Human trophoblast cell cultures

Top PDF Human trophoblast cell cultures:

Stimulation of human trophoblast invasion by placental growth hormone

Stimulation of human trophoblast invasion by placental growth hormone

Transient transfection of primary invasive EVCT and luciferase assay Human EVCT isolated and purified as described above (see EVCT isolation and purification) were plated (5 ⫻ 10 5 cells per well) on Matrigel- coated Falcon six-well cultures dishes. Transfection was performed after 18 h of culture by using the TransIT-LT1 transfection reagent (Mirus, Madison, WI) as recommended by the manufacturer. Briefly, the cells were cotransfected in complete medium containing 1% FCS for 6 h with 3 ␮g of the reporter gene and 2 ␮g of pCH110 (␤-galactosidase expres- sion vector, Pharmacia Biotech, Inc., Piscataway, NJ). The reporter gene carries the luciferase coding sequence under the control of a six-repeat sequence of the lactogenic hormone response element (LHRE), followed by the minimal thymidine kinase promoter (45, 46). The sequence of a single LHRE DNA element is 5⬘-CTGCAGTGTGGACTTCTTGGAAT- TAAGGGACTTTTGCTGCAG-3 ⬘, with the Stat5 consensus binding se- quence underlined. hPGH (100 or 500 ng/ml) was then added for an additional 18 h of culture period. Cells were lysed in 200 ␮l lysing buffer [25 mm Tris/H 3 PO 4 (pH 7.8), 10 mm MgCl 2 , 1 mm EDTA, 15% glycerol, 1% Triton X-100], and luciferase and ␤-galactosidase activities were measured as follows. For luciferase, 400 ␮l lysing buffer (without Triton) containing 1 mm dithiothreitol was added to 100 ␮l cell lysate, and luminescence was measured after addition of 100 ␮l of the same buffer containing 200 mm ATP and 100 ␮g/ml sodium luciferin (Sigma) using a luminometer (Lumat LB 9501, Berthold Evry, France). To measure ␤-galactosidase activity, 250 ␮l of 60 mm Na 2 HPO 4 (2 H 2 O), 40 mm NaH 2 PO 4, 10 mm KCl, 1 mm MgSO 4 (7 H 2 O) (pH 7) containing 3.5␮l/ml ␤-mercaptoethanol was added to 80 ␮l cell lysate and incubated at 37 C for 2 h with 100 ␮l ortho-nitrophenyl galactopyranoside (4 mg/ml) in 100 mm phosphate buffer (pH 7) before reading the OD at 420 nm. Arbitrary luciferase units were normalized for ␤-galactosidase activity, and results were expressed relative to untreated controls.
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Involvement of connexin 43 in human trophoblast cell fusion and differentiation.

Involvement of connexin 43 in human trophoblast cell fusion and differentiation.

In this study, we show that Cx43 expression is also involved in cell fusion. Gap junction have been implicated in placental development (for review see Winterhager et al., 2000). Ultrastructural studies have detected gap junctions between the trophoblastic layers in placentas (de Virgiliis et al., 1980, Firth et al., 1980, Malassiné and Leiser, 1984). Furthermore, gap junctions are present during cytotrophoblastic cell fusion in the guinea-pig placenta (Firth et al., 1980). In human trophoblast, we have previously demonstrated that Cx 26, 32, 33,40 and 45 are not detected, whereas Cx 43 mRNA and protein are present. Furthermore, using gap-FRAP we have demonstrated the presence of a functional gap junctional inter-trophoblastic communication preceeding trophoblastic fusion. The low occurrence of coupled cells observed (5.3% after 2 days of culture) argues for a brief duration or paucity of this gap junctional communication. This is in accordance with the fact that in trophoblast primary cultures, gap junctions are only observed in a low number of cells with transmission electron microscopy (Cronier et al., 1994).
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Annexin-A5 organized in 2D-network at the plasmalemma eases human trophoblast fusion OPEN

Annexin-A5 organized in 2D-network at the plasmalemma eases human trophoblast fusion OPEN

desmoplakin immunostaining (Fig. 2A). By contrast, trophoblasts treated with recombinant AnxA5 presented discontinuous desmoplakin immunostaining already after 24 h of culture, indicating the presence of syncytia (Fig. 2A). AnxA5 triggered cytotrophoblasts fusion in a concentration dependent manner (Supplementary mate- rial Fig. S1D). Interestingly, cells cultured with AnxA5-2Dmut behaved like control cultures, which suggests that AnxA5 organized in a 2D-arrays drives the fusion process (Fig. 2A). In a search for exposed PS, cells were subjected to an AnxA5-FITC binding assay. Cells were treated with EGTA, which chelate calcium and dissociate putative preexisting AnxA5 bound to exposed-PS (Fig. 2B). It is noteworthy that we did not observe significant changes in trophoblast fusion after EGTA treatment (Supplementary material Fig. S1E). After 24 h of culture, we noticed that a few PSs were exposed by cytotrophoblasts as evident from weak AnxA5-FITC staining and corresponding histograms from control and EGTA-treated cells (Fig. 2B). This suggests that few cytotrophoblasts exposed PS. In contrast, at 72 h of culture, EGTA-treated syncytiotrophoblasts revealed a large exposition of PS that was masked in untreated cells by endogenous AnxA5 (Fig. 2B; p < 0.001). These indicate that trophoblast fusion is associated with an increase in PS exposition masked by endogenous AnxA5. These data are in agreement with our differential labelling experiments (Fig. 1C). Subsequently, trophoblast cultures were induced for fusion with recombinant AnxA5 (10 μ g/ml) and subjected to AnxA5-FITC binding assay (Fig. 2C). As evident from histograms and immunostaining, recombinant AnxA5 induced syncytia formation with a large exposition of PS masked by AnxA5 (Fig. 2C; p < 0.001). It is noteworthy that AnxA5 bound to exposed-PS is often associated with early apoptosis. Thus, the level of apoptosis was evaluated during cell fusion with or without addition of recombi
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Opposite Effects of Transforming Growth Factor-β Activation and Rho-Associated Kinase Inhibition on Human Trophoblast Migration in a Reconstituted Placental-Endometrial Coculture System

Opposite Effects of Transforming Growth Factor-β Activation and Rho-Associated Kinase Inhibition on Human Trophoblast Migration in a Reconstituted Placental-Endometrial Coculture System

We also tested the effect of an inhibition of the Rho-ROCK signaling pathway, using the Y27632 inhibitor. Quite inter- estingly, both treatments induced an enhanced superficial trophoblastic migration that appeared to occur at the expense of interstitial invasion of the fibroblast layer. Indeed, extra- villous cytotrophoblast migration exhibited a lesser depen- dence on the fibroblast orientation patterns as EVCTs were also able to migrate across transversally oriented endome- trial fibroblasts (Fig. 5A, bottom panels). Y27632-dependent inhibition of ROCK was linked to an elongated EVCT cell morphology, as shown by cytokeratin 7 staining (Fig. 5B). In addition, confocal analysis of cocultures of prestained EVCTs and endometrial fibroblasts showed that in presence of Y27632, migration of EVCTs had a lesser impact on the en- dometrial fibroblast layer integrity, compared with control conditions or TGF!1-treated cultures (Fig. 5C). Whereas TGF!1 treatment led to the disappearance of endometrial fibroblasts after passage of EVCTs, in Y27632 conditions, the cell morphology of the endometrial fibroblasts apparently remained unchanged. Because of the variability between villi, inherent to the use of human specimens, we extended our survey of the effects of TGF!1 and Y27632 treatments to a total of five different placentas, analyzing about 40 villi for each of the three conditions (TGF!1, Y27632, or control) (Fig. 6). For each placenta considered, we evaluated the effect of the above-mentioned treatments on two main characteristics of the cocultures: 1) EVCT migration capacity (Fig. 6, A–C) and 2) size of lacunes made by EVCTs within the endometrial fibroblast layer (Fig. 6D). As previously observed (see Figs. 1, 2, and 4), EVCT migration in the control conditions took place preferentially along the fibroblast longer orientation axis (migration dependent on the fibroblast orientation pat- tern). Treatment with TGF! appeared to frequently block EVCT migration, whereas, on the contrary, Y27632 treatment seemed to induce EVCT migration, whatever the fibroblast orientation pattern (migration independent of the fibroblast orientation pattern).
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An overview of molecular events occurring in human trophoblast fusion

An overview of molecular events occurring in human trophoblast fusion

involved in trophoblast fusion (in human primary cell cultures and cell lines), as these have already.. 42.[r]

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Trophoblast production of a weakly bioactive human chorionic gonadotropin in trisomy 21-affected pregnancy.

Trophoblast production of a weakly bioactive human chorionic gonadotropin in trisomy 21-affected pregnancy.

mRNA in trophoblast cells of T21-affected placenta. This was based on a northern analysis performed with cells cultured for 24hours. In this study the secretion of hCG at 24 hours is extremely variable from one T21 cell culture to another one. In addition the secretion of hCG at 24 hours in normal cells or in T21 cells is higher than the one observed at 72h and 96h. This suggests a contamination of cytotrophoblast cell cultures by fragments of syncytiotrophoblast, which explains the highest transcript levels of hCG. Indeed it is now well established that hCG transcript levels and hCG secretion are higher in the syncytiotrophoblast as compared to the cytotrophoblasts (9, 21-24). Jauniaux et al (25) and Newby et al (26) have compared the levels of hCG and/or of its free subunits, detected by immunoassay in placental total homogenate extracts. This is a different methodological approach, focused on the protein levels and thereby depending upon the specificity of the antibodies. In addition total placental extracts are contaminated by maternal blood which may lead to artefactual increase in hCG levels and therefore do not directly reflect the trophoblast synthesis of hCG. In addition, Newby et al, did not find an increase in hCG by immunohistochemistry.
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Bacteremia after hematopoietic stem cell transplantation: incidence and predictive value of surveillance cultures.

Bacteremia after hematopoietic stem cell transplantation: incidence and predictive value of surveillance cultures.

SUMMARY We studied 622 transplants between 1982 and 2001 to (1) determine the incidence, timing and etiology of bacteremias, and (2) examine the ability of routine surveillance cultures to predict bacteremias. A total of 404 episodes (0.65 episode per patient) occurred in 248 patients, due to coagulase-negative staphylococci (n=171, 42%), gram-negative bacteria (n=129, 32%), streptococci (n=48, 12%), other gram-positive bacteria (n=33, 8%), anaerobes (n=9, 2%) and fungi (n=14, 3%). Bacteremias were more frequent in allogeneic (0.96 episode/patient) compared to autologous (0.44) transplants (p<0.0001). The overall incidence decreased from 0.92 episode/patient until 1990 to 0.66 in 1991-1996 and 0.55 in 1997- 2001 (p<0.0001), but this was only observed in autologous transplants. Among them, 212 (53%) occurred before hospital discharge and 192 (47%) thereafter. This proportion was lower for coagulase- negative staphylococci, other gram-positive bacteria and gram-negative bacteria compared to other agents (p=0.001). In 50% of the cases, the agent responsible for the bacteremic episode was present in routine surveillance cultures before. In conclusion: (1) bacteremias remain a frequent complication, particularly in allogeneic transplantation, even long after hospital discharge; (2) routine surveillance cultures can predict bacteremias in 50% of the cases but the practical impact of this observation is limited in view of the costs.
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Levels of polyunsaturated fatty acids correlate with growth rate in plant cell cultures.

Levels of polyunsaturated fatty acids correlate with growth rate in plant cell cultures.

whereas 18:1 strongly increased, suggesting that the rate of desaturation of 18:1 was reduced during the fast-growing period. We therefore sought to correlate in Arabidopsis the growth rate in standard complete medium with the 18C unsaturation level, exploiting the variability that occurs between different culture cycles. In Fig.  2A,B, we plotted the unsaturated level of 18C versus the growth rate for more than 10 different cultures grown in the same environment and harvested 3 days after renewing the media. We observed a significant correlation between the growth rate and the proportions of 18:1 and 18:2+ 18:3: with higher growth rates there were higher levels of 18:1 and lower levels of 18:2+ 18:3. Detailed analyses of the lipid composition in fast growing and slow growing cell cultures show that slow growing cells displayed 25–30% less lipids, but an unchanged distribution of the main glycerolipids (Fig.  2C,D). However, the levels of 18:1 and PUFAs were modified in PC and PE as it was observed in the total FA analysis (Fig.  2E). Diacylglycerols (DAGs) also were affected but not triacylglycerols (TAGs), suggesting that the synthesis of TAGs from DAG backbones favor PUFA rich species. The fact that DAG and PC were similarly affected illustrates the dynamic acyl exchange between these two lipids via the phosphatidylcho- line:diacylglycerol choline phosphotransferase (PDCT). Together with acyl editing, this process might also play a central role in the regulation of glycerolipid desaturation 25 . Interestingly, the PUFA levels
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Immersive Analysis of 3D Multi-Cellular In-Vitro and In-Silico Cell Cultures

Immersive Analysis of 3D Multi-Cellular In-Vitro and In-Silico Cell Cultures

II. R ELATED W ORK Eliceiri et al. [9] provide a review of the state-of-the- art in biological imaging software tools. Among the tools available, Fiji [6] is one of the standard tools for the analysis of cell structures. It provides an easy-to-set-up distribution of ImageJ [10], making the image processing pipeline for multi- dimensional data accessible. Fiji and ImageJ are extensible through a plugin architecture and enjoy large community support. However, 3D visualization – especially immersive visualization – is not a focus of the platform. BioImageXD [11] is a visual analysis platform for the exploration of 3D image data with a focus on 3D interactive visualization. It further includes segmentation, filtering, and tracking features and aims to be user-friendly by not requiring programming skills. However, its code repository appears not to have been updated since 2012. Vaa3D [12] also provides spatio-temporal image visualization and analysis tools with a focus on 3D rendering. It supports virtual reality (VR) hardware and large image data sets. Furthermore, it includes various analysis tools such as segmentation, annotation, and quantitative mea- surements. It is still under active development. In contrast to the aforementioned tools, we aim to make simulation a first-class citizen of our applications. VTK [13] is a proven visualization toolkit for scientific applications, providing a framework for the development of many state-of-the-art appli- cations. It is open-source and has a vivid community. Recently, some competition emerged from game engine technology [14]. Game engines are often significantly less efficient at rendering large-scale data sets and volumetric data and at providing data processing pipelines and built-in scientific visualizations. However, they typicslly provide accessible, GUI-driven in- tegrated development environments, special features for the creation of rich user interfaces, and support for state-of-the- art consumer hardware. D¨aschinger et al. [15] presented a prototype for a full workflow-oriented pipeline for integrated modeling and simulation of cell-centered biological assays based on the Unreal game engine. VisNEST [8], [16] is a visual analysis tool for neuroscientists to explore the results of a multi-scale simulation of a macaque monkey brain. [17] used VR head-mounted displays (HMD) to visualize molecular dynamics, using ray tracing algorithms for optimized perfor- mance and quality. [18] provided a case study on analysing the structure and function of blood vessels using 3D and immersive visualization, highlighting the benefits of VR for understanding spatial relations.
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Genetic analysis of cell size homeostasis in human cells

Genetic analysis of cell size homeostasis in human cells

iii Abstract All organisms are essentially structured and fitness defined by cell number, type and size. Composed of around 30 trillion cells, humans have cells with remarkably varied functions and size, ranging from a neuron that can reach one meter in length to a lymphoid cell that is around 16 µm in diameter. At a fundamental level, size is determined by the balance between cell growth and cell division. The molecular networks that control and maintain optimal cell size are yet to be deciphered. The mTORC1 pathway is a major regulator of cell growth that plays a central role in integrating intra- and extra-cellular stimuli. This study addresses the investigation and characterization of the molecular players and processes that orchestrate cell size in human cells, as determined by chemical-genetic size screens and epistasis analysis. I undertook a CRISPR/Cas9 extended-knockout (EKO) genome-wide library screen in the NALM-6 pre-B lymphoma cell line, followed by cell size fractionation by counter flow elutriation in the presence of the mTOR inhibitor rapamycin, and compared the screen data to a similar screen performed in the absence of rapamycin. The analysis indicates that upstream of mTOR, the loss of genes that are related to nutrient sensing, results in size changes in the presence of mTOR inhibition. Also, several gene knockouts in ribosome biogenesis and calcium homeostasis led to size alterations, suggesting a possible a pivotal role of these processes in cell size control in a mTOR-dependent fashion. This study provides insights into the genetic networks that regulate cell size in a mTOR- dependent fashion and establishes new hypotheses for future experimental tests.
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Immersive Analysis of 3D Multi-Cellular In-Vitro and In-Silico Cell Cultures

Immersive Analysis of 3D Multi-Cellular In-Vitro and In-Silico Cell Cultures

Scenario B: Fluorescence Microscopy Images of Organoids: Early on during mammalian embryo development, cells of the central part of the embryo (inner cell mass, ICM), differ- entiate into embryonic or extra-embryonic tissue. In mouse, the common model system, this occurs around day 3.5 after fertilisation. The decision of the cells can be inferred from the expression of the two transcription factors NANOG and GATA6 [22]. Quantification of the 3D spatial pattern which arises during cell differentiation is of major interest, as the results provide a basis for further studies of the mechanisms underlying cell differentiation. Current approaches of quan- tifying the pattern include 3D fluorescence microscopy, 3D segmentation of the cell nuclei and measuring the intensities of NANOG and GATA6 in the segmented nuclei [21], see Figure 1. As a result of the image analysis pipeline, more than 30 features for the individual cells and the whole organoid can be extracted [19], a selection of which you can find in Table II. For ICM organoids, the quantitative analysis indicates a local clustering of cells expressing NANOG and GATA6. The details of this clustering and its underlying mechanisms need further investigation. Simulations rely on agent-based models [23], [24]. These models incorporate physical interactions of the cells, cell signalling, cell division and cellular rearrange- ment. Testing and further improvement of these models relies on comparison of the modeling results with a quantitative description of the 3D protein expression patterns.
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Bisphenol S Impaired Human Granulosa Cell Steroidogenesis In Vitro

Bisphenol S Impaired Human Granulosa Cell Steroidogenesis In Vitro

Abstract: Bisphenol S (BPS) is a structural analog of the endocrine disruptor bisphenol A (BPA); it  is the main BPA replacement in the plastics industry. Previous studies have shown that BPA and  BPS  exhibit  similar  effects  on  reproduction  in  fish  and  rodent  species.  BPS  reportedly  alters  steroidogenesis in bovine granulosa cells. Luteinised granulosa cells collected from 59 women who  were undergoing an in vitro fertilization procedure were cultured for 48 h in the presence or absence  of  BPS  (10  nM,  100  nM,  1  μM,  10  μM  or  50  μM).  BPS  exposure  was  investigated  by  assessing  follicular fluids from these 59 women for their BPS content. Culture medium, cells, total messenger  RNA (mRNA) and total protein extracted from the luteinised granulosa cells were examined for  oestradiol  and  progesterone  secretion,  cellular  proliferation,  viability,  gene  expression,  steroidogenic enzyme expression and cell signaling.  BPS was measured in follicular fluids using  mass spectrometry. Exposure of granulosa cells to 10 or 50 μM BPS for 48 h induced a 16% (p =  0.0059) and 64% (p < 0.0001) decrease, respectively, in progesterone secretion; 50 μM BPS decreased  oestradiol  secretion  by  46%  (p  <  0.0001).  Ten  μM  BPS  also  tended  to  reduce  CYP11A1  protein  expression by 37% (p = 0.0947) without affecting HSD3B1 and CYP19A1 expression. Fifty μM BPS  increased ERR expression. Environmental levels of BPS (nanomolar range) did not induce changes  in steroidogenesis in human granulosa cells. The effects of BPS were observed after only 48 h of BPS  exposure. These acute effects might be similar to chronic effects of physiological BPS levels. 
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Regulation of trophoblast development in the bovine embryo

Regulation of trophoblast development in the bovine embryo

development in embryos produced by somatic ceil nuclear transfer (SCNT) is associated with a high number of pregnancy losses. Several studies have described morphological development of [r]

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Vimentin Contributes to Human Mammary Epithelial Cell Migration

Vimentin Contributes to Human Mammary Epithelial Cell Migration

DISCUSSION Association of vimentin with epithelial cell migration In the present study, vimentin expression was examined in human mammary epithelial MCF10A cells as a function of their migratory status. Using an in vitro wound healing model, we clearly showed that vimentin was specifically induced in the subpopulation of MCF10A cells at the wound’s edge. Vimentin protein and mRNA were induced in this cell subpopulation as assessed by immunolabeling and in situ hybridization, respectively. In support of our in situ hybridization results, the data obtained with MCF10A cells transfected with the vimentin promoter controlling the GFP reporter gene revealed a specific activation of the vimentin promoter in cells located at the wound’s edge. These results are in agreement with our and other’s previous results which have described vimentin expression in epithelial cells involved in wound healing (SundarRaj et al., 1992; Buisson et al., 1996) or other processes requiring cell migration (Savagner et al., 1994; Guarino, 1995; Hay, 1995; Gilles and Thompson, 1996). Our present data obtained with a cellular model in which vimentin expression could be induced and studied in relation with cell migration nevertheless allowed to establish for the first time a direct relationship between these two functions. The time-lapse video recordings indeed demonstrated that the vimentin-expressing subpopulation of MCF10A cells at the wound’s edge are actively migrating. Also, proliferation studies excluded the possibility that vimentin expression could be associated to cell proliferation rather than cell migration. In addition, the observation that, among cells at the wound’s edge, those expressing vimentin migrated faster than the scarce vimentin-negative ones further strengthened the concept that vimentin is associated with the migratory status. We accordingly demonstrated that cells distant from the lesion did not express vimentin, moved erratically without an orientated trajectory and remained essentially stationary. Furthermore, MCF10A cells at the wound’s edge formed lamellipodia and their actin filaments were reorganized, events typical of a migratory phenotype. Actin reorganization in migrating cells at the leading front is indeed a key-element in the extension of lamellipodia associated with cell movements (Welch et al., 1997). In MCF10A cells at the wound’s edge, actin clearly
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Stromal cell contribution to human follicular lymphoma pathogenesis.

Stromal cell contribution to human follicular lymphoma pathogenesis.

Keywords: B cells, bone marrow, lymph nodes, cell interactions, stromal cells B CELL LYMPHOMA MICROENVIRONMENT Human mature B cell lymphomas represent a heterogeneous group of neoplasias characterized by recurrent genetic abnormal- ities and pathway dependencies. Each lymphoma subtype could be assigned to a peculiar stage of normal B cell differentiation, as judged by gene expression profiling and phenotype ( Shaffer et al., 2012 ). In some of them, malignant B cell proliferation, survival, and drug resistance are strongly dependent on a com- bination of external stimuli delivered by the microenvironment within specific niches in invaded lymph nodes (LN) and bone marrow (BM; Burger et al., 2009 ). This is particularly true in fol- licular lymphoma (FL), the most frequent indolent lymphoma, which results from the malignant transformation of germinal cen- ter (GC)-derived B cells (Figure 1). Whereas over 90% of FL cases display a BCL2/IGH translocation, this early event, occurring as a mistake of V(D)J recombination in the BM, could be detected at low frequency within recirculating post-GC memory B cells of most healthy individuals ( Roulland et al., 2011 ). These t(14;18) pos cells exhibit additional characteristics that stand as hallmarks of FL cells; i.e., CD10 expression, unleashed AID activity, persistence of surface IgM despite active class-switch recombination on the translocated allele; are thus called follicular-lymphoma like cells. Given that the actual prevalence of FL is around 0.03%, it is clear that FL pathogenesis requires additional oncogenic events as well as a progressive modification of the composition and organization of tumor microenvironment ( Bende et al., 2007 ). Among the recurrent complementary hits identified in FL patients, several alterations target the transcriptional and epigenetic path- ways including inactivation of CREBBP/EP300 acetyltransferases,
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Neuropeptides activate human mast cell degranulation and chemokine production

Neuropeptides activate human mast cell degranulation and chemokine production

mary cultured mast cells, generated from CD34 + progenitors in the pres- ence of stem cell factor and interleukin-6 (IL-6), and human cultured mast cells (LAD2) were stimulated with these and other stimuli (gastrin, conca- navalin A, radiocontrast media, and mannitol) and their degranulation and chemokine production was assessed. VIP and SP stimulated primary human mast cells and LAD cells to degranulate; gastrin, concanavalin A, radiocontrast media, mannitol, CGRP and NGF did not activate degranula- tion. While anti-IgE stimulation did not induce significant production of chemokines, stimulation with VIP, SP or compound 48/80 potently induced production of monocyte chemoattractant protein-1, inducible pro- tein-10, monokine induced by interferon-c (MIG), RANTES (regulated on activation, normal, T-cell expressed, and secreted) and IL-8. VIP, SP and compound 48/80 also activated release of tumour necrosis factor, IL-3 and granulocyte–macrophage colony-stimulating factor, but not IL-4, inter- feron-c or eotaxin. Human mast cells expressed surface neurokinin 1 receptor (NK1R), NK2R, NK3R and VIP receptor type 2 (VPAC2) but not VPAC1 and activation of human mast cells by IgE/anti-IgE up-regulated expression of VPAC2, NK2R, and NK3R. These studies demonstrate the pattern of receptor expression and activation of mast cell by a host of G-protein coupled receptor ligands and suggest that SP and VIP activate a unique signalling pathway in human mast cells. These results are likely to have direct relevance to neuronally induced inflammatory diseases.
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Endometrial Tumor Microenvironment Alters Human NK Cell Recruitment, and Resident NK Cell Phenotype and Function

Endometrial Tumor Microenvironment Alters Human NK Cell Recruitment, and Resident NK Cell Phenotype and Function

The recruitment of immune cells, including NK cells is regulated by tissue produced chemokines, which, in our study, may explain the lack of NK cells in the tumor. Indeed, we could find that the production of various chemokines was modified in the tumor compared to the adjacent healthy tissue. IP-10, also known as CXCL10, is a chemoattractant for NK cells, and can activate NK cells leading to the lysis of tumor cells ( 37 – 39 ). Interestingly, we showed here that IP-10 is strongly produced in the tumor compared to the adjacent tissue, suggesting that an immune response is occurring in the tumor. Though, considering the results we observed from the functional tests, it may not be sufficient to recruit NK cells and to maintain an efficient NK cell-driven cytolytic response. CCL27, is also a NK cell chemoattractant molecule, and is, in endometrial cancer, less produced in the tumor compared to the healthy adjacent tissue. Similarly, CCL21, which has been described to enhance the recruitment of immune cells in the tumor and to participate to the establishment of a potent immune cellular response ( 40 – 42 ), is significantly reduced in the tumor microenvironment compared to the adjacent tissue. The lower levels of NK cell chemoattractants (CCL27, CCL21) in the tumor microenvironment may participate to the immunosuppressive mechanism of the tumor by limiting cytolytic cell recruitment.
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Dental cell type atlas reveals stem and differentiated cell types in mouse and human teeth

Dental cell type atlas reveals stem and differentiated cell types in mouse and human teeth

Fig. 3 Identi fication of previously unrecognized cell types in dental epithelium and stem cells. a–e In situ hybridization (Igfbp5, Gjb3) and immunohistochemistry (PIEZO2, EGR1, and CLDN10) validations of selected markers demarcating different progenitor and differentiated states in epithelial layer. Note: Validation of expression of Igfbp5 enables identi fication of outer enamel epithelium clusters on a t-SNE representation (a). Validation of PIEZO2-expressing cells shows sporadic cells inside the ameloblast layer (b). Egr1 + cells are present in the progenitor area on the edge of stellate reticulum and outer enamel epithelium (c). Mapping of Gjb3 on the section tissue consistently reveals the position in stellate reticulum within the labial cervical loop (d). Validation of Cldn10 expression helps to outline all non-ameloblastic parts of epithelial differentiation including developing stratum intermedium and outer enamel epithelium (e). f Acta2 CreERT2 /R26 tdTomato genetic tracing shows signi ficant contribution of Acta2 + cells of the labial cervical loop to more differentiated cell types of dental epithelium including ameloblasts, stellate reticulum, outer enamel epithelium, and stratum intermedium after 3 days, 2 weeks, and 1 month long tracing period. g, h Immunohistochemistry identi fication of the Cuboidal layer of stratum intermedium (expressing THBD) and spatial relation to the neighboring blood vessels submerged into the papillary structure of stratum intermedium. COL4 expression characterizes the blood vessels on left panel. h Papillary structure of stratum intermedium with submerged blood vessels (COL4) and CDH1 expressing ameloblasts and cells from stratum intermedium. Note. Cuboidal layer characterized by THBD expression (g) forms subpopulation of stratum intermedium cells (h).
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Cripto is essential to capture mouse epiblast stem cell and human embryonic stem cell pluripotency

Cripto is essential to capture mouse epiblast stem cell and human embryonic stem cell pluripotency

during embryogenesis 17 . Finally, Cdx2-positive cells also express the TE maker Gata3 (ref. 36). Concomitant to induction of TE markers, expression of the DNA methyltransferase Dnmt3b is reduced in F/A Cripto KO cells. Interestingly, Dnmt-deficient and therefore hypomethylated ESCs/embryos show transdifferentiation to the extraembryonic trophoblast lineage 47 . In line with these findings, morphological and molecular analyses indicate that Cripto KO ESCs facilitate the generation of TSCs in vitro. Interestingly, several evidence indicate that BMP signalling pathway plays a crucial role in TE differentiation of pluripotent stem cells. Specifically, Cdx2 expression is directly regulated by the BMP–Smad1/5 pathway 48 , which in turn is repressed by Smad2 (ref. 37). Accordingly, Cripto KO inhibits Smad2 activation and conversely induces BMP-dependent Smad1/5 phosphorylation, suggesting that derepression of BMP signalling may account for TE differentiation of F/A-induced Cripto KO cells. Most remarkably, according to the idea that Cripto deficiency attenuates the normal restriction of mouse ESCs to embryonic lineages, Cripto KO ESCs gain the unique property to colonize TE in blastocyst chimera, which is absent in WT ESCs 40 . However, analysis of E6.5 chimeric embryos generated with Cripto KO ESCs indicates that these cells efficiently contribute to the embryonic but not to the ectoplacental cone. Although we cannot rule out the possibility that Cripto KO ESCs that colonize the TE fail to differentiate properly and thus to efficiently contribute to the trophoblast derivatives, we speculate that a non-cell autonomous activity of Cripto in the WT environment may rescue, at least in part, the mutant phenotype. Although further investigations are required to directly address this issue, this hypothesis is consistent with co-culture data showing that non- cell autonomous Cripto is able to rescue F/A Cripto KO EpiSCs. Intriguingly, to date, no defects in EPI specification/maintenance have been reported in Cripto-null mutants that indeed are defective in anterior–posterior axis formation 14 . Of note, data in
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Peroxisome proliferator‐activated receptor gamma‐ligand‐binding domain mutations associated with familial partial lipodystrophy type 3 disrupt human trophoblast fusion and fibroblast migration

Peroxisome proliferator‐activated receptor gamma‐ligand‐binding domain mutations associated with familial partial lipodystrophy type 3 disrupt human trophoblast fusion and fibroblast migration

In conclusion, the evidence presented here clearly shows that a single missense or nonsense mutation in the LBD of PPARG signifi- cantly inhibits cell fusion and migration processes. F I G U R E 4   R262G and L339X mutations induce morphological changes that reduce fibroblast migration. Wild-type and PPARG-mutated fibroblasts were seeded at equal densities into a 96-well plate (n = 3-5 wells/condition), cultured to confluency, mechanically wounded by scratching and then monitored for 24 h using IncuCyte Zoom. A, Representative images of fibroblast migration at 24 h. The blue region denotes the area of the initial wound (light blue line) covered by advancing cells. B, Time course of wound closure expressed as RWD (%). Values are represented as mean ± SEM of the percentage. C, Comparison between wild-type and PPARG-mutated fibroblasts is shown by AUC analysis of the replicates. Values are represented as mean + SEM. Statistical analysis was performed with paired t test to compare wild-type controls. ****P < .0001. D, Representative images showing immunofluorescent staining for F-actin (red) in upper panels, and vimentin (VIM, green) and vinculin (VINC, red) in lower panels. Cell nuclei were also stained with DAPI (blue). Scale bar: 50 µm. Insets depict a cytoskeleton architecture model of wild-type (WT, Green) and PPARG-mutated (red) fibroblasts
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