Cervical Lavage Research Articles

Female genital schistosomiasis, human papilloma virus infection, and cervical cancer in rural Madagascar: a cross sectional study

BackgroundWomen’s health in resource-limited settings can benefit from the integrated management of high-burden diseases, such as female genital schistosomiasis (FGS) and human papilloma virus (HPV)-related cervical cancer. In schistosomiasis-endemic countries such as Madagascar, data on FGS and HPV prevalence are lacking as well as preventive measures for both conditions. This study aims to estimate the prevalence of FGS and HPV in rural Madagascar, and to examine associated risk factors to identify opportunities for improving women’s health.MethodsAfter initial community outreach activities, interested women aged 18–49 years were recruited consecutively in 2021 at three primary health care centers in the district of Marovoay. FGS was detected by colposcopy. Colposcopy images were double-blind reviewed by two independent specialists. A Luminex bead-based assay was performed on cervical vaginal lavage specimens for HPV typing. Crude (CPR) and adjusted prevalence ratios (APR) of associations between selected factors and FGS and HPV positivity were estimated using univariable and multivariable binary Poisson regression with 95% confidence intervals (CIs).ResultsAmong 500 women enrolled, 302 had complete information on FGS and HPV diagnosis, and were thus eligible for analysis. Within the sample, 189 (62.6%, 95% CI: 56.9–68.1) cases of FGS were detected. A total of 129 women (42.7%, 95% CI: 37.1–48.5) tested positive for HPV. In total, 80 women (26.5%, 95% CI: 21.6–31.8]) tested positive for both conditions. No association was observed between FGS and HPV positivity, while previous pregnancy (APR = 0.65, 95% CI: 0.43–0.78) and older age (APR = 0.59, 95% CI: 0.42–0.81) are showing a negative association with HPV infection compared to no previous pregnancy and younger age groups.ConclusionsThe results of the study show that FGS and HPV are highly prevalent in rural Madagascar. The concurrent prevalence of these two conditions requires urgent adaptations of public health strategies to improve women’s health, such as integrated services at primary level of care.Graphical

Abstract 712: Cytokine and TGF-β levels are associated with changes in cervicovaginal microbiota in a cohort of Caribbean women

Abstract Recent studies suggest that the cervical microbiome can strongly influence inflammation and pre-cancerous lesion progression. However, research focused on understanding the role of microbial communities in the progression of pre-cancerous lesions to cancer in Latino women is scarce. We aimed to investigate the relationship between the cervicovaginal microbiome and inflammation while considering cervical neoplasia and HPV infection in Puerto Rican women. We collected cervical swabs and lavages from 142 participants coming to colposcopy clinics in San Juan, Puerto Rico. Genomic DNA was extracted from swabs, and 16S rDNA V4 region genes were amplified and sequenced by Illumina MiSeq. Inflammatory (IL-1β, TNFa, IFNg, IL-6), anti-inflammatory (IL-4, IL-10, TGFβ1), and trafficking (IL-8, MIP1a, MCP1, IP10) cytokines were measured from cervical lavages, using Luminex MAGPIX technology. Cytokines were related to microbes via an inflammation scoring index based on the quartile and tercile distribution of the cytokine’s concentration. IL-10 (p value= 0.0455) was significantly different when evaluating HPV risk, while IL1-β (p value= 0.0005) and INF-γ (p value= 0.0258) were significant when evaluating cervical disease. We found significant differences in diversity and composition of the microbiota among HPV risk, cervical disease, pro-inflammatory, anti-inflammatory, and trafficking cytokine abundances. The increasing concentration of IL1-β, IL-10, and INF-γ, associated with a decrease in Lactobacillus communities. While contrarily, bacteria associated with dysbiosis such as Gardnerella, Prevotella, Atopobium increased. This study also revealed that the most dominant community state types (CST) among Puerto Rican women regardless of lesion or HPV status, are CST3 and 4 featuring high diversity and anaerobic bacteria typical of vaginosis in Caucasians. These CSTs are especially abundant with ~ 90% dominance in participants with high grade disease (HGSIL) and high-risk HPV. Our study evidence that the cervical microbiota of Puerto Ricans is characteristically diverse and that the joint host-microbe interaction analyses via cytokine signaling and microbiota in pre-cancerous lesions has great translational potential. Citation Format: Eduardo Lemuel Tosado Rodríguez, Anelisse Dominicci-Maura, Loyda Mendez, Stephanie Dorta, Josefina Romaguera, Filipa Godoy-Vitorino. Cytokine and TGF-β levels are associated with changes in cervicovaginal microbiota in a cohort of Caribbean women [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 712.

The Role of IL-2, IL-4, IL-10 and IFN-<i>γ</i> Cytokines Expression in the Microenvironment of Cervical Intraepithelial Neoplasia

Objective: To investigate the role of IL-2, IL-4, IL-10 and IFN-γ cytokines in the microenvironment of cervical intraepithelial neoplasia. Methods: 180 patients participating were enrolled in this trial, where 50 cases are in control group, 50 cases are in low-grade squamous intraepithelial lesion (LSIL) group, 50 cases are in high-grade squamous intraepithelial lesion (HSIL) group, and 30 cases have cervical carcinoma. ELISA methods are used to detect the expression of IL-2, IL-4, IL-10 and IFN-γ in the lavage fluid, and all data is analyzed using one-way analysis of variance. Results: The expression of IL-2 and IFN-γ in cervical lavage fluid gradually decreases with the aggravation of the lesion. The expression of IL-4 and IL-10 increases by the aggravation of pathological changes. There were statistically significant differences in IL-2 level among the four groups. IFN-γ levels are significantly different between the cervical cancer group and the other groups, also between HSIL and LSIL group, and between HSIL and the control group, but no statistically significant difference was observed in IFN-γ e between LSIL and the control group. The expressions of IL-4 and IL-10 between the cervical cancer group and the other groups were significantly different, and also between HSIL and the control group. But there was no statistically significant difference between LSIL and the control group, HSIL and LSIL. Conclusion: The cytokines of IL-2, IL-4, IL-10 and IFN-γ in the microenvironment of the cervix play an important role in the occurrence and development of cervical intraepithelial neoplasia. This study may provide the evidence for diagnosis and treatment of cervical intraepithelial neoplasia.

Abstract 2913: Contributions of the rare microbial biosphere, HPV infections and BMI to shifts in metabolic profiles in the cervical lavage of Hispanics

Abstract The vagina and cervix have the lowest biodiversity of the human microbiota, with a colonized epithelium dominated by protective Lactobacilli. Changes in the cervicovaginal microbes are therefore a predicted cause of malignancies, revealing a tremendous potential of microbiome-related processes to be used in early cancer diagnostics. We hypothesized that bacterial communities and their metabolic profiles change in association to HPV infections, BMI and cervical disease. We characterized the microbiota and the metabolomic profiles of 47 Puerto Rican women coming to San Juan OB/Gyn clinics using 16S rRNA sequencing and Gas Chromatography coupled to Mass Spectrometry respectively. Samples compared between those negative for intraepithelial lesion or malignancy (NILM) and high-grade squamous intraepithelial lesions (HGSIL) samples, and grouped according to HPV status (positive and negative) followed by body mass indexes (BMI) of normal weight (NW) and overweight (OW). The microbiota was analyzed at first with Qiita, using the greengenes database as taxonomic reference, and community analyses were performed in QIIME and R. The MIMOSA2 framework was the approach to identify the key species contributors to the metabolite concentrations and their correspondent producing enzymes annotated using KEGG database. After that, we applied BURRITO framework to pair taxonomic and functional information obtained by MIMOSA2.The analysis of NILM (HPV+ and NW) and NILM (HPV+ and OW) showed a strong influence of HPV infections on microbiome contributions to metabolic shifts related to amino acid metabolism, and they were not overlapped in the metabolite identities and functions. In particular, degradation of alanine and synthesis of alanyl-tRNAAla concomitant with the activity of alanyl-tRNA synthetase which was associated primarily with A. vaginae, Gardnerella, L. inners and to a lesser extent by L. crispatus. Simultaneously, A. vaginae and Gardnerella displayed an antagonistic relationship for alanine production, associated with cysteine desulfurase. NILM HPV positive groups displayed enrichments in metabolites that belong to the tryptophan catabolic pathway. The HGSIL (HPV+ and OW) group triggered the most complex metabolic shift attributed to pyruvate, purine metabolism, glycine, serine and threonine metabolism, and aminoacyl-tRNA biosynthesis. Taken together, our data suggest that HPV infections trigger different changes in the dynamic equilibrium of the cervical lavage microbiota at the compositional and functional levels. These changes alter the metabolic phenotype of the host and are strongly associated with BMI. Sponsored by PRSTRT ARG# 2020-00112; NIGMS P20596GM103475, NIMHD S21MD001830. Citation Format: Natalyia Chorna, Josefina Romaguera, Filipa Godoy-Vitorino. Contributions of the rare microbial biosphere, HPV infections and BMI to shifts in metabolic profiles in the cervical lavage of Hispanics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2913.

HIV-Exposed Seronegative Sex Workers Express Low T-Cell Activation and an Intact Ectocervical Tissue Microenvironment.

Immunological correlates of natural resistance to HIV have been identified in HIV-exposed seronegative (HESN) individuals and include a low-inflammatory genital mucosal status. The cervicovaginal epithelium has not been studied for such correlates despite constituting an important barrier against sexual HIV transmission. To fill this gap in knowledge, we collected samples of blood, cervical mononuclear cells, cervicovaginal lavage, and ectocervical tissue from Kenyan HESN sex workers (n = 29) and controls (n = 33). The samples were analyzed by flow cytometry, protein profiling, 16S rRNA gene sequencing, in situ image analysis, and tissue-based RNA sequencing. A significantly higher relative proportion of regulatory T cells in blood (B7+CD25hiFoxP3+CD127loCD4+ and B7+Helios+FoxP3+CD4+), and a significantly lower proportion of activated cervical T cells (CCR5+CD69+CD4+ and CCR5+CD69+CD8+), were found in the HESN group compared with the controls. In contrast, there were no statistically significant differences between the study groups in cervicovaginal protein and microbiome compositions, ectocervical epithelial thickness, E-cadherin expression, HIV receptor expression, and tissue RNA transcriptional profiles. The identification of an intact ectocervical microenvironment in HESN individuals add new data to current knowledge about natural resistance to sexual transmission of HIV.

Diverse vaginal microbiome was associated with pro-inflammatory vaginal milieu among pregnant women in Uganda

Vaginal microbiota has been postulated as a key contributor to the disproportionately higher HIV acquisition risk in women. The commensal bacterial communities in the vaginal tract have been implicated in HIV pathogenesis, with strict anaerobes such as Gardnerella and Prevotella causing inflammation and increased frequency of HIV target cells. Young African women are up to six times more likely to be infected with HIV than their male counterparts. The underlying biological mechanisms for increased susceptibility to HIV infection are not fully known, particularly among pregnant women who are also at risk of transmitting the infection to their unborn babies.We characterized the vaginal microbiome of pregnant women receiving antenatal care. Using 16S rRNA sequencing, we analyzed the richness and abundances of the commensal bacterial communities within the female genital tract. Data was analyzed using qiime2 version 2018, Dada2 plugin, and Naive-Bayes classifier for Taxonomic assignment.We report that 19% (35/179) of pregnant women had a Lactobacillus-dominant vaginal microbiota profile. Our findings show that the main cervicotypes (“CTs”) were CT1 which was predominantly non-iners Lactobacillus (6%, 11/179), CT2 which was dominated by L. iners (13%, 24/179), CT3 that was Gardnerella dominant (49%, 87/179) and CT4, a mixed CT co-dominated by L. iners, Gardnerella and Atopobium. Cervical lavage of women with non-Lactobacillus CT had significantly higher levels of inflammatory cytokines IL-1beta, TNF-alpha, and chemokines IL-6 and IL-8.Highly diverse cervicotype (CT4) was associated with inflammation, a known catalyst of HIV acquisition and transmission, within pregnant women regardless of HIV sero-status.

Using safe, affordable and accessible non-steroidal anti-inflammatory drugs to reduce the number of HIV target cells in the blood and at the female genital tract.

IntroductionAt its basic level, HIV infection requires a replication‐competent virus and a susceptible target cell. Elevated levels of vaginal inflammation has been associated with the increased risk of HIV infection as it brings highly activated HIV target cells (CCR5+CD4+ T cells; CCR5+CD4+CD161+ Th17 T cells) to the female genital tract (FGT) where they interact with HIV. Decreased HIV risk has been associated with a phenotype of decreased immune activation, called immune quiescence, described among Kenyan female sex workers who were intensely exposed to HIV yet remain uninfected. Current prevention approaches focus on limiting viral access. We took the novel HIV prevention approach of trying to limit the number of HIV target cells in the genital tract by reducing inflammation using safe, affordable and globally accessible anti‐inflammatory drugs.MethodsWe hypothesized that the daily administration of low doses of acetylsalicylic acid (ASA 81 mg) or hydroxychloroquine (HCQ 200 mg) would reduce inflammation thereby decreasing HIV target cells at the FGT. Low‐risk HIV seronegative women from Nairobi, Kenya were randomized for six weeks therapy of ASA (n = 37) or HCQ (n = 39) and tested to determine the impact on their systemic and mucosal immune environment.ResultsThe results showed that HCQ use was associated with a significant reduction in the proportion of systemic T cells that were CCR5+CD4+ (p = 0.01) and Th17 (p = 0.01). In the ASA arm, there was a 35% and 28% decrease in the proportion of genital T cells that were CD4+CCR5+ (p = 0.017) and Th17 (p = 0.04) respectively. Proteomic analyses of the cervical lavage showed ASA use was associated with significantly reduced amount of proteins involved in the inflammatory response and cell recruitment at the mucosa, although none of the individual proteins passed multiple comparison correction. These changes were more apparent in women with Lactobacillus dominant microbiomes.ConclusionTogether, these data indicate that taking low‐dose ASA daily was associated with significant reduction in HIV target cells at the FGT. This study provides proof‐of‐concept for a novel HIV‐prevention approach that reducing inflammation using safe, affordable and globally accessible non‐steroidal anti‐inflammatory agents is associated with significant reduction in the proportion of HIV‐target cells at the FGT.

Reduced levels of genital tract immune biomarkers in postmenopausal women: implications for HIV acquisition

Rates of HIV infections are increasing in older adults. Although it is known that the HIV/AIDS epidemics affects women disproportionately, little is known regarding immune functions in the genital tract of postmenopausal women, as relevant to HIV susceptibility. The objective of the study was to compare levels of female reproductive tract immune mediators that are important for HIV-associated immune responses as well as intrinsic anti-HIV activity in the cervical vaginal lavages collected from HIV-negative pre- and postmenopausal women. Cervical vaginal lavage from 20 premenopausal and 20 postmenopausal women were assayed for interleukin-6, interleukin-8, tumor necrosis factor-α, secretory leukocyte protease inhibitor, elafin, human β-defensin-2, and macrophage inflammatory protein-3α using standard enzyme-linked immunosorbent assays. Anti-HIV activity of cervical-vaginal lavage was measured using TZM-bl indicator cells against HIV-1 IIIB and BaL. Whereas each postmenopausal woman provided only 1sample, each premenopausal woman provided 3 samples, during proliferative, ovulatory, and secretory stages, based on menstrual dates. We observed significantly lower levels of tumor necrosis factor-α, MIP-3α, secretory leukocyte protease inhibitor, elafin, and human β-defensin-2 in cervical vaginal lavage from postmenopausal women compared with premenopausal women. Inhibition of HIV-1 infection was observed for both pre- and postmenopausal women, but cervical vaginal lavage from postmenopausal women showed significantly higher inhibition against HIV-1 BaL after adjusting for total protein concentration, genital pH, and reproductive tract infections. No change in mediators or HIV inhibition was observed through the stages of menstrual cycle. In addition, we observed that postmenopausal women with reproductive tract infections had significantly higher levels of tumor necrosis factor-α and significantly lower levels of interleukin-8, which were not observed in premenopausal women. Our findings suggest that female reproductive tract immune microenvironment is distinct in HIV-negative postmenopausal women. Further studies are needed to assess the risk of HIV acquisition/transmission in this population.

Low prevalences of HIV infection and HSV genital shedding in the general adult female population in Senegal.

Herpes simplex virus (HSV) is the main co-factor for heterosexual transmission of the human immunodeficiency virus (HIV) in sub-Saharan Africa, and could be involved in the dynamics of the HIV epidemic in Senegal. Genital shedding of HSV was evaluated in adult females who had visited the provincial healthcare centres in Diass, Louga, and Kebemer in Senegal. Study subjects were interviewed by a healthcare worker for sociodemographic characteristics and sexual behavior, and HIV serology was offered. In addition, cervical secretion lavage samples were evaluated for HSV DNA by real-time polymerase chain reaction (PCR), the melting curve analysis of which permitted distinction between HSV type 1 (HSV-1) and HSV type 2 (HSV-2). Among 302 women (mean age, 40 years) enrolled, none were infected by HIV. The mean age at first sexual intercourse was 20 years, and the mean number of sexual partners in the previous year was 1.3 (range, 1-7). Only 6 of 302 (1.9%) women had cervico-vaginal secretions positive for HSV DNA. No association between HSV DNA shedding and any sociodemographic or biological variables was found. Surprisingly, genital shedding of HSV-1 was found in two (0.7%) women, representing 33% of herpes-shedding women, and HSV-2 in four (1.5%) women. Taken together, our observations indicate a low prevalence of HSV DNA genital shedding in adult Senegalese women.

The Association of Human Cytomegalovirus with Biomarkers of Inflammation and Immune Activation in HIV-1-Infected Women

Three groups of cytomegalovirus (CMV)-seropositive women (total n = 164) were selected from the Chicago Women's Interagency HIV-1 Study to investigate the association between CMV coinfection and immune activation: (1) HIV-1 viremic, (2) HIV-1 aviremic, and (3) HIV-1 uninfected. Quantitative measures of CMV serum IgG, CMV DNA, and serum biomarkers interleukin (IL)-6, soluble CD163 (sCD163), soluble CD14 (sCD14), and interferon gamma-induced protein (IP10) were obtained. Levels of CMV IgG and the serum biomarkers were significantly higher in the HIV-1 viremic group compared to the aviremic and uninfected groups (p < 0.001). No significant associations with CMV IgG levels were found for HIV-uninfected women. When each of the HIV-infected groups was analyzed, sCD14 levels in the viremic women were significantly associated with CMV IgG levels with p < 0.02 when adjusted for age, CD4 count, and HIV viral load. There was also a modest association (p = 0.036) with IL-6 from plasma and cervical vaginal lavage specimens both unadjusted and adjusted for CD4 count and HIV viral load. The association of CMV IgG level with sCD14 implicates the monocyte as a potential site for interaction of the two viruses, which eventually may lead to non-AIDS-defining pathological conditions.

Increased expression of antimicrobial peptides in the cervix during Trichomonas vaginalis infection (MUC2P.923)

Abstract The lower genital tract is a unique mucosal environment and further studies are needed to define the types of immune responses that occur during genital infections. We investigated the expression of antimicrobial proteins/peptides (AMP) and cytokines produced in response to Trichomonas vaginalis (TV) infection. A cohort of women (30 TV+, 30 TV-) aged 18-45 that was at high risk of acquiring HIV was studied. mRNA was isolated from cervical cytobrush samples, reverse transcribed, and real time PCR used to determine AMP and cytokine expression. Cytokines were measured by ELISA in cervical vaginal lavage (CVL). TV+ samples had significantly higher mRNA levels of S100A7, S100A8, S100A9 and Muc1 than TV- samples. In contrast, lactoferrin was lower in the TV+ group. For defensins (HBDs), mRNA for HBD4 was significantly higher in TV+ samples, while HBD103 was lower in TV+ samples, but there was no difference in HBD1. Cathelicidin was not different between TV+ and TV- samples. mRNA for RegIIIγ was detected in only four samples. Although a role in inducing expression of AMPs has been shown for IL-22 and IL-17A, these two cytokines, as well as IFNγ, were not different in TV+ and TV- groups. However, IL-17 protein, but not IL-22 was higher in TV+ CVL samples (p &amp;lt; 0.05). Our data identify AMPs that have altered expression in the cervix during TV infection suggesting that these AMPs might have an important role in immunity against TV infection.

Analysis of Griffithsin (GRFT) Binding by Proteins in Cervical Vaginal Lavage and the Influence of Vaginal Microflora

Analysis of Griffithsin (GRFT) Binding by Proteins in Cervical Vaginal Lavage and the Influence of Vaginal Microflora

Examining IL-33 expression in the cervix of HPV-infected patients: a preliminary study comparing IL-33 levels in different stages of disease and analyzing its potential association with IFN-γ.

Research has shown the essential role of interleukin-33 (IL-33) in driving protective anti-viral immunity. IFN-γ has been reported to improve IL-33 expression in cultured epithelial cells. The development of cervical intraepithelial neoplasia (CIN) and carcinogenesis was closely related to human papilloma virus (HPV) infection and defective anti-viral immunity. The aim of this study was to investigate IL-33 expression alternation during the disease progress and its association with IFN-γ in HPV-positive patients. IL-33 was detected in endothelial cells and populations of epithelial cells in cervix. Though there was no statistically significant difference of IL-33 levels in cervical lavage and serum among different stages of disease (P > 0.05), the IL-33 protein and mRNA levels in cervical tissues were significantly lower in severe CIN patients than that of mild CIN or no CIN patients (P < 0.05). In addition, IL-33 protein levels were positively correlated with IFN-γ mRNA levels in all groups except cervical cancer (CA) group (r = 0.546, P < 0.01). In vitro, IFN-γ was also found to upregulate IL-33 expression in human epidermal keratinocytes (NHEKs) in a dose-dependent manner. However, CA tissues did not show further reduced IL-33 protein and mRNA levels compared with severe CIN tissues (P > 0.05). IFN-γ mRNA levels were even higher in CA tissues than in severe CIN tissues (P < 0.05). Therefore, in cervical precancerous tissues, IL-33 levels were lower in more severe lesions and that may be related to diminished local IFN-γ.

Age and menopause affect the expression of specific cytokines/chemokines in plasma and cervical lavage samples from female sex workers in Nairobi, Kenya

BackgroundAging of the immune system, known as immunosenescence, is associated with profound changes in both innate and adaptive immune responses, resulting in increased susceptibility to infection and a decreased ability to respond to vaccination. The purpose of this study was to investigate the effect of age and menopause on the expression of 22 different cytokines/chemokines in both plasma and cervical lavage samples from female sex-worker cohort from Nairobi, Kenya (age range 20–65).ResultsCytokine/chemokine levels were measured using a Miliplex multiplex assay (Millipore). We found that age positively correlated with MCP-1 (p = 0.0002) and IP-10 (p = 0.03) systemic cytokine expression, and that women over 50 expressed the highest levels of these cytokines, but also had elevated expression of MIG (ANOVA p = 0.0096) and MIP-3β(ANOVA p = 0.0434). We also found that IL-8 (p = 0.047) and sCD40L (p = 0.01) systemic expression negatively correlated with age. Further, MIG (p = 0.0081) and MCP-1 (p = 0.0157) were present at higher levels in post-menopausal women suggesting a potential estrogen dependant systemic regulation of these cytokines. In cervical lavage samples, age did not directly correlate with the expression of any of the tested cytokines/chemokines, however sIL-2Rα (ANOVA p = 0.0170) and IL-15 (ANOVA p = 0.0251)were significantly higher in women over 50. Menopause was shown to have a more profound effect on cytokine expression in the cervical mucosa with MIG (p = 0.0256), MIP-3α (p = 0.0245), IL-1β (p = 0.0261), IL-6 (p = 0.0462), IL-8 (p = 0.007), IP-10 (p = 0.0357) and MCP-1 (p = 0.0427) all significantly under-expressed in post-menopausal women.ConclusionsThis study demonstrates that aging and menopause-associated hormonal changes are associated with significant changes in systemic and mucosal cytokine/chemokine expression, which may have implications for the age-related decline in the ability to fight against infections.

Stability and transport of cervical cytobrushes for isolation of mononuclear cells from the female genital tract

Cervical cytobrushing, biopsy, or lavages have previously been used to collect mononuclear cells from the female genital tract. Compared with blood, obtaining cells from the female genital tract is more invasive and generally yields few cells for subsequent immune studies. Because of the value of including mucosal sampling in HIV vaccine trials, standardisation of methods for collection, processing, and analysis of immunity from cells derived from the female genital tract is important. The aim of this study was to assess the effect of transport conditions on the viability, recovery and antigenic responsiveness of cervical T cells. This was investigated in cervical cytobrush specimens collected from 215 chronically HIV-infected women. Cytobrushes were either processed immediately, after cryopreservation, or after 24h at 37°C, 4°C or room temperature. CD3+ T cell numbers were quantified using Guava automated cell counting. Viability was assessed using Trypan and Annexin/PI staining. Intracellular cytokine staining was used to evaluate IFN-γ responses to PMA, PHA and CEF peptides in cytobrush-derived T cells ex vivo and after delayed processing. In vitro polyclonal expansion of thawed cervical lymphocytes was conducted for 14days in the presence of anti-CD3 and IL-2. We found that CD3+ T cell recovery and viability was similar in cytobrushes processed immediately or after 24h irrespective of the conditions at which they were maintained. Fifty percent of the CD3+ T cells could be recovered after cryopreservation of cytobrushes and these could be polyclonally expanded in half of the cryopreserved samples. IFN-γ production following mitogenic stimulation was similar in ex vivo and delayed processing cytobrushes. Maintaining cytobrushes at 37°C prior to processing significantly improved the detection of CEF-specific T cell responses compared to ex vivo. We conclude that cervical cytobrush-derived T cells are robust and can preserve their viability, phenotype and function over 24h of mock transport.

Methylation of HPV16 genome CpG sites is associated with cervix precancer and cancer

Objective.Invasive cervix cancer (ICC) is the second most common malignant tumor in women. Human papillomavirus 16 (HPV16) causes more than 50% of all ICC and is a major cause of cervix intraepithelial neoplasia (CIN). DNA methylation is a covalent modification predominantly occurring at CpG dinucleotides. Such epigenetic modifications are associated with changes in DNA–protein interactions and gene activation. This study examined the association of viral and host genomic methylation patterns and cervix neoplasia. Methods.Exfoliated cervical lavage samples positive for HPV16 from women with and without cytomorphic changes of infection (n=46), CIN2 (n=12), and CIN3+ (n=27) were used to interrogate the methylation patterns of the HPV16 L1 gene and upstream regulatory region (URR), five host nuclear genes (TERT, RARB, DAPK1, MAL, and CADM1), and mitochondrial DNA (mtDNA). DNA isolated from exfoliated cervicovaginal cells was treated with bisulfite, specific regions of the viral and host genome were PCR amplified and CpG methylation was quantified using EpiTYPER and pyrosequencing. Results.Methylation at 14 of the tested CpG sites within the HPV16 L1 region were significantly higher in CIN3+ compared to HPV16 genomes from women without CIN3+. In contrast, 2/16 CpG sites in HPV16 URR, 5/5 in TERT, 1/4 in DAPK1 and 1/3 mtDNA, and 2/5 in RARB were associated with increased methylation in CIN3+. Conclusions.These results indicate that increased methylation of CpG sites in the HPV16 L1 ORF is associated with CIN3+ and, thus, may constitute a potential biomarker for precancerous and cancerous cervix disease.

Association between toll‐like receptor expression and human papillomavirus type 16 persistence

The mechanisms involved in mucosal immune control of cervical human papillomavirus (HPV) infection remain ill defined. Because toll-like receptors (TLRs) are key players in innate immune responses, we investigated the association between TLR expression and viral persistence or clearance in young women with incident infections with oncogenic HPV types 16 or 51. Messenger RNA expression of TLR1, TLR2, TLR3, TLR4, TLR6, TLR7, TLR8 and TLR9 was measured by quantitative reverse transcription-PCR using human endocervical specimens, collected before and after viral acquisition, in a cohort well characterized for HPV infections. Wilcoxon rank sum test was used to compare the change seen from preinfection to incident infection between women who subsequently cleared infection with those who did not. HPV 16 infections that cleared were significantly (p < 0.05) associated with an increase in expression of the four viral nucleic acid-sensing TLRs (TLR3, TLR7, TLR8 and TLR9) as well as TLR2 upon viral acquisition. Similar associations were not observed for HPV 51. In women who subsequently cleared their HPV 16 infection, changes in TLR1, TLR3, TLR7 and TLR8 expression levels between preincident and incident visits were significantly correlated with parallel changes in the levels of interferon-α2, measured by immunoassay in cervical lavage specimens. This study suggests that dampened TLR expression in the cervical mucosa is a type-specific mechanism by which HPV 16 interferes with innate immune responses, contributing to viral persistence, and that TLR upregulation and resultant cytokine induction is important in subsequent viral clearance.

Detection of cervical neoplasia by DNA methylation analysis in cervico-vaginal lavages, a feasibility study

ObjectiveTo explore the feasibility of DNA methylation analysis for the detection of cervical neoplasia in self-obtained cervico-vaginal lavages. MethodsLavages collected by a self-sampling device and paired cervical scrapings were obtained from 20 cervical cancer patients and 23 patients referred with an abnormal cervical smear (15 with high-grade cervical intraepithelial neoplasia (CIN2+) and 8 without CIN). All lavages and scrapings were analyzed by liquid based cytology (LBC), Hybrid Capture II (HC-II) for hr-HPV DNA detection and by DNA methylation analysis (JAM3, TERT, EPB41L3 and C13ORF18). Concordance between lavages and scrapings was measured by Cohen's Kappa (k). ResultsIn lavages and scrapings from cervical cancer patients (n=20), methylation analysis was positive in 19 (95%) and 19 (95%), HC-II in 16 (80%) and 15 (75%) and LBC in 15 (75%) and 19 (95%), respectively. In lavages and scrapings from CIN2+ patients (n=15), methylation analysis was positive in 10 (67%) and 12 (80%), HC-II in 15 (100%) and 15 (100%) and LBC in 11 (73%) and 12 (80%), respectively. Concordance between cervical scrapings and lavages (n=43) was for LBC k=0.522 (p<0.001), hr-HPV testing k=0.551 (p<0.001) and DNA methylation analysis k=0.653 (p<0.001). ConclusionsDNA methylation analysis in cervico-vaginal lavages obtained by a self-sampling device is feasible and its diagnostic performance appears to be at least comparable to the detection of cervical neoplasia by cytomorphology and hr-HPV. Our pilot study suggests that detection of cervical neoplasia by DNA methylation analysis in cervico-vaginal lavages warrants exploration of its use in large prospective studies.

The impact of highly active antiretroviral therapy on prevalence and incidence of cervical human papillomavirus infections in HIV-positive adolescents

BackgroundThe implementation of highly active antiretroviral therapy (HAART) among HIV-positive patients results in immune reconstitution, slower progression of HIV disease, and a decrease in the occurrence of opportunistic infections. However, the impact of HAART on cervical human papillomavirus (HPV) infection, clearance, and persistence in high-risk adolescents remains controversial.MethodsHIV-positive and high-risk HIV-negative female adolescents were enrolled in the Reaching for Excellence in Adolescent Care and Health (REACH) longitudinal cohort study. At each semi-annual clinical visit, cervical lavage samples were tested for 30 HPV types. Type-specific and carcinogenic risk-specific HPV prevalence and incidence were compared in 373 eligible participants: 146 HIV-negative female adolescents with a median follow-up of 721.5 [IQR: 483-1301] days and 227 HIV-positive female adolescents. Of the 227 HIV-positive participants, a fixed set (n = 100) were examined both before and after HAART initiation; 70 were examined only before HAART initiation; and 57 were examined only after HAART initiation, with overall median follow-up of 271 [IQR: 86.5-473] and 427.25 [IQR: 200-871] days respectively for before and after HAART initiation.ResultsOf the 373 eligible participants, 262 (70%) were infected with at least one type of HPV at baseline, and 78 of the remaining 111 (70%) became infected with at least one type of HPV by the end of the study. Overall, the incidence and prevalence of HPV types 58, 53/66, 68/70, and 31/33/35 were much higher than the established carcinogenic and HPV vaccine types 16 and 18, especially in HIV-positive females both before and after HAART initiation. Baseline prevalence for individual high-risk HPV types ranged, depending on type, from 0.7-10%, 1-17%, and 1-18% in the HIV-negative group, the HIV-positive before HAART initiation group, and the HIV-positive after HAART initiation group, respectively. Likewise, the incidence ranged, depending on HPV type, from 0.64-9.83 cases/100 PY, 3.00-12.80 cases/100 PY, and 1.49-17.05 cases/100 PY in the three groups, respectively. The patterns of each HPV type infection, clearance, and persistence did not differ considerably before or after the introduction of HAART and were clearly independent of CD4+ change within the short post-HAART follow-up period.ConclusionsHAART did not immediately affect the incidence of type-specific HPV infections within a short-period follow-up; however, future studies are warranted in larger populations to evaluate HAART's impact over longer periods.

Analysis of Multitag Pyrosequence Data from Human Cervical Lavage Samples

We have been using the Roche GS-FLX sequencing platform to produce tens of thousands of sequencing reads from samples of both bacterial communities (microbiome) and fungal communities (mycobiome) of stool, gut mucosa, vaginal washes, and oral washes from a large number of subjects. This vast volume of data from diverse sources has necessitated the development of an analysis pipeline in order to systematically and rapidly identify the taxa within the samples and to correlate the sample data with clinical and environmental features. Specifically, we have developed automated analytical tools for data tracking, taxonomical analysis, and feature clustering of bacteria in the human microbiome and demonstrate the pipeline using Cervical Vaginal Lavage (CVL) samples. This analysis pipeline will not only provide insight to our specific CVL dataset, but is applicable to other microbiome samples and will ultimately broaden our understanding of how the microbiome influences human health.