L-Selectin and P-Selectin Are Novel Biomarkers of Cervicovaginal Inflammation for Preclinical Mucosal Safety Assessment of Anti-HIV-1 Microbicide

Abstract

A major obstacle thwarting preclinical development of microbicides is the lack of a validated biomarker of cervicovaginal inflammation. Therefore, the present study aims to identify novel noninvasive soluble markers in a murine model for assessment of microbicide mucosal safety. By performing cytokine antibody array analysis, we identified two adhesion molecules, L-selectin and P-selectin, which significantly increased when mucosal inflammation was triggered by nonoxynol-9 (N9), an anti-HIV-1 microbicide candidate that failed clinical trials, in a refined murine model of agent-induced cervicovaginal inflammation. We found that patterns of detection of L-selectin and P-selectin were obviously different from those of the two previously defined biomarkers of cervicovaginal inflammation, monocyte chemotactic protein 1 (MCP-1) and interleukin 6 (IL-6). The levels of these two soluble selectins correlated better than those of MCP-1 and IL-6 with the duration and severity of mucosal inflammation triggered by N9 and two approved proinflammatory compounds, benzalkonium chloride (BZK) and sodium dodecyl sulfate (SDS), but not by two nonproinflammatory compounds, carboxymethyl celluose (CMC; microbicide excipients) and tenofovir (TFV; microbicide candidate). These data indicated that L-selectin and P-selectin can serve as additional novel cervicovaginal inflammation biomarkers for preclinical mucosal safety evaluation of candidate microbicides for the prevention of infection with HIV and other sexually transmitted pathogens.

Antigen replacement of domains D2 and D3 in flagellin promotes mucosal IgA production and attenuates flagellin-induced inflammatory response after intranasal immunization

“…RayBio mouse cytokine antibody array III (Ray-Biotech, Inc., Norcross, GA) was used for cytokine antibody assay as described previously. 51 Signal intensities were quantified directly with a chemiluminescence imaging system (FluorChem HD2; Alpha Innotech) and analyzed with its with 10% FBS (GIBCO) and 100 U/ml of penicillin/streptomycin at 37°C in 5% CO 2 , and used as previously described. 30 For the cytokine release assays, the cells were seeded at a density of 200,000 per well into 24-well polystyrene plates (Costar) and used 5 d later.…”

“…c57BL/6 mice were treated intranasally with 250 μg of p24, KFD-p24 3D or KF-p24 in 25 μl pBs. (A) Template showing the location of cytokine antibodies spotted in duplicate onto the RayBio mouse cytokine antibody array III 51. (B) Representative photographs of cytokine arrays using pooled serum samples.…”

Frontline Science: Nasal epithelial GM-CSF contributes to TLR5-mediated modulation of airway dendritic cells and subsequent IgA response

“…RayBio Mouse Cytokine Antibody Array C1 (RayBiotech Norcross, GA, USA) was used for the cytokine antibody assay, as described previously [31]. Signal intensities were quantified and analyzed with a chemiluminescence imaging system (FluorChem HD2; Alpha Innotech, Santa Clara, CA, USA).…”

Flagellins of <b><i>Salmonella</i></b> Typhi and Nonpathogenic <b><i>Escherichia coli</i></b> Are Differentially Recognized through the NLRC4 Pathway in Macrophages

“…Proteins were precipitated with 10% trichloroacetic acid, washed with ethanol and subjected to SDS gel electrophoresis and Western blotting analysis with anti-caspase-1 p10 (Santa Cruz Biotechnology, Santa Cruz, Calif., USA), anti-mouse NLRC4 (Millipore, Billerica, Mass., USA), anti-mouse IL-1β (R&D, Minneapolis, Minn., USA), anti-β actin (Sigma, St. Louis, Mo., USA), or rabbit polyclone antibody to both KF and SF. Signal intensities were quantified and analyzed with a chemiluminescence imaging system software (FluorChem HD2; Alpha Innotec) as described previously [25,26]. To determine the relative amount of protein/β-actin in each sample, bands were digitized into pixel densities and were exported into an Excel program for analysis.…”