Veronica Zaga-Clavellina

In vitro secretion profiles of interleukin (IL)-1beta, IL-6, IL-8, IL-10, and TNF alpha after selective infection with Escherichia coli in human fetal membranes

BackgroundChorioamniotic membranes infection is a pathologic condition in which an abnormal secretion of proinflammatory cytokines halts fetal immune tolerance. The aim of the present study was to evaluate the functional response of human chorioamniotic membranes, as well as the individual contribution of the amnion and choriodecidua after stimulation with Escherichia coli, a pathogen associated with preterm labor.MethodsExplants of chorioamniotic membranes from 10 women (37–40 weeks of gestation) were mounted and cultured in a Transwell system, which allowed us to test the amnion and choriodecidua compartments independently. Escherichia coli (1 × 10 6 CFU/mL) was added to either the amniotic or the choriodecidual regions or both; after a 24-h incubation, the secretion of IL-1beta, IL-6, TNFalpha, IL-8, and IL-10 in both compartments was measured using a specific ELISA. Data were analyzed by Kruskal-Wallis one-way analysis of variance.ResultsAfter stimulation with Escherichia coli, the choriodecidua compartment showed an increase in the secretion of IL-1beta (21-fold), IL-6 (2-fold), IL-8 (6-fold), and IL-10 (37-fold), regardless of which side of the membrane was stimulated; TNFalpha secretion augmented (22-fold) also but only when the stimulus was applied simultaneously to both sides. When the amnion was stimulated directly, the level of IL-1beta (13-fold) rose significantly; however, the increase in IL-8 secretion was larger (20-fold), regardless of the primary site of infection. TNFalpha secretion in the amnion compartment rose markedly only when Escherichia coli was simultaneously applied to both sides.ConclusionSelective stimulation of fetal membranes with Escherichia coli results in a differential production of IL-1beta, IL-6, TNFalpha, IL-8, and IL-10. These tissues were less responsive when the amnion side was stimulated. This is in agreement with the hypothesis that the choriodecidua may play a primary role during an ascending intrauterine infection, being the main barrier to progression of the infection into the amniotic cavity. Therefore, the tissue-specific capacities for the secretion of these immune modulators could be a determining factor for the degree of severity of the inflammation process in fetal membranes.

In Vitro Secretion Profile of Pro‐Inflammatory Cytokines IL‐1β, TNF‐α, IL‐6, and of Human Beta‐Defensins (HBD)‐1, HBD‐2, and HBD‐3 from Human Chorioamniotic Membranes After Selective Stimulation with Gardnerella vaginalis

Citation Zaga‐Clavellina V, Martha RV‐M, Flores‐Espinosa P. In vitro secretion profile of pro‐inflammatory cytokines IL‐1β, TNF‐α, IL‐6, and of human beta‐defensins (HBD)‐1, HBD‐2, and HBD‐3 from human chorioamniotic membranes after selective stimulation with Gardnerella vaginalis. Am J Reprod Immunol 2012; 67: 34–43

Amniotic Membrane is an Immunosuppressor of Peripheral Blood Mononuclear Cells

Amniotic membrane (AM) is the inner layer of the placenta, which is in contact with the fetus; it has been used for transplantation in ocular surface diseases. It has been reported that amniotic membrane promotes epithelialization, inhibits angiogenesis and diminishes ocular inflammation. A persistent epithelial defect is the delay in epithelial wound healing caused by infiltrating inflammatory cells into the cornea and amniotic membrane transplantation has been successfully used in its treatment, however the mechanism of action in inhibiting inflammation it is not well understood. This study was aimed at determining whether denuded amniotic membrane (dAM) induces anti-inflammatory effects on peripheral blood mononuclear cells. Methods: Human peripheral blood mononuclear cells (PBMC) were cultured on dAM. Proliferation and apoptosis assays were performed on PBMC; and synthesis and secretion of pro-inflammatory cytokines by these cells was analyzed. Results: dAM induced apoptosis and inhibited cell proliferation of PBMC; and abolished the synthesis and the secretion of pro-inflammatory cytokines even when they were LPS stimulated. In contrast, when PBMC were cultured on hydrophilic membrane cell culture inserts, apoptosis was not significantly induced, cell proliferation was conserved, and synthesis and secretion of pro-inflammatory cytokines were not affected. Conclusions: Taken together, these results could explain the anti-inflammatory in vivo effects observed when the amniotic membrane is used as a transplant.

Progesterone Elicits an Inhibitory Effect upon LPS-Induced Innate Immune Response in Pre-Labor Human Amniotic Epithelium

Infection of human fetal membranes elicits secretion of pro‐inflammatory modulators through its innate immune capacities. We investigated the effect of lipopolysacharide (LPS) and progesterone (P4) upon expression of TLR‐4/MyD88, TNFα, IL‐6, IL‐8, IL‐10, and HBD2 on the human amniotic epithelium.

Tissue-specific human beta-defensins (HBD)1, HBD2, and HBD3 secretion from human extra-placental membranes stimulated with Escherichia coli

BackgroundDuring an ascending infection along the reproductive tract, the extra-placental membranes must act as a selective and competent barrier against pathogens. Human beta defensins (HBD)1, HBD2, and HBD3 are key elements of innate immunity that are secreted to neutralize/control the progression of infection.MethodsFull-thickness membranes were mounted on a Transwell device, constituted by two independent chambers, 1 × 10(6) CFU/ml of Escherichia coli were added to either the amnion (AMN) or the choriodecidual (CHD) face or to both. Secretion profiles of HBD1, HBD2, and HBD3 to the culture medium were quantified by ELISA.ResultsIn comparison with basal conditions, the secretion profile of HBD1 remained without significant changes; HBD2 level in CHD and AMN increased 1.9- and 1.4-times, respectively, after stimulation with bacteria. HBD3 secretion level increased significantly (7.8 +/- 1.9 pg/micrograms) in the CHD but only if the stimulus was applied on the AMN side.ConclusionsSelective stimulation of extra-placental membranes with E. coli, results in a tissue specific secretion of HBD1, HBD2, and HBD3 mainly in the CHD, which is the first infected region during an ascending infection.

In vitro secretion and activity profiles of matrix metalloproteinases, MMP-9 and MMP-2, in human term extra-placental membranes after exposure to Escherichia coli

BackgroundPremature rupture of fetal membranes (PROM) complicated with intrauterine infection has been associated to alterations of the extracellular matrix (ECM) homeostasis. The aim of this work was to evaluate the integral/functional response of the amnion (AMN) and choriodecidua (CHD) to synthesis, secretion, and activity of MMP-2 and MMP-9 and of their inhibitors TIMP-1, -2, and -4, after stimulation with Escherichia coli.MethodsFull-thickness membranes were mounted on a Transwell device, constituting two independent chambers, Escherichia coli (1×10 (6) CFU/mL) were added to either the amniotic or the choriodecidual face or to both. Secretion profiles of MMP-2, MMP-9, TIMP-1, TIMP-2, and TIMP-4 were quantified by ELISA and gelatinolytic activity by zymography. Immunoreactivity for MMP-2 and MMP-9 was revealed by immunohistochemistry and the collagen content was assessed by the hydroxyproline assay.ResultsLevels of MMP-9 in CHD and AMN increased 4- and 8-fold, respectively, after simultaneous infection. MMP-2 secreted to the medium by CHD increased a mean of 3 times after direct stimulation. Secretion profiles of TIMP-1, TIMP-2, and TIMP-4 remained without significant changes. Collagen content was significantly decreased (4-fold) in infected membranes, and was associated with loss of structural continuity and co-localization with immunoreactive forms of MMP-2 and MMP-9.ConclusionsInfection of chorioamniotic membranes with E. coli induces an increase in the secretion of inactive forms and an association to ECM of active forms of MMP-2 and MMP-9 without changes in TIMP-1, -2, and -4. These changes could explain the significant decrease of collagen content and loss of structural continuity.

Evidence of in vitro differential secretion of human beta-defensins-1, -2, and -3 after selective exposure to Streptococcus agalactiae in human fetal membranes

Abstract Objective. The aim of this work was to characterize the individual contribution of the amnion (AMN) and choriodecidua (CHD) regions to the secretion of human beta defensins (HBD)-1, -2, and -3, after stimulation with Streptococcus agalactiae. Methods. Full-thickness membranes were mounted on a Transwell device, constituted by two independent chambers; 1 × 106 CFU/ml of S. agalactiae were added to either the AMN or CHD face or to both. Secretion profiles of HBD-1, HBD-2, and HBD-3 to the culture medium were quantified by enzyme-linked immunosorbent sandwich assay (ELISA). Results. Secretion profile of HBD-1 remained without significant changes; HBD-2 secretion level by the CHD increased 2.0 (2.73 ± 0.19 pg/μg) and 2.6 (3.62 ± 0.60 pg/μg) times when the stimulus was applied only to the CHD region and simultaneously to both compartments, respectively. The bacterial stimulation in the AMN induced a 2.0 times (2.06 ± 0.29 pg/μg) increase in this region. HBD-3 secretion level increased significantly in the CHD (15.65 ± 2.68 pg/μg) and the AMN (14.94 ± 1.85 pg/μg) only when both regions were stimulated simultaneously. Conclusion. The stimulation of human fetal membranes with S. agalactiae induced a differential and tissue-specific profile of HBD-1, HBD-2, and HBD-3 secretion.

Tissue-specific human beta-defensins (HBD)-1, HBD-2 and HBD-3 secretion profile from human amniochorionic membranes stimulated with Candida albicans in a two-compartment tissue culture system

BackgroundDuring intrauterine infection, amniochorionic membranes represent a mechanical and immunological barrier against dissemination of infection. Human beta defensins (HBD)-1, HBD-2, and HBD-3 are key elements of innate immunity that represent the first line of defense against different pathogen microorganisms associated with preterm labor. The aim of this work was to characterize the individual contribution of the amnion (AMN) and choriodecidua (CHD) regions to the secretion of HBD-1, HBD-2 and HBD-3, after stimulation with Candida albicans.MethodsFull-thickness human amniochorionic membranes were obtained after delivery by elective cesarean section from women at 37-40 wk of gestation with no evidence of active labor. The membranes were cultured in a two-compartment experimental model in which the upper compartment is delimited by the amnion and the lower chamber by the choriodecidual membrane. One million of Candida albicans were added to either the AMN or the CHD face or to both and compartmentalized secretion profiles of HBD-1, HBD-2, and HBD-3 were quantified by ELISA. Tissue immunolocalization was performed to detect the presence of HBD-1, -2, -3 in tissue sections stimulated with Candida albicans.ResultsHBD-1 secretion level by the CHD compartment increased 2.6 times (27.30 [20.9-38.25] pg/micrograms protein) when the stimulus with Candida albicans was applied only on this side of the membrane and 2.4 times (26.55 [19.4-42.5] pg/micrograms protein) when applied to both compartments simultaneously. HBD-1 in the amniotic compartment remained without significant changes. HBD-2 secretion level increased significantly in the CHD when the stimulus was applied only to this region (2.49 [1.49-2.95] pg/micrograms protein) and simultaneously to both compartments (2.14 [1.67- 2.91] pg/micrograms protein). When the stimulus was done in the amniotic compartment HBD-2 remained without significant changes in both compartments. HBD-3 remained without significant changes in both compartments regardless of the stimulation modality. Localization of immune-reactive forms of HBD-1, HBD-2, and HBD-3 was carried out by immunohistochemistry confirming the cellular origin of these peptides.ConclusionSelective stimulation of amniochorionic membranes with Candida albicans resulted in tissue-specific secretion of HBD-1 and HBD-2, mainly in the CHD, which is the first region to become infected during an ascending infection.

Tissue-specific IL-10 secretion profile from term human fetal membranes stimulated with pathogenic microorganisms associated with preterm labor in a two-compartment tissue culture system.

Abstract Objective: Interleukin (IL)-10 is a cytokine with anti-inflammatory properties that plays pivotal roles in immune recognition and maintenance of pregnancy, limiting the harmful effects of pro-inflammatory modulators. The aim of this work was to characterize the contribution of amnion and choriodecidua regions of the human fetal membranes in the production of IL-10 after selective stimulation with Candida albicans, Gardnerella vaginalis and Streptococcus agalactiae. Methods: Pre-labor human fetal membranes were cultured in a two-compartment tissue culture system and stimulated with 1 × 106 CFU/ml of each pathogen added to either the amniotic or choriodecidual region or both. Results: Candida albicans and G. vaginalis were the pathogens most effective in inducing IL-10 secretion, increasing 20 and 10 times, respectively, the levels of this cytokine in the choriodecidual compartment. Stimulation with S. agalactiae was effective only in the choriodecidual region, increasing two times IL-10 concentration. Conclusions: Synthesis and secretion of IL-10 in response to three different pathogens associated with intrauterine infection and preterm birth are differential and depend on the nature of the microorganism and initial contact region.

A POSSIBLE ROLE OF PROGESTERONE RECEPTOR IN MOUSE OOCYTE IN VITRO FERTILIZATION REGULATED BY NORETHISTERONE AND ITS REDUCED METABOLITE

BACKGROUND The contraceptive effect of the progestogen norethisterone (NET) and its main metabolites 5alpha-NET and 3beta,5alpha-NET has been demonstrated in several species, and most studies have focused on the effects of these compounds in the uterus. We previously reported that 5alpha-NET inhibits the progesterone (P(4))-induced acrosome reaction in pig and mouse spermatozoa and induces severe morphological damage in two-cell fertilized mouse oocytes. STUDY DESIGN The main goal of this study was to analyze the possible role of P(4) receptor (PR) in the effects of NET and 5alpha-NET on the oocyte fertilization process. Different steroid treatments were used with or without cumulus-enclosed oocytes. RESULTS It was demonstrated that NET increases the percentage of fertilized oocytes in the same manner as P(4) does, while 5alpha-NET reduces the percentage of fertilized oocytes. This effect was not reversed by P(4) in the same concentrations. CONCLUSION A possible molecular mechanism for the effects of 5alpha-NET may be through a PR localized in the oocyte plasma membrane.