Enhanced differentiation of functional human T cells in NSGW41 mice with tissue-specific expression of human interleukin-7.

Humanized mouse models have become increasingly valuable tools to study human hematopoiesis and infectious diseases. However, human T-cell differentiation remains inefficient. We generated mice expressing human interleukin-7 (IL-7), a critical growth and survival factor for T cells, under the control of murine IL-7 regulatory elements. After transfer of human cord blood-derived hematopoietic stem and progenitor cells, transgenic mice on the NSGW41 background, termed NSGW41hIL7, showed elevated and prolonged human cellularity in the thymus while maintaining physiological ratios of thymocyte subsets. As a consequence, numbers of functional human T cells in the periphery were increased without evidence for pathological lymphoproliferation or aberrant expansion of effector or memory-like T cells. We conclude that the novel NSGW41hIL7 strain represents an optimized mouse model for humanization to better understand human T-cell differentiation in vivo and to generate a human immune system with a better approximation of human lymphocyte ratios.

Molecular characteristics of established trophoblast-derived cell lines.

INTRODUCTION: Research on human placental development and function lacks a conclusive in vivo model. To investigate the intracellular molecular mechanisms in trophoblast cells, different cell lines have been established during the last decades. So far, none of these accomplishes all features of primary trophoblast, thus their suitability as well as the transferability of the results has been discussed. The aim of this study is to assess molecular markers and features matching different trophoblast subpopulations in trophoblastic cell lines to provide orientation on their suitability and relevance for distinct research questions. METHODS: The commonly used trophoblastic cell lines, BeWo, JEG-3, HTR-8/SVneo, AC1-M59, AC1-M32, ACH-3P and Swan71 were selected. qPCR and immunoblotting were used to determine expression of characteristic molecular markers. C14MC, C19MC and miR-371-3 miRNA expression were investigated by real time PCR. Proliferation, migration and network stabilization assays were performed. Hormone secretion was determined by chemiluminescent-immunoassays. DNA profiles were obtained by Short Tandem Repeat (STR)-genotyping. RESULTS: Immortalized cell lines differ from choriocarcinoma-derived ones in the expression of HLA-G, E-cadherin, N-cadherin, VE-cadherin, cadherin-11, cytokeratin 7, vimentin, ADAM12 and PRG2. Compared to choriocarcinoma-derived cell lines, expression of C19MC and hormone secretion were almost absent in immortalized cell lines. Conversely, they express C14MC and exhibit higher migration and network stabilization. DISCUSSION: The data presented will help justify the use of a cell line to evaluate distinct features of trophoblast biology and pathology. In general, characteristics and markers of choriocarcinoma derived cell lines seem to be more similar to in vivo trophoblast than immortalized cell lines and thus might be regarded as more suitable models.

Role of IL-36 Cytokines in the Regulation of Angiogenesis Potential of Trophoblast Cells.

IL-36 cytokines (the agonists IL-36α, IL-36ß, IL-36γ, and the antagonist IL-36Ra) are expressed in the mouse uterus and associated with maternal immune response during pregnancy. Here, we characterize the expression of IL-36 members in human primary trophoblast cells (PTC) and trophoblastic cell lines (HTR-8/SVneo and JEG-3) and upon treatment with bacterial and viral components. Effects of recombinant IL-36 on the migration capacity of trophoblastic cells, their ability to interact with endothelial cells and the induction of angiogenic factors and miRNAs (angiomiRNAs) were examined. Constitutive protein expression of IL-36 (α, ß, and γ) and their receptor (IL-36R) was found in all cell types. In PTC, transcripts for all IL-36 subtypes were found, whereas in trophoblastic cell lines only for IL36G and IL36RN. A synthetic analog of double-stranded RNA (poly I:C) and lipopolysaccharide (LPS) induced the expression of IL-36 members in a cell-specific and time-dependent manner. In HTR-8/SVneo cells, IL-36 cytokines increased cell migration and their capacity to interact with endothelial cells. VEGFA and PGF mRNA and protein, as well as the angiomiRNAs miR-146a-3p and miR-141-5p were upregulated as IL-36 response in PTC and HTR-8/SVneo cells. In conclusion, IL-36 cytokines are modulated by microbial components and regulate trophoblast migration and interaction with endothelial cells. Therefore, a fundamental role of these cytokines in the placentation process and in response to infections may be expected.

[Pregnancy after Obesity and Metabolic Surgery - Risks and Complications]. / Schwangerschaft nach Adipositas- und metabolischer Chirurgie ­ spezielle Risiken und mögliche Komplikationen.

The increasing prevalence of morbid obesity in Germany is associated with an increasing number of metabolic surgical interventions. Short-term surgical and long-term metabolic complications - such as nutrient deficiencies - are the main risks of metabolic surgery and the resulting malabsorption. Obesity, especially morbid obesity, is associated with a high incidence of female infertility. One important cause of female infertility in obese women is the polycystic ovary syndrome, with 6 - 10%. Metabolic surgery significantly increases the fertility of obese women. The positive effect of obesity surgery on weight loss, remission of comorbidities, psychological outcome and fertility (in comparison with the effect of conservative treatment) has led to an increase in the number of metabolic operations. Nutrient deficiencies after restrictive, combined and malabsorptive procedures must be considered. Prophylaxis of these deficiencies during pregnancy after obesity surgery must be based on intensive interdisciplinary treatment. The aim of this overview is to characterise the metabolic complications and their prophylaxis, which are specific for the various bariatric procedures and which, subsequently, require temporary or permanent surveillance and supplementation.

PP057. ENOSI4 and EPHX1 polymorphisms affect maternal susceptibility topreeclampsia - Analysis of five polymorphisms predisposing tocardiovascular disease in 279 caucasian and 241 african women.

OBJECTIVES: Clinical studies have documented a familiar tendency to develop preeclampsia and patients with impaired endothelial health are at increased risk. We studied genetic polymorphisms associated to cardiovascular disease and impaired endothelial function in women with and without preeclampsia. METHODS: 241 African and 279 Caucasian women were recruited for genetic testing of the polymorphisms epoxide hydrolase 1 (EPXH1), endothelial nitric oxide synthase (NOS3) on exon 7 and variable nucleotide tandem repeats in intron 4 (NOS3I4a/b), angiotensinogen and the estrogen receptor1 polymorphism in intron 1. RESULTS: Of 241 African women, 95 developed preeclampsia and out of the 279 Caucasian women 81 had preeclampsia. Carriers of the NOS3I4a/b polymorphism had a 1.7-fold increased risk to develop preeclampsia. There was no difference in distribution of the other tested polymorphism. Furthermore we could show a fourfold reduction to encounter severe course of preeclampsia (defined as occurrence of HELLP-syndrome or eclampsia) in carriers of the EPHX1 polymorphism encoding histidine. CONCLUSIONS: Our data reveal a highly significant association of the NOS3I4a/b polymorphism with increased risk to develop preeclampsia in pregnancy. The shown association of EPXH1 polymorphism with the severity of preeclampsia strengthen the concept, that individual susceptibility determines the capability of the maternal organism to deal with the pregnancy derived agents causing preeclampsia.

17 beta-estradiol transiently disrupts adherens junctions in endothelial cells.

Interendothelial junctions are important regulators of endothelial cell functions such as migration and proliferation, major features in angiogenesis, and endothelial cell monolayer wound healing. 17beta-estradiol regulates these functions in vivo and in vitro and also increases endothelial monolayer permeability as it results from impaired monolayer integrity and intercellular adhesion. We hypothesized that 17beta-estradiol affects these cell adhesion-dependent functions in endothelial cells by targeting the adherens junction complex. Here, we show that 17beta-estradiol increases uterine microvascular endothelial cell monolayer permeability and transiently redistributes interendothelial junction-forming proteins in endothelial cells. Concomitantly, adherens junction proteins are disconnected from the cytoskeleton and alpha-catenin, which links VE-cadherin to the cytoskeleton, is redistributed from the membrane and the adherens junction complex. Furthermore, 17beta-estradiol increased tyrosine phosphorylation of the adherens junction complex. These effects were inhibited by the estrogen receptor antagonist ICI 182,780 but could be provoked using non-cell membrane-permeable 17beta-estradiol-BSA in all cells tested, including EA.hy 926 cells, which have been shown unable to stimulate 17beta-estradiol-dependent gene transcription. Additionally, 17beta-estradiol treatment enhanced the angiogenic effect of vascular endothelial growth factor in an in vitro angiogenesis model, as a potential implication of the adherens junction disruption. Cotreatment with the Src-family kinase inhibitor PP2 prevented the redistribution and phosphorylation of the adherens junction proteins. Taken together, our data show that adherens junctions in endothelial cells are a downstream target of membrane-associated 17beta-estradiol signaling, possibly through Src-family kinases.