Multi-omics analysis of the cervical epithelial integrity of women using depot medroxyprogesterone acetate.

Depot medroxyprogesterone acetate (DMPA) is an injectable hormonal contraceptive used by millions of women worldwide. However, experimental studies have associated DMPA use with genital epithelial barrier disruption and mucosal influx of human immunodeficiency virus (HIV) target cells. We explored the underlying molecular mechanisms of these findings. Ectocervical biopsies and cervicovaginal lavage (CVL) specimens were collected from HIV-seronegative Kenyan sex workers using DMPA (n = 32) or regularly cycling controls (n = 64). Tissue samples were assessed by RNA-sequencing and quantitative imaging analysis, whereas protein levels were measured in CVL samples. The results suggested a DMPA-associated upregulation of genes involved in immune regulation, including genes associated with cytokine-mediated signaling and neutrophil-mediated immunity. A transcription factor analysis further revealed DMPA-associated upregulation of RELA and NFKB1 which are involved in several immune activation pathways. Several genes significantly downregulated in the DMPA versus the control group were involved in epithelial structure and function, including genes encoding keratins, small proline-rich proteins, and cell-cell adhesion proteins. Pathway analyses indicated DMPA use was associated with immune activation and suppression of epithelium development, including keratinization and cornification processes. The cervicovaginal microbiome composition (Lactobacillus dominant and non-Lactobacillus dominant) had no overall interactional impact on the DMPA associated tissue gene expression. Imaging analysis verified that DMPA use was associated with an impaired epithelial layer as illustrated by staining for the selected epithelial junction proteins E-cadherin, desmoglein-1 and claudin-1. Additional staining for CD4+ cells revealed a more superficial location of these cells in the ectocervical epithelium of DMPA users versus controls. Altered protein levels of SERPINB1 and ITIH2 were further observed in the DMPA group. Identification of specific impaired epithelial barrier structures at the gene expression level, which were verified at the functional level by tissue imaging analysis, illustrates mechanisms by which DMPA adversely may affect the integrity of the genital mucosa.

Automated detection of vascular remodeling in tumor-draining lymph nodes by the deep-learning tool HEV-finder.

Vascular remodeling is common in human cancer and has potential as future biomarkers for prediction of disease progression and tumor immunity status. It can also affect metastatic sites, including the tumor-draining lymph nodes (TDLNs). Dilation of the high endothelial venules (HEVs) within TDLNs has been observed in several types of cancer. We recently demonstrated that it is a premetastatic effect that can be linked to tumor invasiveness in breast cancer. Manual visual assessment of changes in vascular morphology is a tedious and difficult task, limiting high-throughput analysis. Here we present a fully automated approach for detection and classification of HEV dilation. By using 12,524 manually classified HEVs, we trained a deep-learning model and created a graphical user interface for visualization of the results. The tool, named the HEV-finder, selectively analyses HEV dilation in specific regions of the lymph nodes. We evaluated the HEV-finder's ability to detect and classify HEV dilation in different types of breast cancer compared to manual annotations. Our results constitute a successful example of large-scale, fully automated, and user-independent, image-based quantitative assessment of vascular remodeling in human pathology and lay the ground for future exploration of HEV dilation in TDLNs as a biomarker. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Preserved Mucosal-Associated Invariant T Cells in the Cervical Mucosa of HIV-Infected Women with Dominant Expression of the TRAV1-2-TRAJ20 T Cell Receptor α-Chain.

BACKGROUND: Mucosa-associated invariant T (MAIT) cells are innate-like T cells with specialized antimicrobial functions. Circulating MAIT cells are depleted in chronic human immunodeficiency virus (HIV) infection, but studies examining this effect in peripheral tissues, such as the female genital tract, are lacking. METHODS: Flow cytometry was used to investigate circulating MAIT cells in a cohort of HIV-seropositive (HIV+) and HIV-seronegative (HIV-) female sex workers (FSWs), and HIV- lower-risk women (LRW). In situ staining and quantitative polymerase chain reaction were performed to explore the phenotype of MAIT cells residing in paired cervicovaginal tissue. The cervicovaginal microbiome was assessed by means of 16S ribosomal RNA gene sequencing. RESULTS: MAIT cells in the HIV+ FSW group were low in frequency in the circulation but preserved in the ectocervix. MAIT cell T-cell receptor gene segment usage differed between the HIV+ and HIV- FSW groups. The TRAV1-2-TRAJ20 transcript was the most highly expressed MAIT TRAJ gene detected in the ectocervix in the HIV+ FSW group. MAIT TRAVJ usage was not associated with specific genera in the vaginal microbiome. CONCLUSIONS: MAIT cells residing in the ectocervix are numerically preserved irrespective of HIV infection status and displayed dominant expression of TRAV1-2-TRAJ20. These findings have implications for understanding the role of cervical MAIT cells in health and disease.

Estradiol-mediated enhancement of the human ectocervical epithelial barrier correlates with desmoglein-1 expression in the follicular menstrual phase.

Background: The cervicovaginal epithelial barrier is crucial for defending the female reproductive tract against sexually transmitted infections. Hormones, specifically estradiol and progesterone, along with their respective receptor expressions, play an important role in modulating this barrier. However, the influence of estradiol and progesterone on gene and protein expression in the ectocervical mucosa of naturally cycling women is not well understood. Methods: Mucosal and blood samples were collected from Kenyan female sex workers at high risk of sexually transmitted infections. All samples were obtained at two time points, separated by two weeks, aiming for the follicular and luteal phases of the menstrual cycle. Ectocervical tissue biopsies were analyzed by RNA-sequencing and in situ immunofluorescence staining, cervicovaginal lavage samples (CVL) were evaluated using protein profiling, and plasma samples were analyzed for hormone levels. Results: Unsupervised clustering of RNA-sequencing data was performed using Weighted gene co-expression network analysis (WGCNA). In the follicular phase, estradiol levels positively correlated with a gene module representing epithelial structure and function, and negatively correlated with a gene module representing cell cycle regulation. These correlations were confirmed using regression analysis including adjustment for bacterial vaginosis status. Using WGCNA, no gene module correlated with progesterone levels in the follicular phase. In the luteal phase, no gene module correlated with either estradiol or progesterone levels. Protein profiling on CVL revealed that higher levels of estradiol during the follicular phase correlated with increased expression of epithelial barrier integrity markers, including DSG1. This contrasted to the limited correlations of protein expression with estradiol levels in the luteal phase. In situ imaging analysis confirmed that higher estradiol levels during the follicular phase correlated with increased DSG1 expression. Conclusion: We demonstrate that estradiol levels positively correlate with specific markers of ectocervical epithelial structure and function, particularly DSG1, during the follicular phase of the menstrual cycle. Neither progesterone levels during the follicular phase nor estradiol and progesterone levels during the luteal phase correlated with any specific sets of gene markers. These findings align with the expression of estradiol and progesterone receptors in the ectocervical epithelium during these menstrual phases.

A topical rectal douche product containing Q-Griffithsin does not disrupt the epithelial border or alter CD4+ cell distribution in the human rectal mucosa.

To reduce HIV transmission, locally applied pre-exposure prophylaxis (PrEP) products for anorectal use will be important complements to oral and injectable PrEP products already available. It is critical to preserve an intact rectal epithelium and avoid an influx of mucosal HIV target cells with such product use. In this phase 1 clinical trial, we evaluated application of a topical rectal douche product containing Q-Griffithsin (Q-GRFT). Colorectal tissue samples were obtained via sigmoidoscopy at baseline, 1 and 24 h after single-dose exposure in 15 healthy volunteers. In situ staining for epithelial junction markers and CD4+ cells were assessed as an exploratory endpoint. A high-throughput, digitalized in situ imaging analysis workflow was developed to visualize and quantify these HIV susceptibility markers. We observed no significant differences in epithelial distribution of E-cadherin, desmocollin-2, occludin, claudin-1, or zonula occludens-1 when comparing the three timepoints or Q-GRFT versus placebo. There were also no differences in %CD4+ cells within the epithelium or lamina propria in any of these comparisons. In conclusion, the rectal epithelium and CD4+ cell distribution remained unchanged following topical application of Q-GRFT. In situ visualization of HIV susceptibility markers at mucosal sites could be useful to complement standard product safety assessments.

Remodeling of the Lymph Node High Endothelial Venules Reflects Tumor Invasiveness in Breast Cancer and is Associated with Dysregulation of Perivascular Stromal Cells.

The tumor-draining lymph nodes (TDLNs) are primary sites for induction of tumor immunity. They are also common sites of metastasis, suggesting that tumor-induced mechanisms can subvert anti-tumor immune responses and promote metastatic seeding. The high endothelial venules (HEVs) together with CCL21-expressing fibroblastic reticular cells (FRCs) are essential for lymphocyte recruitment into the LNs. We established multicolor antibody panels for evaluation of HEVs and FRCs in TDLNs from breast cancer (BC) patients. Our data show that patients with invasive BC display extensive structural and molecular remodeling of the HEVs, including vessel dilation, thinning of the endothelium and discontinuous expression of the HEV-marker PNAd. Remodeling of the HEVs was associated with dysregulation of CCL21 in perivascular FRCs and with accumulation of CCL21-saturated lymphocytes, which we link to loss of CCL21-binding heparan sulfate in FRCs. These changes were rare or absent in LNs from patients with non-invasive BC and cancer-free organ donors and were observed independent of nodal metastasis. Thus, pre-metastatic dysregulation of core stromal and vascular functions within TDLNs reflect the primary tumor invasiveness in BC. This adds to the understanding of cancer-induced perturbation of the immune response and opens for prospects of vascular and stromal changes in TDLNs as potential biomarkers.