<i>L. vaginalis</i> Elicits hBD2 Gene Induction from Reproductive Epithelia.

<p>Confluent monolayers of epithelial cells were inoculated with bacteria and coincubated for up to 24 hr, then analyzed by RTqPCR for hBD2 and hBD3 expression. Transcript expression was normalized to mock-inoculated cells, and is shown as the average of three or four independent experiments. Average MOI are: 6.9 for <i>L. acidophilus,</i> 7.3 for <i>L. crispatus,</i> 9.7 for <i>L. gasseri,</i> 7.3 for <i>L. jensenii,</i> 7.3 for <i>L. johnsonii,</i> 3.4 for <i>L. vaginalis,</i> 3.7 for <i>A. vaginae,</i> 3.1 for <i>G. vaginalis,</i> 3.6 for <i>M. curtisii,</i> and 3.9 for <i>P. bivia</i>. Asterisks indicate a significant (p<0.05) increase in expression over mock-treated cells for at least one timepoint.</p

<i>L. vaginalis</i> Initiates an Innate Immune Response from FRT Epithelia.

<p>Confluent monolayers of epithelial cells were inoculated with bacteria and coincubated for 24 hr. Conditioned media were collected, clarified and ELISA was used to quantify hBD2, IL-6 and IL-8 concentrations. MOI is the same as in Figure Five. Analyte concentrations are shown as fold induction compared to mock condition, and are the average of three independent experiments where one (p<0.05), two (p<0.01), or three (p<0.001) asterisks indicate a significant increase over the mock-treated condition.</p

Identification and Characterization of Bacterial Vaginosis-Associated Pathogens Using a Comprehensive Cervical-Vaginal Epithelial Coculture Assay

<div><p>Bacterial vaginosis (BV) is the most commonly treated female reproductive tract affliction, characterized by the displacement of healthy lactobacilli by an overgrowth of pathogenic bacteria. BV can contribute to pathogenic inflammation, preterm birth, and susceptibility to sexually transmitted infections. As the bacteria responsible for BV pathogenicity and their interactions with host immunity are not understood, we sought to evaluate the effects of BV-associated bacteria on reproductive epithelia. Here we have characterized the interaction between BV-associated bacteria and the female reproductive tract by measuring cytokine and defensin induction in three types of FRT epithelial cells following bacterial inoculation. Four BV-associated bacteria were evaluated alongside six lactobacilli for a comparative assessment. While responses differed between epithelial cell types, our model showed good agreement with clinical BV trends. We observed a distinct cytokine and human β-defensin 2 response to BV-associated bacteria, especially <em>Atopobium vaginae</em>, compared to most lactobacilli. One lactobacillus species, <em>Lactobacillus vaginalis</em>, induced an immune response similar to that elicited by BV-associated bacteria, stimulating significantly higher levels of cytokines and human β-defensin 2 than other lactobacilli. These data provide an important prioritization of BV-associated bacteria and support further characterization of reproductive bacteria and their interactions with host epithelia. Additionally, they demonstrate the distinct immune response potentials of epithelial cells from different locations along the female reproductive tract.</p> </div

<i>L. vaginalis</i> Induces a Greater Immune Response than Other Vaginal Lactobacilli.

<p>Confluent monolayers of reproductive epithelia were inoculated with commensal lactobacilli or BVAB, and after 24 hr conditioned media was analyzed for IL-6, IL-8 and hBD2 protein. BVAB are filled black bars, <i>L. vaginalis</i> is hatched, and all other lactobacilli are white bars. MOI are the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050106#pone-0050106-g005" target="_blank">Figure 5</a>. Protein is shown as fold expression compared to a mock-treated condition, and one, two, or three asterisks indicate values that are significantly (p<0.05, p<0.01, or p<0.001, respectively) different from the <i>L. vaginalis</i>-treated condition.</p

Epithelial Coculture Mirrors In Vivo Cytokine Response.

<p>A) Cervicovaginal lavage samples from BV-negative (n = 5) or BV-positive (n = 13) women were analyzed by multiplex cytokine bead array. Fold expression for each cytokine was calculated relative to the average value of the BV-negative samples. One (p<0.05) or two (p<0.01) asterisks indicate a significant increase in cytokine concentration for the BV-positive samples over the BV-negative samples. B-D) Cytokine induction in each epithelial line B) End1, C) Ect1, and D) VK2 in response to <i>L. johnsonii</i> (average MOI = 33) and <i>A. vaginae</i> (average MOI = 13) normalized to their paired mock-inoculated conditions. One (p<0.05), two (p<0.01), or three (p<0.001) asterisks indicate a significant increase in cytokine concentration for the <i>A. vaginae</i>-inoculated conditions over the <i>L. johnsonii</i>-inoculated conditions.</p

<i>A. vaginae</i> Induces Epithelial Expression of Soluble and Cell-Associated hBD2, a Protein That Attracts Primary Lymphocytes.

<p>A) Confluent monolayers of epithelial cells were inoculated with bacteria. Twenty-four hr post-inoculation, conditioned media were collected, clarified, and analyzed by ELISA to quantify concentrations of soluble hBD2 protein. Average MOI are: 5.8 for <i>L. johnsonii</i>, 2.5 for <i>G. vaginalis</i>, and 1.4 for <i>A. vaginae</i>. Results are averaged from 3 independent experiments, and asterisks indicate significant (p<0.001) increase over mock<i>-</i>treated condition. B) Confluent monolayers were inoculated with <i>A. vaginae</i>, or mock-inoculated. 24, 48, and 72 hr post-inoculation, cell monolayers were acid-extracted and analyzed by AU-PAGE western to quantify cell-associated hBD2 protein. A recombinant hBD2 protein standard (shown in ng per lane) was run alongside cell extracts for semi-quantitative comparison. Average MOI is 21. Shown is one example of three independent experiments. C) Unstimulated primary lymphocytes were isolated and evaluated for chemotaxis toward recombinant hBD2 protein. Chemotactic index is the ratio of migrated cells in hBD2-containing wells over vehicle-control wells, and significant increases over the matched vehicle condition are shown by three (p<0.001) asterisks.</p

Analysis of Viral Fitness and Drug Resistance.

<p>To determine if N126K is acting as a compensatory mutation in RC-101, viral fitness in culture over a 7 day period was determined for wild-type (WT) and drug-resistant mutants in the presence or absence of RC-101 and ENF. An MOI of 0.003, as determined by TCID50, was used for the initial inoculation of PM1 cells. Fitness was determined by p24 concentration measured in cell supernatants. Error bars represent SEM. Black lines indicate comparisons between conditions (N = 3; * = <i>p</i><0.05).</p

Synthesis and Characterization of BaL <i>env</i> Molecular Clones.

<p>To study the effect of gp41 mutations identified in the BaL envelope, the dominant <i>env</i> genotypes from untreated and RC-101-passaged virus were used to generate the pNBaL molecular clone containing the complete BaL envelope within pNL43 (<b>A</b>). Molecular clones possessing the envelope proteins cloned from untreated BaL (WT) or from RC-101-passaged BaL (RCres) were treated with either RC-101 (<b>B</b>) or ENF (<b>C</b>). Error bars represent SEM. Differences in percent inhibition were determined between WT and RCres at each drug concentration (N = 3; ** = <i>p</i><0.01, *** = <i>p</i><0.001).</p

Correlation of HIV neutralizing activity and levels of cationic polypeptides in the study groups.

<p>CVS were collected from HIV seropositive women (HIV pos) as well as from HIV seronegative women living with either an HIV infected man (HESN) or an HIV uninfected man (Low-risk). HIV neutralization capacity (NT) of IgA-depleted CVS: The study samples were sorted by whether neutralization occurred (NT) or not (no NT). Levels of (a) HNP1–3 (HIV-pos no NT <i>n = 26</i>, HIV-pos NT <i>n = 15</i>, HESN no NT <i>n = 124</i>, HESN NT <i>n = 27</i>, low-risk no NT <i>n = 43</i>, low-risk NT <i>n = 14</i>), (b) LL-37 (HIV-pos no NT <i>n = 28</i>, HIV-pos NT <i>n = 15</i>, HESN no NT <i>n = 123</i>, HESN NT <i>n = 27</i>, low-risk no NT <i>n = 41</i>, low-risk NT <i>n = 15</i>) and (c) SLPI (HIV-pos no-NT <i>n = 29</i>, HIV-pos NT <i>n = 17</i>, HESN no NT <i>n = 73</i>, HESN NT <i>n = 12</i>, low-risk no NT <i>n = 25</i>, Ctrl NT <i>n = 11</i>). Assay detection level was 0.16 ng/ml for HNP1–3, 0.14 for LL-37 and 0.06 for SLPI, results below cut-off were assigned the value y = 0.1. Solid line indicates median concentration, whiskers indicate 5–95 percentile. <i>*p<0.05</i>.</p

Entry Kinetics of RC-101-Resistant Virus.

<p>Viral entry was observed over one hour to determine differences in entry kinetics between wild type (WT) and RC-101-resistant virus. Equal concentrations of infectious virus were used to infect reporter cells and infection was halted at specific time-points. Entry kinetics is shown as percent of total entry and graphed using cubic splines. Linear regression curves were fit to data and slopes were compared between the 15 and 30-minute time intervals. Error bars represent SEM. Both wild type (WT) and Q66R+N126K had significantly greater slopes than the Q66R mutant (N = 4; <i>p</i><0.05).</p