Diffusion of Immunoglobulin G in Shed Vaginal Epithelial Cells and in Cell-Free Regions of Human Cervicovaginal Mucus

Human cervicovaginal mucus (CVM) is a viscoelastic gel containing a complex mixture of mucins, shed epithelial cells, microbes and macromolecules, such as antibodies, that together serve as the first line of defense against invading pathogens. Here, to investigate the affinity between IgG and different mucus constituents, we used Fluorescence Recovery After Photobleaching (FRAP) to measure the diffusion of IgG in fresh, minimally modified CVM. We found that CVM exhibits substantial spatial variations that necessitate careful selection of the regions in which to perform FRAP. In portions of CVM devoid of cells, FRAP measurements using different IgG antibodies and labeling methods consistently demonstrate that both exogenous and endogenous IgG undergo rapid diffusion, almost as fast as in saline, in good agreement with the rapid diffusion of IgG in mid-cycle endocervical mucus that is largely devoid of cells. This rapid diffusion indicates the interactions between secreted mucins and IgG must be very weak and transient. IgG also accumulated in cellular debris and shed epithelial cells that had become permeable to IgG, which may allow shed epithelial cells to serve as reservoirs of secreted IgG. Interestingly, in contrast to cell-free regions of CVM, the diffusion of cell-associated IgG was markedly slowed, suggesting greater affinity between IgG and cellular constituents. Our findings contribute to an improved understanding of the role of IgG in mucosal protection against infectious diseases, and may also provide a framework for using FRAP to study molecular interactions in mucus and other complex biological environments

Vaginal Glycogen, Not Estradiol, Is Associated With Vaginal Bacterial Community Composition in Black Adolescent Women

Purpose The purpose of this study was to characterize the composition of vaginal bacterial communities in a cohort of Black adolescent women and to determine how the species composition of these communities correlate with levels of estradiol, glycogen, and stress. Methods Twenty-one Black adolescent women were sampled longitudinally. The composition of their vaginal communities was determined by analyzing the sequences of the Vl-V3 region of l6S rRNA genes and they were grouped based on patterns in species abundances. The relationships between estradiol, glycogen, psychosocial stress, and the composition of these communities were assessed. Results Vaginal communities could be distinguished and classified into three groups that differed in the abundances of Lactobacillus. Eighty-one percent of study participants had communities dominated by species of Lactobacillus. Glycogen levels were higher in communities dominated by one or multiple species of Lactobacillus as compared to those having low proportions of Lactobacillus. Estradiol and psychosocial stress measurements did not differ among the three groups, while estradiol and glycogen exhibited a weak positive relationship that was not statistically significant. Conclusions The findings of this pilot study suggest that glycogen levels are associated with vaginal community composition in young Black women; however, estradiol and psychosocial stress are not. Additionally, the results suggest there is no simple relationship between levels of estradiol and the production of vaginal glycogen

Mechanisms Driving Bacterial Community Dynamics in the Human Vagina

Five community state types have been identified in the vaginal microbiota of reproductive age women. Of these, four are dominated by one Lactobacillus species, namely L. crispatus, L. gasseri, L. iners, and L. jensenii, and these communities encompass 70% of women. The composition of vaginal communities can change over time. With this, we often see changes in the relative abundances of lactobacilli, for example, a community once dominated by L. iners may transition to being dominated by L. crispatus. We posit that changes in the relative abundances of vaginal lactobacilli are driven by competition for resources. Resource competition will be measured in co-culture of the aforementioned Lactobacillus species during exponential phase growth on media containing one carbon source. We will evaluate the outcome of competition in co-culture and compare growth patterns to those observed in vaginal communities. Additionally, we will evaluate growth inhibition between strains on agar and spent media. Preliminary data suggests that the species do not inhibit one another, which supports our hypothesis of resource competition being the main driver of changes in community composition. This research will provide a better understanding of the factors that drive changes in community composition towards states that promote vaginal health

FRAP analysis of Zenon in pH-neutralized or native, acidic human CVM.

<p>(A,B) Normalized fluorescence intensities over time for photobleached ROIs of (A) Zenon in pH-neutralized human CVM (“Zenon pH 7”) and (B) Zenon in native, acidic human CVM (“Zenon pH 4”). ROIs were selected within cellular regions of the samples. Thin grey lines represent individual measurements of distinct ROIs, while thick black lines represent the average. Dashed lines represent a normalized fluorescence intensity of 1, i.e. the starting intensity prior to photobleaching. t = 0 s is defined as the start of fluorescence recovery. (C) Ratio of diffusivity in CVM (D_cvm) to diffusivity in PBS (D_pbs). (D) Unrecovered fraction of fluorescence within the time scale of measurement normalized to the initial bleached fraction. Data represent 7–9 repeated measurements per condition. * indicates a statistically significant difference (p < 0.05).</p

FRAP analysis of Zenon (“Z”), IVIG pre-mixed with Zenon (“Z/I”), and DM-IgG pre-mixed with Zenon (“Z/D”) in pH-neutralized human CVM.

<p>(A) Ratio of diffusivity in CVM (D_cvm) to diffusivity in PBS (D_pbs). (B) Unrecovered fraction of fluorescence within the time scale of measurement normalized to the initial bleached fraction. ROIs are distinguished based on whether they are in cell-free regions of the sample (“Cell-Free”) or within cells (“Cell”). Data represent 7–9 repeated measurements per condition. * indicates a statistically significant difference (p < 0.05) compared to the same molecule in Cell-Free ROIs; ** indicates a statistically significant difference (p < 0.05) compared to Zenon in ROIs of the same type.</p

FRAP analysis of human intravenous immunoglobulin (“IVIG”) and donor-matched purified IgG (“DM-IgG” or “DM”) in pH-neutralized human CVM.

<p>Normalized fluorescence intensities over time for photobleached ROIs of FITC-labeled (A,B) IVIG and (C,D) DM-IgG in human CVM. ROIs are distinguished based on whether they are (A,C) in cell-free regions of the sample (“Cell-Free ROI”) or (B,D) within cells (“Cell ROI”). Thin grey lines represent individual measurements of distinct ROIs, while thick black lines represent the average. Dashed lines represent a normalized fluorescence intensity of 1, i.e. the starting intensity prior to photobleaching. t = 0 s is defined as the start of fluorescence recovery. (E) Ratio of diffusivity in CVM (D_cvm) to diffusivity in PBS (D_pbs). (F) Unrecovered fraction of fluorescence within the time scale of measurement normalized to the initial bleached fraction. Data represent 7–9 repeated measurements per condition. * indicates a statistically significant difference (p < 0.05) compared to the same molecule in Cell-Free ROIs.</p

Representative confocal image of FITC-labeled human intravenous immunoglobulin (IVIG) in pH-neutralized human CVM.

<p>(A) Blue, (B) green, (C) DIC and (D) composite channel images, with sample “Cell-Free” (“CF”) and “Cell” (“C”) ROIs measuring approximately 10 μm x 10 μm indicated. An example of an intact epithelial cell (“IEC”) without absorbed antibody is also shown. Arrows indicate a subset of lactobacilli. Cells are identified from their morphology and blue auto-fluorescence. Intact epithelial cells do not absorb antibody and thus appear dark in the green channel. “Cell” ROIs are selected within cells that have absorbed antibody and thus appear green at comparable or greater intensity than the surrounding mucus. “Cell-Free” ROIs are selected outside of cells, where the green-fluorescent antibody is relatively uniformly distributed. For comparison, a representative confocal image of CVM without FITC-labeled antibody is presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158338#pone.0158338.s001" target="_blank">S1 Fig</a>.</p

Representative blue and green channel confocal images of Zenon in human CVM.

<p>(A) Zenon added directly to pH-neutralized CVM (“Zenon pH 7”), (B) Zenon added directly to native, acidic CVM (“Zenon pH 4”), and (C) DM-IgG pre-mixed with Zenon in pH-neutralized CVM (“Zenon + DM-IgG pH 7”). Sample “Cell-Free” (“CF”) vs. “Cell” (“C”) ROIs measuring 10 μm x 10 μm are indicated, along with intact epithelial cells (“IEC”). Zenon added directly to CVM associates preferentially with cellular material, an effect that is enhanced under acidic pH, as evident by green-fluorescent cell structures against the dark background of the surrounding medium. In contrast, pre-mixed Zenon-IgG exhibits a much more uniform distribution throughout the sample, with the exception of within IEC that do not appreciably absorb IgG.</p

Anti-PEG antibodies alter the mobility and biodistribution of densely PEGylated nanoparticles in mucus

Antibodies that specifically bind polyethylene glycol (PEG) can lead to rapid elimination of PEGylated therapeutics from the systemic circulation. We have recently shown that virus-binding IgG can immobilize viruses in mucus via multiple low-affinity crosslinks between IgG and mucins. However, it remains unclear whether anti-PEG antibodies in mucus may also alter the penetration and consequently biodistribution of PEGylated nanoparticles delivered to mucosal surfaces. We found that both anti-PEG IgG and IgM can readily bind nanoparticles that were densely coated with PEG polymer to minimize adhesive interactions with mucus constituents. Addition of anti-PEG IgG and IgM into mouse cervicovaginal mucus resulted in extensive trapping of mucus-penetrating PEGylated nanoparticles, with the fraction of mobile particles reduced from over 95% to only 34% and 7% with anti-PEG IgG and IgM, respectively. Surprisingly, we did not observe significant agglutination induced by either antibody, suggesting that particle immobilization is caused by adhesive crosslinks between mucin fibers and IgG or IgM bound to individual nanoparticles. Importantly, addition of corresponding control antibodies did not slow the PEGylated nanoparticles, confirming anti-PEG antibodies specifically bound to and trapped the PEGylated nanoparticles. Finally, we showed that trapped PEGylated nanoparticles remained largely in the luminal mucus layer of the mouse vagina even when delivered in hypotonic formulations that caused untrapped particles to be drawn by the flow of water (advection) through mucus all the way to the epithelial surface. These results underscore the potential importance of elucidating mucosal anti-PEG immune responses for PEGylated therapeutics and biomaterials applied to mucosal surfaces