Modeling an ascending infection by Ureaplasma parvum and its cell signaling and inflammatory response at the feto-maternal interface.

PROBLEM: Ascending bacterial infection is associated with ∼ 40% of spontaneous preterm birth (PTB), and Ureaplasma spp. is one of the most common bacteria isolated from the amniotic fluid. Developing novel in vitro models that mimic in vivo uterine physiology is essential to study microbial pathogenesis. We utilized the feto-maternal interface organ-on-chip (FMi-OOC) device and determined the propagation of Ureaplasma parvum, and its impact on cell signaling and inflammation. METHOD OF STUDY: FMi-OOC is a microphysiologic device mimicking fetal membrane/decidua interconnected through microchannels. The impact of resident decidual CD45+ leukocytes was also determined by incorporating them into the decidual chamber in different combinations with U. parvum. We tested the propagation of live U. parvum from the decidual to the amniochorion membranes (immunocytochemistry and quantitative PCR), determined its impact on cytotoxicity (LDH assay), cell signaling (JESSTM Western Blot), cellular transition (immunostaining for vimentin and cytokeratin), and inflammation (cytokine bead array). RESULTS: U. parvum transversed the chorion and reached the amnion epithelium after 72 hours but did not induce cell signaling kinases (p38MAPK and JNK) activation, or cellular transition (epithelial-mesenchymal), regardless of the presence of immune cells. The inflammatory response was limited to the choriodecidual interface and did not promote inflammation in the amnion layer. CONCLUSIONS: Our data suggest that U. parvum is poorly immunogenic and does not produce massive inflammatory changes at the feto-maternal interface. We speculate that the presence of U. parvum may still compromise the feto-maternal interface making it susceptible to other pathogenic infection.

Cervicovaginal Gardnerella sialidase-encoding gene in persistent human papillomavirus infection.

Disturbed vaginal microbiota have a role in the persistence of high-oncogenic-risk human papillomavirus (hrHPV) and Gardnerella spp. is closely related with this condition. Such bacteria are the major source of cervicovaginal sialidases, important for microbiota alterations. The sialidase-encoding gene nanH3 is account for their sialidase activity. Thus, a subset of 212 women positive for hrHPV at the first visit were included in the analysis of the current study aiming to compare the loads of nanH3 in cervicovaginal fluid (CFV) of women with persistent hrHPV infection and with those cleared the infection after a year. Participants were assigned to two study groups named "persistence" (n = 124, 53.22%) or "clearance" (n = 88, 37.77%), according to the HPV status upon enrollment and follow-up. Absolute quantification of nanH3 gene was performed using quantitative real-time PCR (qPCR). Persistence and clearance group did not show statistical difference in the load of nanH3 gene (p = 0.19). When considering the subset of women with HPV16, differences in number of copies of nanh3 gene was observed between the persistent (7.39E+08 copies/µL) and clearance group (2.85E+07 copies/µL) (p = 0.007). Therefore, baseline loads of nanH3 gene is increased in women that persist with cervical HPV16 infection after 12 months.

Vitamin D Receptor and Retinoid X Receptor Alpha in Melanocytic Benign Lesions and Melanoma.

ABSTRACT: Vitamin D receptor (VDR) exerts its biological effects when it heterodimerizes to a nuclear receptor of the retinoid family called retinoid X receptor α (RXRα), stimulating or inhibiting DNA transcription. VDR stimulation by vitamin D analogs led to in vitro antiproliferative effects, and experimental RXRα knockout led to loss of proliferation control in melanoma cells. The aim of this study was to determine VDR and RXRα positivity in melanocytic lesions, compared with normal skin species. By immunohistochemistry assays, nuclear VDR, cytoplasmic VDR, and RXRα and RXRα in keratinocytes surrounding melanocytes were evaluated in 77 controls, 92 intradermal nevi, 54 dysplastic nevi, and 83 melanomas in this retrospective cross-sectional study. Nuclear VDR, cytoplasmic VDR, and RXRα were less expressed in exposed areas ( P < 0.001, P = 0.0006, and P < 0.001, respectively) than covered areas. All melanocytic lesions had loss of VDR and RXRα comparing with the control group. In the melanoma group, nuclear VDR tended to inversely correlate with the Breslow index (r = -0.11, P = 0.29) but directly correlated with histological regression ( P = 0.0293). RXRα inversely correlated with mitosis (r = -0.245; P = 0.0263). We can suggest that sun exposure affected VDR and RXRα immunopositivity. Nuclear VDR tendency of inverse correlation with the Breslow index showed that worse melanomas have a greater loss of VDR. RXRα inversely correlated with mitosis, indicating that RXRα can have a role in proliferation control. VDR and RXRα may participate in the development of melanocytic lesions and be a future target of new studies and directed therapies.

Factors associated with Trichomonas vaginalis infection in reproductive-aged women attending cervical screening in southeast of Brazil.

BACKGROUND: Sexually Transmitted Infections (STIs) can be caused by viruses, bacteria, and parasites. The World Health Organization estimated more than 300 million new global cases of curable STIs among individuals of reproductive age. Infection by Trichomonas vaginalis is one of the most prevalent curable STI. Despite the current treatments available, the diagnosis of T. vaginalis can be difficult, and the resistance to the treatment increased concern for the healthcare system. OBJECTIVES: The aim of this study was to determine the prevalence and factors associated with Trichomonas vaginalis infection among women of reproductive age attending community-based services for cervical screening. PATIENTS AND METHODS: A total of 1477 reproductive-aged women attending 18 Primary Health Care Units in Botucatu, Brazil, from September to October 2012, were enrolled. A structured questionnaire was used for individual face-to-face interviews for obtaining data on sociodemographic, gynecologic, and obstetrics history, sexual and hygiene practices, among others. Cervicovaginal samples were obtained for detection of T. vaginalis by culture using Diamond's medium and microscopic vaginal microbiota classification according to Nugent. A multivariable logistic regression analysis was carried out to estimate Odds Ratios (OR) and 95% Confidence Intervals (95% CI) for the association between participants' sociodemographic, behavioral factors, and clinical factors with T. vaginalis infection. RESULTS: Median age of study participants was 33 years (ranging from 18 to 50). The overall prevalence of T. vaginalis infection was 1.3% (n = 20). Several factors were independently associated with T. vaginalis infection, such as self-reporting as black or Pardo for ethnicity (OR = 2.70; 95% CI 1.03‒7.08), smoking (OR=3.18; 95% CI 1.23‒8.24) and having bacterial vaginosis (OR = 4.01; 95%CI = 1.55-10.38) upon enrollment. A protective effect of higher educational level (having high school degree) was observed (OR = 0.16; 95% CI 0.05‒0.53). CONCLUSIONS: Our data suggest that screening programs to correctly detect T. vaginalis infection can be helpful to guide prevention strategies to the community. Our study supports an association between abnormal vaginal microbiota and T. vaginalis infection.

Condyloma acuminata: An evaluation of the immune response at cellular and molecular levels.

Condyloma acuminata (CA) is a benign proliferative disease mainly affecting in non-keratinized epithelia. Most cases of CA are caused by low-risk human papillomavirus (HPV), mainly HPV 6 and 11. The aim of the current study was to highlight the candidate genes and pathways associated with immune alterations in individuals who did not spontaneously eliminate the virus and, thus, develop genital warts. Paraffin-embedded condyloma samples (n = 56) were analyzed by immunohistochemistry using antibodies against CD1a, FOXP3, CD3, CD4, CD8, and IFN-γ. The immunomarkers were chosen based on the evaluation of the innate and adaptive immune pathways using qPCR analysis of 92 immune-related genes, applying a TaqMan Array Immune Response assay in HPV 6 or HPV 11 positive samples (n = 27). Gene expression analysis revealed 31 differentially expressed genes in CA lesions. Gene expression validation revealed upregulation of GZMB, IFNG, IL12B, and IL8 and downregulation of NFATC4 and IL7 in CA samples. Immunohistochemical analysis showed increased FOXP3, IFN-γ, CD1a, and CD4 expression in CA than in the control tissue samples. In contrast, CD3 and CD8 expression was decreased in CA lesion samples. Increased levels of pro-inflammatory cytokines in HPV-positive patients compared with HPV-negative patients seem to reflect the elevated immunogenicity of HPV-positive CA lesions. Host defense against HPV begins during the early stages of the innate immune response and is followed by activation of T lymphocytes, which are mainly represented by CD4+ and regulatory T cells. The low CD8+ T cell count in CA may contribute to this recurrent behavior. Additional studies are needed to elucidate the mechanism of host defense against HPV infection in CA.

Factors associated with bacterial vaginosis in women with homosexual, bisexual and heterosexual practices.

BACKGROUND: The factors associated with bacterial vaginosis in women with homosexual, bisexual and heterosexual practices are still poorly explored. Thus, the aim of this study was to analyze the factors associated with bacterial vaginosis in women with different sexual practices. METHODS: Cross-sectional study that included 453 women, 149 Women with Homosexual practice (WSW); 80 bisexual Women (WSWM) and 224 Women with heterosexual practice (WSM). The diagnosis of bacterial vaginosis was performed by microscopic examination of the vaginal smears stained by Gram method and classified according to the Nugent et al. (1991) score. Data analysis was performed by Cox multiple regression. RESULTS: Bacterial vaginosis was associated to years of education among WSW (0.91 [95% CI 0.82‒0.99]; p = 0.048) and non-white skin color (2.34 [95% CI 1.05‒5.19]; p = 0.037) between WSWM. Changing partners in the last 3-months (2.09 [95% CI 1.14‒3.82]; p = 0.017), inconsistent use of condoms (2.61 [95% CI 1.10‒6.20]; p = 0.030) and positive diagnosis of Chlamydia trachomatis (2.40 [95% CI 1.01‒5.73]; p = 0.048) were associated with bacterial vaginoses only in WSH. CONCLUSIONS: The factors associated to bacterial vaginosis differ between different sexual practices, suggesting that the type of sexual partner may influence the risk of developing this classic dysbiosis.

Cervicovaginal loads of Gardnerella spp. are increased in immunocompetent women with persistent high-risk human papillomavirus infection.

Introduction. Two high-oncogenic-risk human papilomavirus (hrHPV) genotypes - HPV16 and HPV18 - cause most of the cases of cervical cancer worldwide. Bacterial vaginosis is associated with increased hrHPV persistence, although the mechanism underlying this association remains unclear. Gardnerella spp. are detected in nearly all cases of bacterial vaginosis and are the major source of cervicovaginal sialidases. The NanH1 gene is present in virtually all Gardnerella sialidase-producing strains and has been proposed as a potential marker for persistent hrHPV infection.Hypothesis. Gardnerella spp. load and the NanH1 gene are associated with hrHPV persistence.Aim. To compare the cervicovaginal load of Gardnerella spp. and the frequency of the NanH1 gene between women with persistent HPV16 and/or HPV18 infection and those who cleared the infection after 11 months.Methodology. Among a population of 1638 HPV screened, we detected 104 with positive HPV16 and/or HPV18 results. Samples were obtained at two time points (baseline and at a median of 11 months at follow-up) and tested using the Linear Array HPV Genotyping kit (Roche Molecular Systems, Pleasanton, CA, USA). Based on their HPV16/HPV18 status at enrolment and follow-up, participants were assigned to 'persistence' or 'clearance' groups. We used cervicovaginal fluid samples obtained upon enrolment to determine the load of the 23 s rRNA gene of Gardnerella spp. and the presence of the NanH1 gene using real-time polymerase chain reaction (PCR). We compared Gardnerella spp. loads and NanH1 frequency between the groups by, respectively, Mann-Whitney and chi-squared tests, with a P-value <0.05 considered to be significant.Results. Of the 104 participants who were positive for HPV16/HPV18, 73 (70.2 %) persisted with at least 1 of the baseline genotypes at follow-up, while 31 (29.8 %) cleared the infection in this time frame. Participants in the persistence group had significantly higher loads of Gardnerella spp. [5.8E+02 (0-3.0E+05) copies µl-1] than those in the clearance group [9.9E+01 (0-7.7E+04) copies µl-1] (P=0.03). The baseline frequency of NanH1 was higher in the persistence' (n=46, 63.0 %) than in the clearance (n=14, 45.2 %) group, although this was not statistically significant (P=0.09).Conclusion. These findings reinforce the negative effect of vaginal microbiota for the clearance of hrHPV and indicate a possible association between sialidase-producing species with hrHPV persistence.

In vitro effect of triamcinolone and platelet-rich plasma on cytokine levels of elbow lateral epicondylitis-derived cells.

BACKGROUND: The pathogenesis and treatment of lateral elbow epicondylitis (LEE) are still controversial. The purpose of the current study was to evaluate the production of inflammatory cytokines by LEE-derived cells and to compare the anti-inflammatory effect of triamcinolone acetonide with platelet-rich plasma (PRP) on cytokines production in primary culture of these cells. METHODS: Third passage cells from primary cultures of LEE were assessed for the production of the cytokines IL-1ß, IL-6, IL-8, IL-10 and TNF-α by immune-enzymatic assay (ELISA), after the treatment with 1, 10 and 100 µM triamcinolone compared to no treated controls at the time points 6, 12, 18, 24, 48, 72 and 96 h, and to PRP at 48, 72 and 96 h. RESULTS: The cytokines IL-6 and IL-8 were produced in high concentrations by LEE cells. One, 10 and 100 µM triamcinolone induced significant decrease in the production of IL-6 and IL-8 at 48, 72 and 96 h, adding the time point 12 h for IL-8. Compared to controls, PRP caused a significant increase in the production of IL-6 and IL-8 and there was a significant increase in IL-10 production with the use of 100 µM triamcinolone at 48 h. The production of IL1-ß and TNF-α was very low and did not change when the cultures were treated with triamcinolone or PRP. CONCLUSION: LEE-derived cells produce IL-6 and IL-8, confirming the inflammatory nature of this condition. While triamcinolone inhibited the production of IL-6 and IL-8 by LEE cells, PRP induced an increase in these cytokines compared with controls.

Vaginal Microbiome Components as Correlates of Cervical Human Papillomavirus Infection.

BACKGROUND: Interplay between vaginal microbiome and human papillomavirus (HPV) remains unclear, partly due to heterogeneity of microbiota. METHODS: We used data from 546 women enrolled in a cross-sectional study in 5 Brazil. We genotyped vaginal samples for HPV and sequenced V3-V4 region of 16S rRNA gene for vaginal microbiome analysis. We used stepwise logistic regression to construct 2 linear scores to predict high-risk HPV (hrHPV) positivity: one based exclusively on presence of individual bacterial taxa (microbiome-based [MB] score) and the other exclusively on participants' sociodemographic, behavioral, and clinical (SBC) characteristics. MB score combined coefficients of 30 (of 116) species. SBC score retained 6 of 25 candidate variables. We constructed receiver operating characteristic curves for scores as hrHPV correlates and compared areas under the curve (AUC) and 95% confidence intervals (CI). RESULTS: Overall, prevalence of hrHPV was 15.8%, and 26.2% had a Lactobacillus-depleted microbiome. AUCs were 0.8022 (95% CI, .7517-.8527) for MB score and 0.7027 (95% CI, .6419-.7636) for SBC score (P = .0163). CONCLUSIONS: The proposed MB score is strongly correlated with hrHPV positivity-exceeding the predictive value of behavioral variables-suggesting its potential as an indicator of infection and possible value for clinical risk stratification.

The role of nuclear factor erythroid 2-related factor 2 (NRF2) in normal and pathological pregnancy: A systematic review.

OBJECTIVE: A homeostatic balance between reactive oxygen species production and the antioxidant redox system is an important component of normal pregnancy. Nuclear Factor Erythroid 2-Related Factor 2 (Nrf2) preserves cellular homeostasis by enhancing the cell's innate antioxidant status to reduce oxidative stress and inflammatory damage to the cell during pregnancy. Active Nrf2, in the nucleus of the cell, transactivates various antioxidant genes. The objective of this systematic review was to synthesize evidence on the role of Nrf2 in various adverse pregnancy outcomes (APOs). METHODS: We conducted a systematic review of the role of Nrf2 in pregnancy. Articles written in English, Portuguese, and Spanish were obtained from three different databases from inception until January 2021. The titles, abstracts and full text were reviewed independently by six reviewers. The quality of the included studies was assessed using a quality assessment tool developed to assess basic science and clinical studies. Nrf2 expression (gene and protein), functional contributions, and association with APOs were assessed. RESULTS: A total of 747 citations were identified; 80 were retained for full review. Most studies on Nrf2 have been carried out using placental tissues and placenta-derived cells. Limited studies have been conducted using fetal membranes, uterus, and cervix. Nuclear translocation of Nrf2 results in transactivation of antioxidant enzymes, including glutathione peroxidase, hemeoxygenase-1, and superoxide dismutase in gestational cells during pregnancy. This antioxidant response maintains cellular homeostasis during pregnancy. This promotes trophoblast cell survival and prevents cell death and abnormal angiogenesis in the placenta. Excessive and insufficient Nrf2 response may promote oxidative and reductive stress, respectively. This Nrf2 dysregulation has been associated with APOs including gestational diabetes mellitus, intrauterine growth restriction, reproductive toxicity, preeclampsia, and preterm birth. CONCLUSION: Several studies have localized and reported an association between Nrf2's differential expression in reproductive tissues and the pathogenesis of APOs. However, a comprehensive functional understanding of Nrf2 in reproductive tissues is still lacking. Nrf2's activation and functions are complex, and therefore, current in vitro and in vivo studies are limited in their experimental approaches. We have identified key areas for future Nrf2 research that is needed to fill knowledge gaps.

Sialidase Activity in the Cervicovaginal Fluid Is Associated With Changes in Bacterial Components of Lactobacillus-Deprived Microbiota.

INTRODUCTION: Sialidase activity in the cervicovaginal fluid (CVF) is associated with microscopic findings of bacterial vaginosis (BV). Sequencing of bacterial 16S rRNA gene in vaginal samples has revealed that the majority of microscopic BV cases fit into vaginal community-state type IV (CST IV), which was recently named "molecular-BV." Bacterial vaginosis-associated bacterial species, such as Gardnerella spp., may act as sources of CVF sialidases. These hydrolases lead to impairment of local immunity and enable bacterial adhesion to epithelial and biofilm formation. However, the impact of CVL sialidase on microbiota components and diversity remains unknown. OBJECTIVE: To assess if CVF sialidase activity is associated with changes in bacterial components of CST IV. METHODS: One hundred forty women were cross-sectionally enrolled. The presence of molecular-BV (CST IV) was assessed by V3-V4 16S rRNA sequencing (Illumina). Fluorometric assays were performed using 2-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid (MUAN) for measuring sialidase activity in CVF samples. Linear discriminant analysis effect size (LEfSe) was performed to identify the differently enriched bacterial taxa in molecular-BV according to the status of CVF sialidase activity. RESULTS: Forty-four participants (31.4%) had molecular-BV, of which 30 (68.2%) had sialidase activity at detectable levels. A total of 24 bacterial taxa were enriched in the presence of sialidase activity, while just two taxa were enriched in sialidase-negative samples. CONCLUSION: Sialidase activity in molecular-BV is associated with changes in bacterial components of the local microbiome. This association should be further investigated, since it may result in diminished local defenses against pathogens.

Telomere-Related Disorders in Fetal Membranes Associated With Birth and Adverse Pregnancy Outcomes.

Telomere disorders have been associated with aging-related diseases, including diabetes, vascular, and neurodegenerative diseases. The main consequence of altered telomere is the induction of the state of irreversible cell cycle arrest. Though several mechanisms responsible for the activation of senescence have been identified, it is still unclear how a cell is indeed induced to become irreversibly arrested. Most tissues in the body will experience senescence throughout its lifespan, but intrinsic and extrinsic stressors, such as chemicals, pollution, oxidative stress (OS), and inflammation accelerate the process. Pregnancy is a state of OS, as the higher metabolic demand of the growing fetus results in increased reactive oxygen species production. As a temporary organ in the mother, senescence in fetal membranes and placenta is expected and linked to term parturition (>37 weeks of gestation). However, a persistent, overwhelming, or premature OS affects placental antioxidant capacity, with consequent accumulation of OS causing damage to lipids, proteins, and DNA in the placental tissues. Therefore, senescence and its main inducer, telomere length (TL) reduction, have been associated with pregnancy complications, including stillbirth, preeclampsia, intrauterine growth restriction, and prematurity. Fetal membranes have a notable role in preterm births, which continue to be a major health issue associated with increased risk of neo and perinatal adverse outcomes and/or predisposition to disease in later life; however, the ability to mediate a delay in parturition during such cases is limited, because the pathophysiology of preterm births and physiological mechanisms of term births are not yet fully elucidated. Here, we review the current knowledge regarding the regulation of telomere-related senescence mechanisms in fetal membranes, highlighting the role of inflammation, methylation, and telomerase activity. Moreover, we present the evidences of TL reduction and senescence in gestational tissues by the time of term parturition. In conclusion, we verified that telomere regulation in fetal membranes requires a more complete understanding, in order to support the development of successful effective interventions of the molecular mechanisms that triggers parturition, including telomere signals, which may vary throughout placental tissues.

Inflammation, but not infection, induces EMT in human amnion epithelial cells.

A non-reversible state of epithelial to mesenchymal transition (EMT) at term accumulates proinflammatory mesenchymal cells and predisposes fetal membrane to weakening prior to delivery at term. We investigated the induction of EMT in amnion epithelial cells (AEC) in response to inflammation and infection associated with spontaneous preterm birth (SPTB). For this, membranes from SPTB were screened for EMT markers. Primary AEC in culture were treated with TNF-α (10 and 50 ng/mL) and LPS (50 and 100 ng/mL) for 72 h. Cell shape index (SI) was determined based on morphological shift (microscopy followed by ImageJ software analysis). Immunocytochemistry and Western blot assessed changes in epithelial markers (cytokeratin-18 and E-cadherin) and mesenchymal markers (vimentin and N-cadherin). Involvement of transforming growth factor beta (TGF-ß) in EMT induction and EMT associated inflammation was tested using specific markers (Western blot) and by measuring MMP9 (ELISA), respectively. We report that PTB is associated with fetal membrane EMT. TNF-α produced dose- and time-dependent induction of EMT; within 24 h by 50 ng/mL and after 72 h by 10 ng/mL. AEC showed mesenchymal morphology, lower E-cadherin, higher vimentin and N-cadherin and higher MMP9 compared to control. TNF-α-induced EMT was not associated with canonical TGF-ß pathway. LPS, regardless of dose or time, did not induce EMT in AEC. We conclude that PTB with intact membranes is associated with EMT. Our data suggest that inflammation, but not infection, is associated with non-canonical activation of EMT and inflammation that can predispose membrane to undergo weakening.

Profile of pro-inflammatory cytokines in colostrum of nursing mothers at the extremes of reproductive age.

Gestations at the extremes of reproductive age are characterized as high-risk pregnancies, conditions that might influence colostrum composition. This first milk secretion contains nutrients necessary for the development and immunity of the newborn; therefore, this study aims to compare adolescent and advanced maternal age mothers regarding sociodemographic, gestational, and perinatal characteristics and the colostrum levels of pro-inflammatory cytokines in these groups of study. This cross-sectional study has compared sociodemographic, gestational and perinatal data from adolescent mothers (between 10 and 24 years old) (n = 117), advanced maternal age mothers (over 35 years of age) (n = 39) and mothers considered a control group (25 to 34 years old) (n = 58). Additionally, colostrum samples were obtained from the studied and control group subjects by manual milking, between 48 and 72 hours postpartum, and the samples were analyzed for cytokine concentrations by enzyme-linked immunosorbent assay (ELISA). The majority of the studied mothers reported living a stable union, and 81.2% of the adolescent mothers did not carry out any paid activity. Mothers with advanced maternal age mainly delivered by cesarean section and presented a higher body mass index (BMI). Neonatal weight and Apgar score were not different between the groups. The concentrations of interleukin (IL)-1ß and IL-6 were higher in the colostrum of mothers with advanced age compared to adolescent mothers, but did not differ from the control group. The concentrations of IL-8 and tumor necrosis factor alpha did not differ between the three groups. Therefore, our data demonstrated that maternal age influenced the sociodemographic and gestational characteristics as well as the composition of colostrum cytokines.

Telomere Length and Telomerase Activity in Foetal Membranes from Term and Spontaneous Preterm Births.

The reduction of telomere length, the protective cap structures of chromosomes, is one of the biomarkers of senescence (a mechanism of ageing), and ageing of foetal gestational tissues is associated with both term and preterm parturition. A mechanism regulating telomere length is the activity of telomerase, an enzyme that adds telomere fragments during DNA replication and cell division; however, its role in regulating telomere length is not well studied in gestational tissues. The objective of this study is to correlate telomere length and telomerase activity in foetal membranes from term and spontaneous preterm births. Foetal membrane samples were collected from pregnant women experiencing term labour (TL), term not in labour (TNL), preterm premature rupture of membranes (pPROM) and spontaneous preterm labour (PTL) with intact membranes (n = 20/group). Telomere length and telomerase activity were analyzed by relative quantification (T/S), real-time PCR and PCR-based fluorometric detection, respectively. Data were analyzed by ANOVA or the Kruskal-Wallis test. Demographic variables were not statistically different among the groups. Foetal membranes from the TL group showed telomere length reduction compared with those from the others (p < 0.0002). Telomerase activity did not change in foetal membranes irrespective of pregnancy outcome. Telomere shortening in foetal membranes is suggestive of senescence associated with triggering of labour at term; however, this is likely independent of telomerase activity, while prematurity may be associated with senescence, but due to other mechanisms than telomere length reduction in foetal membranes.

Reduction in the inflammatory markers CD4, IL-1, IL-6 and TNFα in dogs with keratoconjunctivitis sicca treated topically with mesenchymal stem cells.

Keratoconjunctivitis sicca (KCS) is of predominantly immune-mediated origin. Dogs are an excellent model for understanding this disease, as the origin of KCS in dogs is like that in humans. The objective of this study was to localize and quantify immunological markers, such as CD4 lymphocytes, interleukin (IL)-1, IL-6 and tumor necrosis factor alpha (TNFα), before and after topical treatment with mesenchymal stem cells (MSCs). Twenty-two dogs positive for KCS were topically treated with 50 µL (1 × 106 MSCs) in the conjunctival sac and were evaluated for 6 months. The levels of the markers CD4, IL-6, IL-1 and TNFα were analyzed in conjunctival biopsy and cytology of the third eyelid gland by immunohistochemistry and immunocytochemistry. The results showed that before treatment, there was marked expression of all the markers (CD4, IL-6, IL-1 and TNFα), and after 6 months, there were significant (p < .05) reductions in the expression levels of all the markers. These results demonstrated that topical MSC treatment promotes a significant decrease in the expression levels of these inflammatory markers and could be used as adjuvant therapy in the treatment of KCS in dogs and humans. In addition, these markers can be excellent tools for diagnosing and analyzing the progression of KCS.

Dexamethasone induces primary amnion epithelial cell senescence through telomere-P21 associated pathway†.

Dexamethasone (Dex), a corticosteroid hormone, is used during the perinatal period to help fetal lung and other organ development. Conversely, Dex-induced cell proliferation has been associated with accelerated aging. Using primary amnion epithelial cells (AECs) from term, not in labor, fetal membranes, we tested the effects of Dex on cell proliferation, senescence, and inflammation. Primary AECs treated with Dex (100 and 200 nM) for 48 h were tested for cell viability (crystal violet dye exclusion), cell cycle progression and/or type of cell death (flow cytometry), expression patterns of steroid receptors (glucocorticoid receptor, progesterone receptor membrane component 1&2), inflammatory mediators (IL-6 and IL-8), and telomere length (quantitative RT-PCR). Mechanistic mediators of senescence (p38MAPK and p21) were determined by western blot analysis. Dex treatment did not induce AEC proliferation, cell cycle, influence viability, or morphology. However, Dex caused dependent telomere length reduction and p38MAPK-independent but p21-dependent (confirmed by treatment with p21 inhibitor UC2288). Senescence was not associated with an increase in inflammatory mediators, which is often associated with senescence. Co-treatment with RU486 produced DNA damage, cell cycle arrest, and cellular necrosis with an increase in inflammatory mediators. The effect of Dex was devoid of changes to steroid receptors, whereas RU486 increased GR expression. Dex treatment of AECs produced nonreplicative and noninflammatory senescence. Extensive use of Dex during the perinatal period may lead to cellular senescence, contributing to cellular aging associated pathologies during the perinatal and neonatal periods.

Polybacterial stimulation suggests discrete IL-6/IL-6R signaling in human fetal membranes: Potential implications on IL-6 bioactivity.

The polybacterial invasion of the amniotic cavity and risk of preterm birth is often due to cervicovaginal bacteria such as genital mycoplasmas (Mycoplasma hominis and Ureaplasma urealyticum) and Gardnerella vaginalis. The most studied biomarker associated with preterm birth is interleukin-6 (IL-6), a pleiotropic cytokine that performs different functions based on classical or trans-signaling mechanisms. This study evaluated the changes in IL-6 and IL-6 function associated accessory molecules by human fetal membranes to determine the functional availability of IL-6 assessment in an in vitro model of polybacterial infection. Fetal membranes were treated with LPS or heat-inactivated genital mycoplasmas and G. vaginalis alone or in combination. IL-6 and its soluble receptors (sgp130, sIL-6R) were assessed in conditioned medium by immunoassays and membrane-bound receptors were evaluated in the tissue using immunohistochemistry and RT-PCR. Data from protein and gene expression were evaluated using linear mixed effects models. Data from immunohistochemistry were evaluated using one-way analysis of variance followed by the Tukey test. Genital mycoplasmas alone, or in combination, inhibited IL-6 trans-signaling with increased sgp130 production. G. vaginalis activated the classical IL-6 signaling pathway, as did LPS. Polybacterial treatment resulted in a balanced response with neither pathway being favored. The increase in IL-6 production by fetal membranes in response to infection is likely a non-specific innate response and not an indicator of a functional mediator of any labor-inducing pathways. This suggests that correlating the risk of adverse pregnancy outcomes and designing interventions based on IL-6 levels without considering soluble receptors may be an ineffective strategy.