In additional experiments to determine possible postinfection mechanisms of inhibition by Trappin-2/Elafin, TZM cells were infected with HIV-1 IIIB and BaL
at an MOI of 1 and were washed out at 6 and 24 hr postinfection followed by the addition of 10 ng/ml of recombinant Trappin-2/Elafin ITF2357 ic50 (rTrappin-2/Elafin). Assays were developed at 48 hr by addition of the Beta-Glo substrate and measurement of relative light units using a luminometer. Viability of TZM cells upon treatment with Trappin-2/Elafin and CVL was quantified using the CellTiter 96® AQueous One Solution Cell Proliferation assay (Promega) according to the manufacturer’s instructions. Briefly, reagent was added directly to cell cultures B-Raf inhibition and incubated for 1 hr at 37°, after which the absorbance of each well was read at 490 nm in a plate reader. CVL samples from 32 HIV-positive women (12 Black, nine Hispanic and 12 White) were provided by Dr S. Cu-Uvin (Brown University, Providence, RI). Fifteen CVL samples from HIV-negative women (five Black, five Hispanic and five White) were obtained from the Rhode Island HIV Epidemiology Research Study (HERS). All sample collections were carried out in accordance with human experimentation guidelines of Miriam Hospital (Brown University, Providence, RI). CVL from women were catalogued by race based on self-identification.
The HIV-positive and HIV-negative women were in
the same age range (18–50 years). The HIV-positive women were relatively healthy with average CD4 counts of 712 cells/mm3 blood, an average plasma viral load of 12 666 copies/ml and Thiamet G were not on any antiretroviral therapy. Only six out of 32 women showed a detectable genital tract viral load. CVL was collected by washing the cervical-vaginal area with 10 ml of sterile saline (pH 7·0) and collecting the fluid, which was then centrifuged at 10 000 g for 5 min and separated from the cellular fraction. The supernatants were aliquoted and stored frozen at −80° until use. For the HIV-negative samples used in this study, CVL were collected and frozen immediately at −80°. Before assaying the supernatants, samples were thawed, centrifuged at 10 000 g for 5 min and separated from the cellular fraction. A two-tailed paired t-test or a one-way analysis of variance (anova) with Bonferonni’s post-test was performed using GraphPad InStat version 3.0a (GraphPad Software, San Diego, CA). A P-value of < 0·05 was taken as indicative of statistical significance. Epithelial cells were isolated from uterus (UT), Fallopian tube (FT), endocervix (Cx) and ectocervix (Ecx) FRT tissues, grown to confluence and, in the case of epithelial cell (EC) from the upper FRT, high TER (> 500 ohms/well). CM was collected at 24 hr and cells were harvested to isolate RNA.