title: A quantitative multiplex nuclease protection assay reveals immunotoxicity gene expression profiles in the rabbit model for vaginal drug safety evaluation.
abstract: 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.
authors.affiliation: Laboratory of Genital Tract Biology, Department of Obstetrics, Gynecology and Reproductive Biology, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA. Electronic address: rfichorova@rics.bwh.harvard.edu.
authors.forename: Raina N
authors.initials: RN
authors.lastname: Fichorova
authors.affiliation: Laboratory of Genital Tract Biology, Department of Obstetrics, Gynecology and Reproductive Biology, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA.
authors.forename: Kevin
authors.initials: K
authors.lastname: Mendonca
authors.affiliation: Laboratory of Genital Tract Biology, Department of Obstetrics, Gynecology and Reproductive Biology, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA.
authors.forename: Hidemi S
authors.initials: HS
authors.lastname: Yamamoto
authors.affiliation: Laboratory of Genital Tract Biology, Department of Obstetrics, Gynecology and Reproductive Biology, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA.
authors.forename: Ryan
authors.initials: R
authors.lastname: Murray
authors.affiliation: CONRAD, Department of Obstetrics and Gynecology, Eastern Virginia Medical School, Norfolk, VA, USA.
authors.forename: Neelima
authors.initials: N
authors.lastname: Chandra
authors.affiliation: CONRAD, Department of Obstetrics and Gynecology, Eastern Virginia Medical School, Norfolk, VA, USA.
authors.forename: Gustavo F
authors.initials: GF
authors.lastname: Doncel
mesh_terms: D000225:Adenine / Q000008:administration & dosage / Q000009:adverse effects / Q000031:analogs & derivatives; D000818:Animals; D019380:Anti-HIV Agents / Q000008:administration & dosage / Q000009:adverse effects; D004195:Disease Models, Animal; D004305:Dose-Response Relationship, Drug; D004338:Drug Combinations; D004353:Drug Evaluation, Preclinical; D005069:Evaluation Studies as Topic; D005260:Female; D054884:Host-Pathogen Interactions; D007150:Immunohistochemistry; D007249:Inflammation / Q000473:pathology; D053583:Interleukin-1beta / Q000235:genetics / Q000378:metabolism; D016209:Interleukin-8 / Q000235:genetics / Q000378:metabolism; D009092:Mucous Membrane / Q000187:drug effects / Q000378:metabolism; D017137:Nonoxynol / Q000008:administration & dosage / Q000009:adverse effects; D023282:Nuclease Protection Assays / Q000379:methods*; D009842:Oligopeptides / Q000235:genetics / Q000378:metabolism; D063065:Organophosphonates / Q000008:administration & dosage / Q000009:adverse effects; D011817:Rabbits; D000068698:Tenofovir; D051197:Toll-Like Receptor 4 / Q000235:genetics / Q000378:metabolism; D059467:Transcriptome; D014621:Vagina / Q000187:drug effects* / Q000473:pathology
chemical_list: D019380:Anti-HIV Agents; D004338:Drug Combinations; D053583:Interleukin-1beta; D016209:Interleukin-8; D009842:Oligopeptides; D063065:Organophosphonates; D051197:Toll-Like Receptor 4; C543187:valyl-seryl-tryptophyl-phenylalanyl-phenylalanyl-glutamic acid; D017137:Nonoxynol; D000068698:Tenofovir; D000225:Adenine
keywords: Anti-HIV microbicides; BPI; EMP-1; Inflammation; RVI; Toll-like receptors