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People living with HIV (PLWH) who are immune nonresponders (INRs) are at greater risk of comorbidity and mortality than are immune responders (IRs) who restore their CD4+ T cell count after antiretroviral therapy (ART). INRs have low CD4+ T cell counts (

ProblemPregnant women are at increased risk of HIV acquisition, but the biological mechanisms contributing to this observation are not well understood.Method of studyHere, we assessed host immune and microbiome differences in the vaginal mucosa of healthy pregnant and non-pregnant women using a metaproteomics approach. Cervicovaginal lavage (CVL) samples were collected from 23 pregnant and 25 non-pregnant women.ResultsMass spectrometry analysis of CVL identified 550 human proteins and 376 bacterial proteins from 11 genera. Host proteome analysis indicated 56 human proteins (10%) were differentially abundant (P

ProblemPregnant women are at increased risk of HIV acquisition, but the biological mechanisms contributing to this observation are not well understood.Method of studyHere, we assessed host immune and microbiome differences in the vaginal mucosa of healthy pregnant and non-pregnant women using a metaproteomics approach. Cervicovaginal lavage (CVL) samples were collected from 23 pregnant and 25 non-pregnant women.ResultsMass spectrometry analysis of CVL identified 550 human proteins and 376 bacterial proteins from 11 genera. Host proteome analysis indicated 56 human proteins (10%) were differentially abundant (P

Gastrointestinal (GI) mucosal dysfunction predicts and likely contributes to non-infectious comorbidities and mortality in HIV infection and persists despite antiretroviral therapy. However, the mechanisms underlying this dysfunction remain incompletely understood. Neutrophils are important for containment of pathogens but can also contribute to tissue damage due to their release of reactive oxygen species and other potentially harmful effector molecules. Here we used a flow cytometry approach to investigate increased neutrophil lifespan as a mechanism for GI neutrophil accumulation in chronic, treated HIV infection and a potential role for gastrointestinal dysbiosis. We report that increased neutrophil survival contributes to neutrophil accumulation in colorectal biopsy tissue, thus implicating neutrophil lifespan as a new therapeutic target for mucosal inflammation in HIV infection. Additionally, we characterized the intestinal microbiome of colorectal biopsies using 16S rRNA sequencing. We found that a reduced Lactobacillus: Prevotella ratio associated with neutrophil survival, suggesting that intestinal bacteria may contribute to GI neutrophil accumulation in treated HIV infection. Finally, we provide evidence that Lactobacillus species uniquely decrease neutrophil survival and neutrophil frequency in vitro, which could have important therapeutic implications for reducing neutrophil-driven inflammation in HIV and other chronic inflammatory conditions.

Lichen sclerosus is a chronic inflammatory condition of unknown etiology that affects the genital and extragenital skin, which can lead to sexual dysfunction and has been associated with vulvar cancer. The vaginal microbiome has a critical role in gynecologic health, but little is known about the microbiome in lichen sclerosus. This study investigated the vaginal and vulvar microbiomes of 27 post-menopausal women with lichen sclerosus. The most abundant genera detected in the vaginal microbiome were Lactobacillus, Gardnerella, and Anaerococcus, while Lactobacillus, Anaerococcus, and Staphylococcus were the most abundant in the vulvar microbiome. The vaginal samples clustered into two main groups, Lactobacillus dominant (n = 6, > 50% microbiome Lactobacillus) and polymicrobial (n = 21) with no dominant genus. The vulvar samples were mainly polymicrobial (n = 25). Actinomyces, Anaerococcus, and Ezakiella in the vaginal microbiome and Actinomyces and Ezakiella in the vulvar microbiome were significantly associated with lichen sclerosus symptoms (adjusted p < 0.05). In this population of post-menopausal women with lichen sclerosus the majority have diverse, non-Lactobacillus dominant microbiomes, which is considered less optimal for gynecologic health based on studies of pre-menopausal women. Actinomyces, Ezakiella, and Anaerococcus were associated with lichen sclerosus symptoms. Understanding the role of these bacteria in lichen sclerosus pathogenesis will be an essential future investigation. [ABSTRACT FROM AUTHOR]

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 correlatedwith 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 proteinexpressionwithestradiol levels inthe 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. [ABSTRACT FROM AUTHOR]

Risk of HIV acquisition varies, and some individuals are highly HIV-1-exposed, yet, persistently seronegative (HESN). The immunologic mechanisms contributing to this phenomenon are an area of intense interest. As immune activation and inflammation facilitate disease progression in HIV-1-infected persons and gastrointestinal-associated lymphoid tissue is a highly susceptible site for transmission, we hypothesized that reduced gut mucosal immune reactivity may contribute to reduced HIV-1 susceptibility in HESN men with a history of numerous rectal sexual exposures. To test this, we used ex vivo mucosal explants from freshly acquired colorectal biopsies from healthy control and HESN subjects who were stimulated with specific innate immune ligands and inactivated whole pathogens. Immune reactivity was then assessed via cytokine arrays and proteomic analysis. Mucosal immune cell compositions were quantified via immunohistochemistry. We found that explants from HESN subjects produced less proinflammatory cytokines compared with controls following innate immune stimulation; while noninflammatory cytokines were similar between groups. Proteomic analysis identified several immune response proteins to be differentially expressed between HIV-1-stimulated HESN and control explants. Immunohistochemical examination of colorectal mucosa showed similar amounts of T cells, macrophages, and dendritic cells between groups. The results of this pilot study suggest that mucosal innate immune reactivity is dampened in HESN versus control groups, despite presence of similar densities of immune cells in the colorectal mucosa. This observed modulation of the rectal mucosal immune response may contribute to lower risk of mucosal HIV-1 transmission in these individuals.

The mechanism(s) by which bacterial communities impact susceptibility to infectious diseases, such as HIV, and maintain female genital tract (FGT) health are poorly understood. Evaluation of FGT bacteria has predominantly been limited to studies of species abundance, but not bacterial function. We therefore sought to examine the relationship of bacterial community composition and function with mucosal epithelial barrier health in the context of bacterial vaginosis (BV) using metaproteomic, metagenomic, and in vitro approaches. We found highly diverse bacterial communities dominated by Gardnerella vaginalis associated with host epithelial barrier disruption and enhanced immune activation, and low diversity communities dominated by Lactobacillus species that associated with lower Nugent scores, reduced pH, and expression of host mucosal proteins important for maintaining epithelial integrity. Importantly, proteomic signatures of disrupted epithelial integrity associated with G. vaginalis-dominated communities in the absence of clinical BV diagnosis. Because traditional clinical assessments did not capture this, it likely represents a larger underrepresented phenomenon in populations with high prevalence of G. vaginalis. We finally demonstrated that soluble products derived from G. vaginalis inhibited wound healing, while those derived from L. iners did not, providing insight into functional mechanisms by which FGT bacterial communities affect epithelial barrier integrity.

Background Adolescent girls and young women aged 15‒24 years in sub-Saharan Africa are at disproportionate risk of human immunodeficiency virus (HIV) infection. Given the known association between vaginal microbial dysbiosis and HIV susceptibility, we performed an age-stratified analysis of the vaginal microbiome in South African women and compared this to their risk of HIV acquisition. Methods Vaginal microbiome data were generated by mass spectrometry–based proteomic analysis of cervicovaginal lavages collected from participants (n = 688) in the Centre for the AIDS Programme of Research in South Africa (CAPRISA) 004 trial. Participants were grouped by age (18–19 years, n = 93; 20–24 years, n = 326; 25–41 years, n = 269). Results Four microbiome types were identified based on predominant taxa, including Lactobacillus crispatus (CST-LC, 12.2%), Lactobacillus iners (CST-LI, 43.6%), Gardnerella vaginalis (CST-GV, 26.6%), or polymicrobial (CST-PM, 15.1%). Women aged 18–19 and 20–24 years had increased CST-PM and a non- Lactobacillus -dominant microbiome compared to those 25–41 years (odds ratio [OR], 3.14 [95% confidence interval {CI}, 1.12–7.87], P =.017; OR, 2.81 [95% CI, 1.07–7.09], P =.038, respectively; and OR, 1.65 [95% CI, 1.02–2.65], P =.028; OR, 1.40 [95% CI, 1.01–1.95], P =.030, respectively). The HIV incidence rate of women with CST-PM microbiome was 7.19-fold higher compared to women with CST-LC (hazard ratio [HR], 7.19 [95% CI, 2.11–24.5], P =.00162), which was also consistent in women aged 20–24 years (HR, 4.90 [95% CI, 1.10–21.9], P =.0375). Conclusions Younger women were more likely to have a higher-risk polymicrobial microbiome suggesting that vaginal microbiota are contributing to increased HIV-1 susceptibility in this group. Clinical Trials Registration NCT00441298. [ABSTRACT FROM AUTHOR]

In Sub-Saharan Africa, young women 15–24 years of age account for nearly 30% of all new HIV infections, however, biological and epidemiological factors underlying this disproportionate infection rate are unclear. In this study, we assessed biological contributors of SIV/HIV susceptibility in the female genital tract (FGT) using adolescent (n = 9) and adult (n = 10) pigtail macaques (PTMs) with weekly low-dose intravaginal challenges of SIV. Immunological variables were captured in vaginal tissue of PTMs by flow cytometry and cytokine assays. Vaginal biopsies were profiled by proteomic analysis. The vaginal microbiome was assessed by 16S rRNA sequencing. We were powered to detect a 2.2-fold increase in infection rates between age groups, however, we identified no significant differences in susceptibility. This model cannot capture epidemiological factors or may not best represent biological differences of HIV susceptibility. No immune cell subsets measured were significantly different between groups. Inflammatory marker MCP-1 was significantly higher (adj p = .02), and sCD40L trended higher (adj p = .06) in vaginal cytobrushes of adults. Proteomic analysis of vaginal biopsies showed no significant (adj p < .05) protein or pathway differences between groups. Vaginal microbiomes were not significantly different between groups. No differences were observed between age groups in this PTM model, however, these animals may not reflect biological factors contributing to HIV risk such as those found in their human counterparts. This model is therefore not appropriate to explore human adolescent differences in HIV risk. Young women remain a key population at risk for HIV infection, and there is still a need for comprehensive assessment and intervention strategies for epidemic control of this uniquely vulnerable population. [ABSTRACT FROM AUTHOR]

The vaginal microbiome's role in risk, progression, and treatment of female cancers has been widely explored. Yet, there remains a need to develop methods to understand the interaction of microbiome factors with host cells and to characterize their potential therapeutic functions. To address this challenge, we developed a systems biology framework we term the Pharmacobiome for microbiome pharmacology analysis. The Pharmacobiome framework evaluates similarities between microbes and microbial byproducts and known drugs based on their impact on host transcriptomic cellular signatures. Here, we apply our framework to characterization of the Anti-Gynecologic Cancer Vaginal Pharmacobiome. Using published vaginal microbiome multi-omics data from the Partners PrEP clinical trial, we constructed vaginal epithelial gene signatures associated with each profiled vaginal microbe and metabolite. We compared these microbiome-associated host gene signatures to post-drug perturbation host gene signatures associated with 35 FDA-approved anti-cancer drugs from the Library of Integrated Network-based Cellular Signatures database to identify vaginal microbes and metabolites with high statistical and functional similarity to these drugs. We found that Lactobacilli and their metabolites can regulate host gene expression in ways similar to many anti-cancer drugs. Additionally, we experimentally tested our model prediction that taurine, a metabolite produced by L. crispatus, kills cancerous breast and endometrial cancer cells. Our study shows that the Pharmacobiome is a powerful framework for characterizing the anti-cancer therapeutic potential of vaginal microbiome factors with generalizability to other cancers, microbiomes, and diseases.

Objectives: Bacterial vaginosis (BV) contributes to poor reproductive health and is characterized by a displacement of Lactobacillus in the vaginal microbiome. However, treatment for BV is limited to antibiotics and half of the women treated experience recurrence within a year. THRIVE (The Study of Host-Bacterial Relationships and Immune Function in Different Vaginal Environments) is a prospective study in Winnipeg, Manitoba, Canada, which is designed to capture the daily variation of the microbiome and host mucosal immunity during treatment. The objective of this study is to identify host and bacterial factors that associate with vaginal microbiome stability to better inform therapeutic interventions., Methods: Women treated for BV, and controls, are followed for 6 months collecting daily vaginal swabs and monthly questionnaires. Comprehensive mucosal sampling, including swabs, cytobrushes, biopsies, and blood are collected at baseline, months 1 and 6 post-enrolment., Results: We performed analysis on the first 52 participants, (19 BV+, 33 BV-). Molecular profiling by 16s RNA sequencing showed 20 women with non-Lactobacillus-dominant microbiomes and 32 with Lactobacillus-dominant microbiomes, with increased microbial diversity in non-Lactobacillus-dominant microbiomes (P = 3.1E-05). A pilot analysis in 2 participants demonstrates that multi-omics profiling of self-collected daily swabs provides high-quality data identifying 73 bacterial species, 1773 mucosal proteins and 117 metabolites. Initial flow cytometry analysis showed an increased cluster of differentiation (CD)4+ T cells and neutrophil activation (CD11b+CD62L neg/dim ) in the positive participant at baseline, while after treatment these shifted and resembled the control participant., Conclusions: This study provides a framework to comprehensively investigate the kinetics of vaginal mucosal microbiome alterations, providing further insight into host and molecular features predicting BV recurrence., (Copyright © 2024 The Author. Published by Elsevier Inc. All rights reserved.)

A substantial percentage of the population remains at risk for cervical cancer due to pre-existing human papillomavirus (HPV) infections, despite prophylactic vaccines. Early diagnosis and treatment are crucial for better disease outcomes. The development of new treatments heavily relies on suitable preclinical model systems. Recently, we established a mouse papillomavirus (MmuPV1) model that is relevant to HPV genital pathogenesis. In the current study, we validated the use of Papanicolaou (Pap) smears, a valuable early diagnostic tool for detecting HPV cervical cancer, to monitor disease progression in the MmuPV1 mouse model. Biweekly cervicovaginal swabs were collected from the MmuPV1-infected mice for viral DNA quantitation and cytology assessment. The Pap smear slides were evaluated for signs of epithelial cell abnormalities using the 2014 Bethesda system criteria. Tissues from the infected mice were harvested at various times post-viral infection for additional histological and virological assays. Over time, increased viral replication was consistent with higher levels of viral DNA, and it coincided with an uptick in epithelial cell abnormalities with higher severity scores noted as early as 10 weeks after viral infection. The cytological results also correlated with the histological evaluation of tissues harvested simultaneously. Both immunocompromised and immunocompetent mice with squamous cell carcinoma (SCC) cytology also developed vaginal SCCs. Notably, samples from the MmuPV1-infected mice exhibited similar cellular abnormalities compared to the corresponding human samples at similar disease stages. Hence, Pap smear screening proves to be an effective tool for the longitudinal monitoring of disease progression in the MmuPV1 mouse model., Importance: Papanicolaou (Pap) smear has saved millions of women's lives as a valuable early screening tool for detecting human papillomavirus (HPV) cervical precancers and cancer. However, more than 200,000 women in the United States alone remain at risk for cervical cancer due to pre-existing HPV infection-induced precancers, as there are currently no effective treatments for HPV-associated precancers and cancers other than invasive procedures including a loop electrosurgical excision procedure (LEEP) to remove abnormal tissues. In the current study, we validated the use of Pap smears to monitor disease progression in our recently established mouse papillomavirus model. To the best of our knowledge, this is the first study that provides compelling evidence of applying Pap smears from cervicovaginal swabs to monitor disease progression in mice. This HPV-relevant cytology assay will enable us to develop and test novel antiviral and anti-tumor therapies using this model to eliminate HPV-associated diseases and cancers., Competing Interests: The authors declare no conflict of interest.