Structural and Functional Equivalency Between Lyopreserved and Cryopreserved Chorions with Viable Cells.

Objective: Clinical studies have demonstrated that the use of cryopreserved amnion or trophoblast (TR)-free chorion, containing viable cells, in the treatment of chronic wounds results in high rate of wound closure. Recently, a new lyopreservation method has been developed for preservation of amnion that also retains the endogenous viable cells. The objective of this study was to use this method for lyopreservation of TR-free chorionic membrane (viable lyopreserved chorionic membrane [VLCM]) and compare it with the viable cryopreserved chorionic membrane (VCCM). A second objective was to investigate the immunogenicity of chorion, an important question that has not been fully addressed. Approach: Chorion immunogenicity was tested in vitro in a mixed lymphocyte reaction and lipopolysaccharide (LPS) challenge assay, and in vivo in a mouse subcutaneous pocket implantation model. VLCM tissue structure was assessed histologically, growth factor content by multiplex assay, and cell viability by LIVE/DEAD cell fluorescent staining. Inhibition of tumor necrosis factor α secretion by LPS-activated THP-1 cells and endothelial cell tubule formation assays were performed to evaluate the anti-inflammatory and proangiogenic properties, respectively. An in vivo rabbit abdominal adhesion model was used to evaluate the antifibrotic properties. Results: Chorionic membrane without trophoblast (CM) was shown to be nonimmunogenic. Tissue architecture, growth factors, and cell viability of fresh CM were maintained in VLCM and VCCM. In vitro studies showed that anti-inflammatory and angiogenic properties were retained in VLCM. Furthermore, VLCM prevents formation of postsurgical adhesions in a rabbit abdominal surgical adhesion model. Innovation: Characterization of structural and functional properties of VLCM is reported for the first time. Conclusion: Similar to VCCM, VLCM retains native components of fresh CM, including collagen-rich extracellular matrix, growth factors, and viable cells. In vitro and in vivo models demonstrate that VLCM is anti-inflammatory, proangiogenic and antifibrotic. Results of this study support the structural and functional equivalency between VLCM and VCCM.

Discordance of Twin Placentas for Multifocal Eosinophilic/T-cell Chorionic Vasculitis.

Eosinophilic/T-cell chorionic vasculitis (ETCV) is an idiopathic placental lesion characterized by chorionic vasculitis composed predominantly of eosinophils and CD3+ T lymphocytes. It usually presents as a unifocal lesion, but a subset have multifocal involvement. We report 4 Di-Di and 2 Di-Mo twins sharing fused placental discs with discordant circulatory involvement by multifocal ETCV. The findings are difficult to explain by sampling alone. The limitation of ETCV to 1 fetus's vascular territory in monozygotic twin pregnancies is difficult to explain but could provide insights into the fetal immune system and the etiology of ETCV.

A Role for the Inflammasome in Spontaneous Labor at Term with Acute Histologic Chorioamnionitis.

Inflammasomes are cytosolic signaling platforms that regulate the activation of caspase (CASP)-1, which induces the maturation of interleukin (IL)-1ß and IL-18. Herein, we determined whether the chorioamniotic membranes from women in spontaneous labor at term with acute histologic chorioamnionitis express major inflammasome components and whether these changes are associated with the activation of CASP-1 and CASP-4 and the release of mature IL-1ß and IL-18. When comparing the chorioamniotic membranes from women in spontaneous labor at term with acute histologic chorioamnionitis to those without this placental lesion, we found that (1) the messenger RNA (mRNA) abundance of NLR family pyrin domain containing 3 ( NLRP3), NLR family CARD domain containing 4 ( NLRC4), absent in melanoma 2 ( AIM2), and nucleotide binding oligomerization domain 2 ( NOD2) was higher; (2) the NLRP3 and NLRC4 protein quantities were increased; (3) the mRNA and protein expressions of CASP-1 and its active forms were greater; (4) CASP-4 was increased at the mRNA level only; (5) the mRNA and protein expressions of IL-1ß and its mature form were higher; and (6) a modest increase in the total protein concentration and abundance of the mature form of IL-18 was observed. In vitro incubation of the chorioamniotic membranes with the CASP-1 inhibitor, VX765, decreased the release of endotoxin-induced IL-1ß and IL-18 (2-fold) but not IL-6 or tumor necrosis factor α. In conclusion, spontaneous labor at term with acute histologic chorioamnionitis is characterized by an upregulation of inflammasome components which, in turn, may participate in the activation of CASP-1 and lead to the release of mature IL-1ß by the chorioamniotic membranes. These results support a role for the inflammasome in the mechanisms responsible for spontaneous labor at term with acute histologic chorioamnionitis.

Differential immunophenotype of macrophages in acute and chronic chorioamnionitis.

AIM: The aim of this study was to investigate the involvement and immunophenotype of macrophages in acute chorioamnionitis (ACA) and chronic chorioamnionitis (CCA), marking amniotic fluid infection and anti-fetal rejection, respectively. METHODS: Chorioamniotic membranes from (1) gestational age-matched cases without chorioamnionitis, (2) cases with ACA, and (3) cases with CCA were studied after immunohistochemical staining using antibodies against CD14, CD68, CD163, and DC-SIGN. RESULTS: Macrophages increased prominently in the chorionic trophoblastic layer of both ACA and CCA cases in contrast to non-inflammatory cases. Macrophages in the decidua and the chorioamniotic membranes of ACA cases expressed CD14. Macrophages in the chorionic trophoblastic layer of CCA cases were characterized by CD68 positivity. DC-SIGN-positive cells were increased in the chorioamniotic mesodermal layer of CCA cases. CONCLUSIONS: Macrophages participate in the inflammatory response in ACA and CCA. The differential immunophenotypes of macrophages in the decidua and chorioamniotic membranes of ACA and CCA cases suggest their disease-specific and region-specific roles at the feto-maternal interface.

Differential Spatiotemporal Patterns of Galectin Expression are a Hallmark of Endotheliochorial Placentation.

PROBLEM: Galectins influence the progress of pregnancy by regulating key processes associated with embryo-maternal cross talk, including angiogenesis and placentation. Galectin family members exert multiple roles in the context of hemochorial and epitheliochorial placentation; however, the galectin prolife in endotheliochorial placenta remains to be investigated. METHOD OF STUDY: Here, we used immunohistochemistry to analyze galectin (gal)-1, gal-3 and gal-9 expression during early and late endotheliochorial placentation in two different species (dogs and cats). RESULTS: We found that during early feline gestation, all three galectin members were more strongly expressed on trophoblast and maternal vessels compared to the decidua. This was accompanied by an overall decrease of gal-1, gal-3 and gal-9 expressions in late feline gestation. In canine early pregnancy, we observed that gal-1 and gal-9 were expressed strongly in cytotrophoblast (CTB) cells compared to gal-3, and no galectin expression was observed in syncytiotrophoblast (STB) cells. Progression of canine gestation was accompanied by increased gal-1 and gal-3 expressions on STB cells, whereas gal-9 expression remained similar in CTB and STB. CONCLUSION: These data suggest that both the maternal and fetal compartments are characterized by a spatiotemporal regulation of galectin expression during endotheliochorial placentation. This strongly suggests the involvement of the galectin family in important developmental processes during gestation including immunemodulation, trophoblast invasion and angiogenesis. A conserved functional role for galectins during mammalian placental development emerges from these studies.

A thicker chorion gives ova of Atlantic salmon (Salmo salar L.) the upper hand against Saprolegnia infections.

Since the ban of malachite green in the fish farming industry, finding alternative ways of controlling Saprolegnia infections has become of utmost importance. Much effort has been made to elucidate the mechanisms by which Saprolegnia invades fish eggs. Little is known about the defence mechanisms of the hosts, making some eggs more prone to infection than others. One clue might lie in the composition of the eggs. As the immune system in the embryos is not developed yet, the difference in infection levels could be explained by factors influenced by the mother herself, by either transferring passive immunity, influencing the physical aspects of the eggs or both. One of the physical aspects that could be influenced by the female is the chorion, the extracellular coat surrounding the fish egg, which is in fact the first major barrier to be overcome by Saprolegnia spp. Our results suggest that a thicker chorion in eggs from Atlantic salmon gives a better protection against Saprolegnia spp. In addition to the identification of differences in sensitivity of eggs in a fish farm set-up, we were able to confirm these results in a laboratory-controlled challenge experiment.

The trichloroethylene metabolite S-(1,2-dichlorovinyl)-l-cysteine but not trichloroacetate inhibits pathogen-stimulated TNF-α in human extraplacental membranes in vitro.

Extraplacental membranes define the gestational compartment and provide a barrier to infectious microorganisms ascending the gravid female reproductive tract. We tested the hypothesis that bioactive metabolites of trichloroethylene (TCE) decrease pathogen-stimulated innate immune response of extraplacental membranes. Extraplacental membranes were cultured for 4, 8, and 24h with the TCE metabolites trichloroacetate (TCA) or S-(1,2-dichlorovinyl)-l-cysteine (DCVC) in the absence or presence of lipoteichoic acid (LTA) or lipopolysaccharide (LPS) to simulate infection. In addition, membranes were cocultured with DCVC and Group B Streptococcus (GBS). DCVC (5-50µM) significantly inhibited LTA-, LPS-, and GBS-stimulated cytokine release from tissue cultures as early as 4h (P≤0.05). In contrast, TCA (up to 500µM) did not inhibit LTA-stimulated cytokine release from tissue punches. Because cytokines are important mediators for host response to infectious microorganisms these findings suggest that TCE exposure could potentially modify susceptibility to infection during pregnancy.

Escherichia coli-induced temporal and differential secretion of heat-shock protein 70 and interleukin-1ß by human fetal membranes in a two-compartment culture system.

INTRODUCTION: Escherichia coli is recognized as an etiological bacteria associated with chorioamnionitis and the preterm premature rupture of fetal membranes. This pathological condition induces pro-inflammatory cytokines and degradative metalloproteinases, which are considered biological markers secreted in an acute stage of infection. Heat-shock proteins (HSPs) are an important component of the innate immunity response and are found in different pathological conditions. They have not been previously measured in human fetal membranes in response to infectious conditions. We hypothesized that the choriodecidual tissue and amniotic epithelium secreted temporal and differential Hsp-60, Hsp-70, and interleukin (IL)-1ß mediated by E. coli infection. METHODS: Fetal membranes were mounted in a two-compartment culture system and infected with two passes of live E. coli at different doses (10², 104, 105, and 106 colony-forming units (CFU)/mL) and intervals of incubation (3, 6, and 24 h). The culture medium was collected, and Hsp-60, Hsp-70, and IL-1ß were assessed using the enzyme-linked immunosorbent assay (ELISA) method. RESULTS: After 3 and 6 h of infection, E. coli induced an increase in Hsp-70 secretion in the choriodecidual tissue. However, after 24 h of incubation, Hsp-70 was downregulated and we observed an increase in IL-1ß secretion. By contrast, E. coli induced a lower Hsp-60 secretion in the amnion compared to Hsp-70. DISCUSSION: Human fetal membranes responded actively to E. coli infection, with an increase in Hsp-70 during the first hours of infection. After 24 h, there was an increase in the liberation of IL-1ß.

Amniotic Fluid Soluble Myeloid Differentiation-2 (sMD-2) as Regulator of Intra-amniotic Inflammation in Infection-induced Preterm Birth.

PROBLEM: TLR4 mediates host responses to pathogens through a mechanism that involves protein myeloid differentiation-2 (MD-2) and its soluble form sMD-2. The role of sMD2 in intra-amniotic inflammation-induced preterm birth has not been previously explored. METHOD OF STUDY: Human amniotic fluid (AF) sMD-2 was studied by Western blotting in 152 AF samples of patients who had an amniocentesis to rule-out infection (yes infection, n = 50; no infection, n = 50) or women with normal pregnancy outcome (second trimester genetic karyotyping, n = 26; third trimester lung maturity testing, n = 26). Histological localization and mRNA expression of MD2 in fetal membranes were studied by immunohistochemistry and RT-PCR. The ability of fetal membrane to release sMD-2 and inflammatory cytokines was studied in vitro. RESULTS: Human AF contains three sMD-2 proteoforms whose levels of expression were lower at term. Intra-amniotic infection upregulated sMD-2. MD-2 mRNA and immunohistochemistry findings concurred. In vitro, LPS and monensin increased, while cycloheximide decreased sMD-2 production. Recombinant sMD-2 modulated TNF-α and IL-6 levels in a dose- and time-dependent fashion. CONCLUSION: sMD2 proteoforms are constitutively present in human AF. The intensity of the intra-amniotic inflammatory response to bacteria or perhaps to other TLR4 ligands may be facilitated through synthesis and release of sMD2 by the amniochorion.

Transplacental total IgG transfer in twin pregnancies.

PROBLEM: In twin pregnancies, factors that influence total umbilical cord IgG concentration and IgG transfer ratio are not well known. METHOD: Blood samples were prospectively collected from 57 twin pregnancies. Stepwise multivariate regression analysis was used to evaluate the association between total IgG levels in the umbilical cord blood and IgG transfer ratio according to serum IgG concentration, pregnancy chorionicity, the presence of abnormal umbilical artery pulsatility index, intrauterine growth restriction, gestational age at delivery (GAD), birthweight, and placental weight. RESULTS: Umbilical cord IgG concentration showed a positive correlation with serum IgG concentration and GAD; levels were significantly lower in monochorionic compared with dichorionic pregnancies. IgG transfer ratio also increased with GAD but was inversely correlated with serum IgG concentration levels. CONCLUSION: In twin pregnancies, besides serum IgG concentration and GAD, chorionicity also influences umbilical cord IgG concentration. Monochorionic twins have lower IgG cord concentration than dichorionic twins.

Pandemic H1N1 influenza A virus induces a potent innate immune response in human chorionic cells.

To understand the mechanistic basis for the reported outcomes of influenza A virus (IAV) infection during pregnancy, the effects of mouse adapted and pandemic (pdm) IAV infection in human choriocarcinoma cells were examined. Both viruses were able to infect and replicate in human placental cells, with pdm IAV being more apoptotic. A strong induction of innate signaling molecules, type I interferon and pro-inflammatory cytokine production, were associated with pdm IAV infection of human placental cells, with implications for adverse immediate and late outcomes during pregnancy.

Choriodecidual cells from term human pregnancies show distinctive functional properties related to the induction of labor.

PROBLEM: Human parturition is associated with an intrauterine pro-inflammatory environment in the choriodecidua. Evidence that some mediators of this signaling cascade also elicit responses leading to labor prompted us to characterize the cellular sources of these mediators in the human choriodecidua. METHOD OF STUDY: Leukocyte-enriched preparations from human choriodecidua (ChL) and intervillous placental blood leukocytes (PL) were maintained in culture. Secretions of inflammatory cytokines, chemokines, and MMP-9 were documented. Leukocyte phenotype of ChL and PL was determined by flow cytometry using specific fluorochrome-conjugated antibodies. RESULTS AND CONCLUSIONS: ChL showed a distinct pro-inflammatory secretion pattern of cytokines and chemokines when compared with PL, including higher amounts of TNF-α and IL-6, and decreased secretions of IL-4 and IL-1ra. ChL also secreted more MIP-1α and MCP-1 and MMP-9 than PL. No significant differences were found in leukocytes subsets between compartments. Based on our findings, we propose that ChL isolated from fetal membranes at term are functionally different from PL and may collaborate to modulate the microenvironment linked to induction and progression of human labor.

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.

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 × 10(6) 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.

Role of inflammatory proteins S100A8 and S100A9 in pathophysiology of recurrent early pregnancy loss.

Altered expression of inflammatory molecule at the maternal fetal interface is associated with early pregnancy loss. S100A8 and S100A9 are inflammatory proteins and they exhibit cytokine like function enhancing leukocyte recruitment to the inflammatory site. Reports from mouse model suggest the role of S100A8 with the vasculature of the decidual tissue and leukocyte recruitment during early pregnancy. Hence we hypothesized that maternal overexpression of S100A8 & S100A9 might increase the recruitment of inflammatory leukocytes in maternal-fetal interface resulting in uteroplacental perfusion deficiency, development of thrombotic events, and placental hypoxia, eventually embryo abortion. In the present study we investigated altered expression of S100A8 and S100A9 in 25 recurrent early pregnancy loss (REPL) patients compared to 40 induced abortion subjects as controls. S100A8 and S100A9 mRNA were evaluated using semi-quantitative RT-PCR and quantitative real-time PCR. To determine if differential expression pattern of these transcripts is translated to protein western blot analysis was performed.S100A8 and S100A9 mRNA and protein level were significantly increased in endometrial decidua tissue (p < 0.05) of REPL patients as compared to controls. This is the first report predicting the role of inflammatory molecules S100A8 & S100A9 in REPL. It opens a new perspective for understanding significance of S100A8 and S100A9 in pregnancy maintenance and outcome.

Effect of oxygen tension on bacteria-stimulated cytokine production by fetal membranes.

AIM: Tissue culture studies indicate that bacterial products stimulate the production of proinflammatory cytokines by reproductive tissues. However, most of these studies have been performed under room air conditions, supplemented with 5% CO2. In this study, we tested whether O2 tension affects bacteria-stimulated cytokine production by extra-placental fetal membranes. METHODS: Cultures of full-thickness membranes, isolated choriodecidua, and isolated amnion were exposed to bacteria and incubated under 21% (room air) or 5% O2 for 18 h. Cytokine concentrations in conditioned medium was quantified by immunoassay. RESULTS: Culture under 5% O2 increased production of interleukin (IL)-1ß and tumor necrosis factor (TNF)-α, but reduced IL-10 and IL-6 production by full membranes. Isolated choriodecidua responded to 5% O2 with increased IL-1ß production and reduced IL-6 production, but had no effect on TNF-α and IL-10 production was not detected. No effect of O2 tension on IL-1ß or IL-6 production by isolated amnion was detected, however, Escherichia coli-stimulated IL-10, TNF-α and IL-8 production was enhanced by culture under 5% O2. CONCLUSIONS: Increased oxygen tension reduces the pro-inflammatory responsiveness of cell cultures to E. coli and promotes an anti-inflammatory cytokine profile. Differential effects of O2 tension on choriodecidua and amnion suggests a network of paracrine factors that regulate cytokine levels in response to changes in O2 tension.

Antimicrobial peptide response to group B Streptococcus in human extraplacental membranes in culture.

OBJECTIVE: Streptococcus agalactiae (GBS) is an important cause of chorioamnionitis. This study characterizes GBS colonization and stimulation of antimicrobial responses in human extraplacental membranes using an ex vivo transwell two-compartment system of full-thickness membranes and live GBS. STUDY DESIGN: Human extraplacental membranes were affixed to transwell frames (without synthetic membranes). Live GBS was added to the decidual side of membranes in transwell cultures, and cocultures were incubated for 4, 8 and 24 h. GBS recovery from homogenized membranes and culture medium was determined by enumerating colony forming units (CFU) on blood agar. Antimicrobial peptide expression was identified using immunohistochemistry and ELISA. GBS killing by HBDs was assessed in vitro by incubating GBS with different human beta defensins (HBDs) for 3 h, then enumerating CFU. RESULTS: GBS recovery from membranes markedly decreased over time (P < 0.05). The antimicrobial peptides HBD-1, HBD-2, HBD-3, and lactoferrin were expressed in both GBS-exposed and non-exposed tissues. Notably, a pattern of localized increased HBD-2 in the amnion of GBS-infected tissue was observed. Moreover, GBS-treated membranes released increased amounts of HBD-2 into the amniotic and decidual compartments of the transwell cultures after 24 h (P < 0.05). In bacterial cultures, HBD-2 decreased GBS viability in a concentration-dependent manner (P < 0.05). CONCLUSION: Innate immune responses in ex vivo human extraplacental membranes suppress GBS growth. HBD-2 was implicated in this GBS suppression with evidence of signal transduction across the tissue. Antimicrobial peptides may be important for innate immune defense against intrauterine GBS infections during pregnancy.

CD106 identifies a subpopulation of mesenchymal stem cells with unique immunomodulatory properties.

Mesenchymal stem cells (MSCs) reside in almost all of the body tissues, where they undergo self-renewal and multi-lineage differentiation. MSCs derived from different tissues share many similarities but also show some differences in term of biological properties. We aim to search for significant differences among various sources of MSCs and to explore their implications in physiopathology and clinical translation. We compared the phenotype and biological properties among different MSCs isolated from human term placental chorionic villi (CV), umbilical cord (UC), adult bone marrow (BM) and adipose (AD). We found that CD106 (VCAM-1) was expressed highest on the CV-MSCs, moderately on BM-MSCs, lightly on UC-MSCs and absent on AD-MSCs. CV-MSCs also showed unique immune-associated gene expression and immunomodulation. We thus separated CD106(+)cells and CD106(-)cells from CV-MSCs and compared their biological activities. Both two subpopulations were capable of osteogenic and adipogenic differentiation while CD106(+)CV-MSCs were more effective to modulate T helper subsets but possessed decreased colony formation capacity. In addition, CD106(+)CV-MSCs expressed more cytokines than CD106(-)CV-MSCs. These data demonstrate that CD106 identifies a subpopulation of CV-MSCs with unique immunoregulatory activity and reveal a previously unrecognized mechanism underlying immunomodulation of MSCs.

Expression of pregnancy-associated glycoprotein family in the epitheliochorial placenta of two Camelidae species (C. dromedarius and C. bactrianus).

This study describes placental morphology and immunolocalization of the placental pregnancy associated glycoprotein-like family (PAGs) identified in two selected taxa of Old-World camels of the Camelidae family: Camelus dromedarius (Cd) and Camelus bactrianus (Cb). Placental tissues of Cd from days 140-293 post-coitum (dpc), term (404 dpc); and of Cb from term (440 dpc) were examined. Histological staining (hematoxylin/eosin and propidium iodine) revealed the development of the placental structure, while chorionic folding increased the feto-placental surface during the progress of pregnancy. The camelid placenta during early pregnancy is similar to the diffuse epitheliochorial type, and during later stages of pregnancy resembles the synepitheliochorial (cotyledonary) type. Placental expression of the PAGs was detected (Alexa 488 - green) within camelid trophectoderm cells (TRD - chorionic epithelium as outer layer of embryonic cells) among all placental cells with nuclei stained by propidium iodide (red). The PAGs, identified in both Camelidae taxa, were named CbPAGs and CdPAGs. Placental CbPAG and CdPAG expression is restricted to the TRD cells, which are differentially developed throughout gestation. Cross-reactivity of polyvalent anti-pPAG polyclonals with the CbPAGs and CdPAGs revealed high structural similarities of the PAG-like epitopes in pigs and camels. This is the first study identifying PAG expression in chorionic cells of the camel placenta.

Expression and co-expression of surface markers of pluripotency on human amniotic cells cultured in different growth media.

OBJECTIVES: Despite constant advances in the field of biology and medical application of human embryonic stem cells, the molecular mechanism of pluripotency remains largely unknown. So far, definitions of pluripotent stem cells (SC) have been based on a limited number of antigenic markers and have not allowed for unambiguous determination of the homogeneity of each subpopulation. Moreover, the use of some crucial pluripotency markers such as SSEA-3 and SSEA-4 has recently been questioned due to the possibility that the pattern of surface glycans may be changed depending on the content of the cell culture medium. AIM: Quantitative analysis of amniotic SC subpopulations cultured in different media, based on the following pluripotency surface markers: SSEA-3, SSEA-4, TRA- 1-60 and TRA- 1-81 expression and co-expression. MATERIAL AND METHODS: Immunofluorescence and fluorescence microscopy were used to identify and localize SC within a normal human placenta at term. The number of SSEA-4+, SSEA-3+, TRA-1-60+ and TRA-1-81+ cells and cells with co-expression of the above mentioned markers, cultured in media containing different protein supplements of animal origin, was counted by flow cytometry RESULTS AND CONCLUSIONS: Cells with characteristics of embryonic SC were identified in the amniotic epithelium and the chorion, but not in the decidua basalis. Amniotic epithelium contained various types of SC, with SSEA-4+ as the most numerous. Disproportion in the number of SSEA-4+, SSEA-3+, TRA-1-60+ and TRA-1-81+ cells and cells characterized by co-expression of these antigens, as well as lack of quantitative differences between SC subpopulations cultured in different media, was observed. In conclusion, the amniotic epithelium is composed of SC at different stages of the development but human amnion might become an alternative source of SSEA-4+ embryonic-like SC. The composition of the evaluated media, characterized by different content of animal-derived proteins, does not influence the number of cells identified within the SC subpopulations.

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.

BACKGROUND: During 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. METHODS: Full-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. RESULTS: HBD-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. CONCLUSION: Selective 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.