LEFTY2 inhibits endometrial receptivity by downregulating Orai1 expression and store-operated Ca²+ entry

Early embryo development and endometrial differentiation are initially independent processes, and synchronization, imposed by a limited window of implantation, is critical for reproductive success. A putative negative regulator of endometrial receptivity is LEFTY2, a member of the transforming growth factor (TGF)-β family. LEFTY2 is highly expressed in decidualizing human endometrial stromal cells (HESCs) during the late luteal phase of the menstrual cycle, coinciding with the closure of the window of implantation. Here, we show that flushing of the uterine lumen in mice with recombinant LEFTY2 inhibits the expression of key receptivity genes, including Cox2, Bmp2, and Wnt4, and blocks embryo implantation. In Ishikawa cells, a human endometrial epithelial cell line, LEFTY2 downregulated the expression of calcium release-activated calcium channel protein 1, encoded by ORAI1, and inhibited store-operated Ca2+ entry (SOCE). Furthermore, LEFTY2 and the Orai1 blockers 2-APB, MRS-1845, as well as YM-58483, inhibited, whereas the Ca2+ ionophore, ionomycin, strongly upregulated COX2, BMP2 and WNT4 expression in decidualizing HESCs. These findings suggest that LEFTY2 closes the implantation window, at least in part, by downregulating Orai1, which in turn limits SOCE and antagonizes expression of Ca2+-sensitive receptivity genes

TWEAK Appears as a Modulator of Endometrial IL-18 Related Cytotoxic Activity of Uterine Natural Killers

BACKGROUND: TWEAK (Tumor necrosis factor like WEAK inducer of apoptosis) is highly expressed by different immune cells and triggers multiple cellular responses, including control of angiogenesis. Our objective was to investigate its role in the human endometrium during the implantation window, using an ex-vivo endometrial microhistoculture model. Indeed, previous results suggested that basic TWEAK expression influences the IL-18 related uNK recruitment and local cytotoxicity. METHODOLOGY/PRINCIPAL FINDINGS: Endometrial biopsies were performed 7 to 9 days after the ovulation surge of women in monitored natural cycles. Biopsies were cut in micro-pieces and cultured on collagen sponge with appropriate medium. Morphology, functionality and cell death were analysed at different time of the culture. We used this ex vivo model to study mRNA expressions of NKp46 (a uNK cytotoxic receptor) and TGF-beta1 (protein which regulates uNK cytokine production) after adjunction of excess of recombinant IL-18 and either recombinant TWEAK or its antibody. NKp46 protein expression was also detailed by immunohistochemistry in selected patients with high basic mRNA level of IL-18 and either low or high mRNA level of TWEAK. The NKp46 immunostaining was stronger in patients with an IL-18 over-expression and a low TWEAK expression, when compared with patients with both IL-18 and TWEAK high expressions. We did not observe any difference for TWEAK expression when recombinant protein IL-18 or its antibody was added, or conversely, for IL-18 expression when TWEAK or its antibody was added in the culture medium. In a pro-inflammatory environment (obtained by an excess of IL-18), inhibition of TWEAK was able to increase significantly NKp46 and TGF-beta1 mRNA expressions. CONCLUSIONS/SIGNIFICANCE: TWEAK doesn't act on IL-18 expression but seems to control IL-18 related cytotoxicity on uNK cells when IL-18 is over-expressed. Thus, TWEAK appears as a crucial physiological modulator to prevent endometrial uNK cytotoxicity in human

Pro-inflammatory profile of preeclamptic placental mesenchymal stromal cells: new insights into the etiopathogenesis of preeclampsia.

The objective of the present study was to evaluate whether placental mesenchymal stromal cells (PDMSCs) derived from normal and preeclamptic (PE) chorionic villous tissue presented differences in their cytokines expression profiles. Moreover, we investigated the effects of conditioned media from normal and PE-PDMSCs on the expression of pro-inflammatory Macrophage migration Inhibitory Factor (MIF), Vascular Endothelial Growth Factor (VEGF), soluble FMS-like tyrosine kinase-1 (sFlt-1) and free β-human Chorionic Gonadotropin (βhCG) by normal term villous explants. This information will help to understand whether anomalies in PE-PDMSCs could cause or contribute to the anomalies typical of preeclampsia. METHODS: Chorionic villous PDMSCs were isolated from severe preeclamptic (n = 12) and physiological control term (n = 12) placentae. Control and PE-PDMSCs’s cytokines expression profiles were determined by Cytokine Array. Control and PE-PDMSCs were plated for 72 h and conditioned media (CM) was collected. Physiological villous explants (n = 48) were treated with control or PE-PDMSCs CM for 72 h and processed for mRNA and protein isolation. MIF, VEGF and sFlt-1 mRNA and protein expression were analyzed by Real Time PCR and Western Blot respectively. Free βhCG was assessed by immunofluorescent. RESULTS: Cytokine array showed increased release of pro-inflammatory cytokines by PE relative to control PDMSCs. Physiological explants treated with PE-PDMSCs CM showed significantly increased MIF and sFlt-1 expression relative to untreated and control PDMSCs CM explants. Interestingly, both control and PE-PDMSCs media induced VEGF mRNA increase while only normal PDMSCs media promoted VEGF protein accumulation. PE-PDMSCs CM explants released significantly increased amounts of free βhCG relative to normal PDMSCs CM ones. CONCLUSIONS: Herein, we reported elevated production of pro-inflammatory cytokines by PE-PDMSCs. Importantly, PE PDMSCs induced a PE-like phenotype in physiological villous explants. Our data clearly depict chorionic mesenchymal stromal cells as central players in placental physiopathology, thus opening to new intriguing perspectives for the treatment of human placental-related disorders as preeclampsia

Dissociated Representations of Pleasant and Unpleasant Olfacto-Trigeminal Mixtures: An fMRI Study

How the pleasantness of chemosensory stimuli such as odorants or intranasal trigeminal compounds is processed in the human brain has been the focus of considerable recent interest. Yet, so far, only the unimodal form of this hedonic processing has been explored, and not its bimodal form during crossmodal integration of olfactory and trigeminal stimuli. The main purpose of the present study was to investigate this question. To this end, functional magnetic resonance imaging (fMRI) was used in an experiment comparing brain activation related to a pleasant and a relatively unpleasant olfacto-trigeminal mixture, and to their individual components (CO2 alone, Orange alone, Rose alone). Results revealed first common neural activity patterns in response to both mixtures in a number of regions: notably the superior temporal gyrus and the caudate nucleus. Common activations were also observed in the insula, although the pleasant mixture activated the right insula whereas the unpleasant mixture activated the left insula. However, specific activations were observed in anterior cingulate gyrus and the ventral tegmental area only during the perception of the pleasant mixture. These findings emphasized for the firs time the involvement of the latter structures in processing of pleasantness during crossmodal integration of chemosensory stimuli

Offspring of xenogeneically-reconstituted scid scid mice are capable of a primary xenogeneic immune response to DNP-KLH

Human peripheral blood leukocyte (PBL) reconstitution of severe combined immunodeficient (SCID) mice has provided a small animal model system (hu-PBL-SCID) useful for the study of the human immune system and disease pathogenesis. Transfer of xenogeneic PBL from donors other than humans has also been successful; however, the controversy remains regarding the capability of xenogeneically engrafted lymphocytes to mount a primary immune response. Human cells have been identified in offspring from hu-PBL-SCID but were not evaluated for a primary immune response, In the present study, offspring of bovine PBL-reconstituted SCID mice (F1-PBL-SCID-bo) were assessed for specific immune function, Sera from all of the F1-PBL-SCID-bo contained relatively low levels of bovine IgG 5 weeks after birth but bovine Ig became undetectable by 14 or 18 weeks. Eight F1-PBL-SCID-bo (23 or 27 weeks of age) were immunized with a single dose of 100 mu g dinitrophenyl-keyhole limpet hemocyanin (DNP-KLH). Individual cells secreting bovine antibody were enumerated using the ELISA-plaque assay. One week after immunization, bovine cells secreting bovine immunoglobulin (IgG) specific for DNP-KLH were identified in the spleens from three of the F1-PBL-SCID-bo at a frequency of one antibody-secreting cell per 9x10(3) to 1x10(6) spleen cells. Thus, xenogeneic lymphocytes, passed from the mother to her offspring, retain the capacity for a primary immune response to DNP-KLH

DBA-lectin Reactivity Defines Natural Killer Cells that have Homed to Mouse Decidua

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)During normal mouse pregnancy, abundant numbers of uterine natural killer (uNK) cells differentiate at implantation sites and contribute to early, post-implantation endometrial angiogenesis and to spiral arterial modification. Mouse uNK cells are confidently recognized by light microscopy in tissue sections stained with special protocols. Classically, mouse uNK cells were identified as lymphocytes containing Periodic Acid Schiff's (PAS) reactive cytoplasmic granules. More recently, Dolichos biflorus lectin (DBA) reactions which stain not only the cytoplasmic granules but also the uNK cell membranes have been widely adopted. No lymphocytes in any tissues of virgin mice or external to the uterus of pregnant mice have DBA lectin reactivity equivalent to that of uNK cells; however, some uNK cells are now recognized as DBA, Here, we describe a PAS/DBA lectin double staining protocol and assess the coincident staining of C57Bl/6 J uNK cells from gestation day (gd)6, the first day of uNK cell abundance, to gd12, the day when Tunel+ nuclear senecence appears widely in uNK cells before their numerical decline. For these gd, PAS+ DBA- and PAS + DBA+ cells but not PAS-DBA+ cells were identified. Dual positive cells increased from 47% at gd6 to 85% at gd12. Transplantation of normal bone marrow into alymphoid mice who were subsequently mated revealed the uterus repopulated by doubly reactive PAS + DBA+ uNK cells (>95%). Thus, in normal pregnancies, most uNK cells appear to arise from progenitor cells that have homed to the uterus. (C) 2009 Elsevier Ltd. All rights reserved.3011968973Natural Sciences and Engineering Council CanadaCanada Research Chairs Program (BAC)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP