| 1. |
- Bradley, Frideborg
(författare)
-
Factors associated with HIV susceptibility in the female genital tract
- 2020
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The majority of new HIV infections in women are transmitted through vaginal intercourse where the female genital tract (FGT) functions as the portal of viral entry. The aim of this thesis was to characterize mucosal factors within the FGT to better understand potential molecular mechanisms associated with altered HIV-susceptibility. The vaginal microbiome and the menstrual cycle are associated with altered HIV-susceptibility, but their collective impact on the cervicovaginal milieu remains largely unknown. In Paper 1, we studied healthy, Swedish women and observed the largest changes of the genital proteome during the estradiol-dominated ovulatory phase. This phase was characterized by a decrease in neutrophil-associated proteins and pathways and an increase in epithelial barrier-promoting proteins, as compared to the progesterone-dominated luteal phase. Menstrual cycle-related changes in epithelial barrier proteins were enhanced in women with a non-Lactobacillus dominated vaginal microbiome. This study showed that female sex hormones modulated genital inflammation and epithelial barrier function and that these changes were further impacted by the vaginal microbiome. Semen can induce an inflammatory response in the FGT, but its role in HIVtransmission is largely unknown and limited due to a lack of adequate experimental models. In Paper 2, we used a genital tissue explant model to show that seminal plasma induced genital inflammation and increased HIV-infectivity, highlighting the importance of including seminal plasma as a factor in HIV-transmission studies. The genital tissue explant model used in this study could also be suitable for studying HIV-transmission and evaluation of microbicides. Studies in female sex workers (FSWs) at high risk of HIV-infection reveal alterations in the genital mucosal milieu. In Paper 3, we used a high-throughput bead-based affinity set-up to evaluate protein expression in genital secretions of another cohort at risk of HIV-infection, namely Kenyan HIV-seronegative women living in HIV-serodiscordant relationships. As compared to HIV-seronegative women in HIV-seroconcordant relationships, we found alterations in genital proteins involved in inflammation and epithelial barrier remodeling pathways. Such phenotype was observed despite low levels of clinical inflammation and high levels of safe sex practices in this cohort. These results suggest that women in HIV-serodiscordant relationships have a unique cervicovaginal environment, and that this may be associated with an altered susceptibility to HIV infection. However further studies would be required to fully elucidate the relationship between the observed phenotype and HIV infection risk. Observational and experimental studies indicate that use of the injectable progestin-based contraceptive depot medroxyprogesterone acetate (DMPA) is associated with increased risk of HIV. In Paper 4, we revealed a transcriptional profile consistent with impaired epithelial integrity and increased immune activation in ectocervical tissues from Kenyan FSWs using DMPA. In situ-based imaging analysis revealed a thinner superficial epithelial layer and an altered distribution of potential HIV-target cells. Collectively, these results suggest that DMPA may weaken the epithelial barrier and contribute to increased HIV-susceptibility. In summary, female sex hormones, living in a HIV-serodiscordant relationship and seminal factors induce changes in the FGT that may alter HIVsusceptibility. Endogenous and exogenous progesterone/progestins reduce epithelial barrier integrity and induce cervicovaginal inflammation. The knowledge gained from these studies can help guide the development of safe contraceptive methods. The aforementioned factors must be taken into consideration when designing and interpreting results from clinical studies in the HIV-prevention field, and can also help guide the development of prophylactic compounds aimed at reducing HIV-transmission.
|
|
| 2. |
- Bradley, Frideborg, et al.
(författare)
-
Multi-omics analysis of the cervical epithelial integrity of women using depot medroxyprogesterone acetate
- 2022
-
Ingår i: PLoS Pathogens. - : Public Library of Science (PLoS). - 1553-7366 .- 1553-7374. ; 18:5
-
Tidskriftsartikel (refereegranskat)abstract
- 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. Author summarySexual transmission accounts for the majority of all new HIV infections in women, and alterations to the mucosal environment of the female genital tract have been associated with an increase in the risk of acquiring HIV. Observational epidemiological studies have implied that the use of the injectable hormonal contraceptive depot medroxyprogesterone acetate (DMPA) may be associated with increased HIV-acquisition. However, a prospective clinical study has not confirmed this association and the controversial findings are currently evaluated in the context of international reproductive health policies. Several studies using various model systems indicate that DMPA affects the integrity of the genital epithelial barrier as well as the mucosal immune system, but the exact mechanisms remain largely unknown. To characterize the effect of DMPA on the genital mucosal environment, we used a multi-omics approach to assess paired genital secretions and cervical tissue samples from long-term regular DMPA users living in Kenya. This unique cohort represents a population at risk of HIV infection in which DMPA is one of the most commonly used hormonal contraceptives. We identified impaired cervical epithelial barrier structures, including DMPA-associated reduction in the expression of cell-cell adhesion molecules, keratins, small proline-rich proteins and a thinner upper epithelial layer with more superficially located CD4(+) cells. Gene set enrichment pathway analyses indicated DMPA use was associated with immune activation and suppression of epithelium development including keratinization and cornification pathways. Protein analysis identified altered levels of selected anti-proteases. Our findings illustrate mechanisms by which DMPA adversely may affect the integrity of the genital mucosa.
|
|
| 3. |
- Edfeldt, Gabriella, et al.
(författare)
-
Distinct cervical tissue-adherent and luminal microbiome communities correlate with mucosal host gene expression and protein levels in Kenyan sex workers
- 2023
-
Ingår i: Microbiome. - : Springer Nature. - 2049-2618. ; 11:1
-
Tidskriftsartikel (refereegranskat)abstract
- Background The majority of studies characterizing female genital tract microbiota have focused on luminal organisms, while the presence and impact of tissue-adherent ectocervical microbiota remain incompletely understood. Studies of luminal and tissue-associated bacteria in the gastrointestinal tract suggest that these communities may have distinct roles in health and disease. Here, we performed a multi-omics characterization of paired luminal and tissue samples collected from a cohort of Kenyan female sex workers.Results We identified a tissue-adherent bacterial microbiome, with a higher alpha diversity than the luminal microbiome, in which dominant genera overall included Gardnerella and Lactobacillus, followed by Prevotella, Atopobium, and Sneathia. About half of the L. iners-dominated luminal samples had a corresponding Gardnerella-dominated tissue microbiome. Broadly, the tissue-adherent microbiome was associated with fewer differentially expressed host genes than the luminal microbiome. Gene set enrichment analysis revealed that L. crispatus-dominated tissue-adherent communities were associated with protein translation and antimicrobial activity, whereas a highly diverse microbial community was associated with epithelial remodeling and pro-inflammatory pathways. Tissue-adherent communities dominated by L. iners and Gardnerella were associated with lower host transcriptional activity. Tissue-adherent microbiomes dominated by Lactobacillus and Gardnerella correlated with host protein profiles associated with epithelial barrier stability, although with a more pro-inflammatory profile for the Gardnerella-dominated microbiome group. Tissue samples with a highly diverse composition had a protein profile representing cell proliferation and pro-inflammatory activity.Conclusion We identified ectocervical tissue-adherent bacterial communities in all study participants of a female sex worker cohort. These communities were distinct from cervicovaginal luminal microbiota in a significant proportion of individuals. We further revealed that bacterial communities at both sites correlated with distinct host gene expression and protein levels. The tissue-adherent bacterial community could possibly act as a reservoir that seed the lumen with less optimal, non-Lactobacillus, bacteria.
|
|
| 4. |
- Edfeldt, Gabriella, et al.
(författare)
-
Regular Use of Depot Medroxyprogesterone Acetate Causes Thinning of the Superficial Lining and Apical Distribution of Human Immunodeficiency Virus Target Cells in the Human Ectocervix
- 2022
-
Ingår i: Journal of Infectious Diseases. - : Oxford University Press. - 0022-1899 .- 1537-6613. ; 225:7, s. 1151-1161
-
Tidskriftsartikel (refereegranskat)abstract
- BackgroundThe hormonal contraceptive depot medroxyprogesterone acetate (DMPA) may be associated with an increased risk of acquiring human immunodeficiency virus (HIV). We hypothesize that DMPA use influences the ectocervical tissue architecture and HIV target cell localization.MethodsQuantitative image analysis workflows were developed to assess ectocervical tissue samples collected from DMPA users and control subjects not using hormonal contraception.ResultsCompared to controls, the DMPA group exhibited a significantly thinner apical ectocervical epithelial layer and a higher proportion of CD4+CCR5+ cells with a more superficial location. This localization corresponded to an area with a nonintact E-cadherin net structure. CD4+Langerin+ cells were also more superficially located in the DMPA group, although fewer in number compared to the controls. Natural plasma progesterone levels did not correlate with any of these parameters, whereas estradiol levels were positively correlated with E-cadherin expression and a more basal location for HIV target cells of the control group.ConclusionsDMPA users have a less robust epithelial layer and a more apical distribution of HIV target cells in the human ectocervix, which could confer a higher risk of HIV infection. Our results highlight the importance of assessing intact genital tissue samples to gain insights into HIV susceptibility factors.
|
|
| 5. |
- Häggmark, Anna, 1985-, et al.
(författare)
-
A High-throughput Bead-based Affinity Assay Enables Analysis of Genital Protein Signatures in Women At Risk of HIV Infection
- 2019
-
Ingår i: Molecular & Cellular Proteomics. - : AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC. - 1535-9476 .- 1535-9484. ; 18:3, s. 461-476
-
Tidskriftsartikel (refereegranskat)abstract
- Women at high risk of HIV infection, including sex workers and those with active genital inflammation, have molecular signatures of immune activation and epithelial barrier remodeling in samples of their genital mucosa. These alterations in the local immunological milieu are likely to impact HIV susceptibility. We here analyze host genital protein signatures in HIV uninfected women, with high frequency of condom use, living in HIV-serodiscordant relationships. Cervicovaginal secretions from women living in HIV-serodiscordant relationships (n = 62) were collected at three time points over 12 months. Women living in HIV-negative seroconcordant relationships (controls, n = 25) were sampled at one time point. All study subjects were examined for demographic parameters associated with susceptibility to HIV infection. The cervicovaginal samples were analyzed using a high-throughput bead-based affinity assay. Proteins involved in epithelial barrier function and inflammation were increased in HIV-serodiscordant women. By combining several methods of analysis, a total of five proteins (CAPG, KLK10, SPRR3, elafin/PI3, CSTB) were consistently associated with this study group. Proteins analyzed using the affinity set-up were further validated by label-free tandem mass spectrometry in a partially overlapping cohort with concordant results. Women living in HIV-serodiscordant relationships thus had elevated levels of proteins involved in epithelial barrier function and inflammation despite low prevalence of sexually transmitted infections and a high frequency of safe sex practices. The identified proteins are important markers to follow during assessment of mucosal HIV susceptibility factors and a high-throughput bead-based affinity set-up could be a suitable method for such evaluation.
|
|
| 6. |
- Rohl, Maria, et al.
(författare)
-
HIV-Exposed Seronegative Sex Workers Express Low T-Cell Activation and an Intact Ectocervical Tissue Microenvironment
- 2021
-
Ingår i: Vaccines. - : MDPI AG. - 2076-393X. ; 9:3
-
Tidskriftsartikel (refereegranskat)abstract
- 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(+)CD25(hi)FoxP3(+)CD127(lo)CD4(+) 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.
|
|
| 7. |
- Winberg, Johanna, et al.
(författare)
-
Mutation Screening and Array Comparative Genomic Hybridization Using a 180K Oligonucleotide Array in VACTERL Association
- 2014
-
Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 9:1, s. e85313-
-
Tidskriftsartikel (refereegranskat)abstract
- In order to identify genetic causes of VACTERL association (V vertebral defects, A anorectal malformations, C cardiac defects, T tracheoesofageal fistula, E esophageal atresia, R renal anomalies, L limb deformities), we have collected DNA samples from 20 patients diagnosed with VACTERL or with a VACTERL-like phenotype as well as samples from 19 aborted fetal cases with VACTERL. To investigate the importance of gene dose alterations in the genetic etiology of VACTERL association we have performed a systematic analysis of this cohort using a 180K array comparative genomic hybridization (array-CGH) platform. In addition, to further clarify the significance of PCSK5, HOXD13 and CHD7 genes in the VACTERL phenotype, mutation screening has been performed. We identified pathogenic gene dose imbalances in two fetal cases; a hemizygous deletion of the FANCB gene and a (9;18)(p24;q12) unbalanced translocation. In addition, one pathogenic mutation in CHD7 was detected, while no apparent disease-causing mutations were found in HOXD13 or PCSK5. Our study shows that although large gene dose alterations do not seem to be a common cause in VACTERL association, array-CGH is still important in clinical diagnostics to identify disease cause in individual cases.
|
|
