. The activation status of T lymphocytes was determined by evaluating CD38+, HLA-DR+, dual expression of CD38+HLA-DR+ and chemokine receptor CCR5+ expression from CD4+ and CD8+ T cells using flow cytometry assay. Results Alum stock solution was acidic with a pH of 2.62 whereas the snuff stock solution was basic with a pH of 9.11. Alum and savlon were found to have high cytotoxicity. Snuff exposed cell resulted in a significantly increased CCR5 chemokine expression in CD4+ T cells when compared to the unexposed cells (p=0.0483) and also when compared to alum exposed cells (p=0.0446). However, snuff exposure did not significantly increase any of the activation markers in CD8+ T cells and it did not change the inflammatory cytokine profile. In CD8+ T lymphocytes the CD38+ biomarker was significantly more expressed in unexposed cells compared to the alum exposed cells (p=0.0185). Alum exposed cells significantly increased expression of HLADR+ (P=0.0348) and also the dual expression of CD38+HLA-DR+in CD8+ T cells (p=0.0208) when compared to the unexposed cells and was also associated with significantly high levels of cytokines IP-10 (p=0.039), MCP-1 (P=0.0024), MIP-1α (p=0.0005), IL-6 (P=0.0005), TNF-α (P=0.0020), IL-7 (P=0.0005) and GM-SCF (P=0.0005) when compared to the unexposed cells. Conclusion This study is the first of its kind to identify a possible link between intravaginal insertion products and inflammation. Alum, in particular, was more inflammatory compared to snuff. These findings may help explain the previous observations of an increased HIV acquisition risk in IVIP users. Future research can extend the current pilot study on an invitro human vaginal epithelial cell model. Knowledge from this work and future studies is crucial in developing new female-initiated interventions for preventing HIV acquisition.


Masters Degree. University of KwaZulu-Natal, Durban.