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The invasion of the uterine wall by extravillous trophoblast is acknowledged as a crucial component of the establishment of pregnancy however, the only part of this process that has been clearly identified is the differentiation of cytotrophoblast (CTB) into the invasive extravillous trophoblast (EVT). The control of invasion, both initiation and termination, have yet to be elucidated and even the mechanism of differentiation is unclear. This review describes our studies which are designed to characterize the intracellular mechanisms that drive differentiation. We have used the over-invasion observed in abnormally invasive placenta (AIP; placenta accreta) to further interrogate this mechanism. Our results show that first trimester CTB to EVT differentiation is accomplished via an epithelial-mesenchymal transition (EMT), with EVT displaying a metastable, mesenchymal phenotype. In the third trimester, while the invasiveness of the EVT is lost, these cells still demonstrate signs of the EMT, albeit diminished. EVT isolated from AIP pregnancies do not however, show the same degree of reduction in EMT shown by normal third trimester cells. They exhibit a more mesenchymal phenotype, consistent with a legacy of greater invasiveness. The master regulatory transcription factor controlling the EMT appears, from the observational data, to be ZEB2 (zinc finger E-box binding protein 2). We verified this by overexpressing ZEB2 in the BeWo and JEG3 trophoblast cell lines and showing that they became more stellate in shape, up-regulated the expression of EMT-associated genes and demonstrated a substantially increased degree of invasiveness. The identification of the differentiation mechanism will enable us to identify the factors controlling invasion and those aberrant processes which generate the abnormal invasion seen in pathologies such as AIP and preeclampsia.

The Stat6VT mouse model of atopic dermatitis (AD) is induced by T cell specific expression of a constitutively active form of the protein signal transducer and activator of transcription 6 (STAT6). Although AD-like lesions are known to develop in Stat6VT mice, this study was designed to determine if these mice develop acute and chronic phases of disease similar to humans. To address this, AD-like lesions from Stat6VT mice were harvested at two different time points relative to their onset. Lesions harvested within 1 week after development were defined as acute lesions, and those present for 1 month or more were defined as chronic lesions. Acute and chronic AD-like lesions from Stat6VT mice exhibited histologic findings and cytokine expression patterns similar to acute and chronic AD lesions in humans. Further analysis revealed increased levels of interleukin (IL)-33 transcripts in AD-like lesions compared to Stat6VT nonlesional and wild type skin controls. Immunofluorescence also revealed increased numbers of IL-33(+) keratinocytes in Stat6VT lesional skin and localized IL-33(+) keratinocytes to a keratin 5(+) subset. Furthermore, AD-like disease was more severe in IL-33-deficient Stat6VT mice compared to IL-33 sufficient Stat6VT mice. These studies suggest Stat6VT mice can serve as a model of acute and chronic AD and that IL-33 may attenuate inflammation in this system.

Poly‐ADP ribose polymerase‐14 (PARP14 or ARTD8) was initially identified as a transcriptional co‐activator for signal transducer and activator of transcription 6 (Stat6), where the presence of interleukin‐4 (IL‐4) and activated Stat6 induces the enzymatic activity of PARP14 that promotes T helper type 2 differentiation and allergic airway disease. To further our understanding of PARP14 in allergic disease, we studied the function of PARP14 in allergic inflammation of skin using mice that express constitutively active Stat6 in T cells (Stat6VT) and develop spontaneous inflammation of the skin. We mated Stat6VT mice to Parp14 −/− mice and observed that approximately 75% of the Stat6VT × Parp14 −/− mice develop severe atopic dermatitis (AD)‐like lesions, compared with about 50% of Stat6VT mice, and have increased morbidity compared with Stat6VT mice. Despite this, gene expression in the skin and the cellular infiltrates was only modestly altered by the absence of PARP14. In contrast, we saw significant changes in systemic T‐cell cytokine production. Moreover, adoptive transfer experiments demonstrated that decreases in IL‐4 production reflected a cell intrinsic role for PARP14 in Th2 cytokine control. Hence, our data suggest that although PARP14 has similar effects on T‐cell cytokine production in several allergic disease models, the outcome of those effects is distinct, depending on the target organ of disease.

The main pathogenesis of type 1 diabetes mellitus (T1DM) is autoimmune-mediated apoptosis of pancreatic islet β cells. We sought to characterize the function of microRNA-203a (miR-203a) on pancreatic islet β cell proliferation and apoptosis. In situ hybridization was used to detect the expression of miR-203a in islet β cells in normal and hyperglycaemic non-obese diabetic (NOD) mice. Cell proliferation was measured by cell counting kit eight and cell apoptosis was detected using flow cytometry. Insulin receptor substrate 2 (IRS2/Irs2) was determined to be a direct target of miR-203a by Luciferase reporter assay. We detected the effects of miR-203a overexpression or inhibition on proliferation and apoptosis of IRS2-overexpressing or IRS2-knockdown MIN6 cells respectively, and preliminarily explored the downstream targets of the IRS2 pathway. NOD mice model was used to detect miR-203a inhibitor treatment for diabetes. Our experiment showed miR-203a was upregulated in pancreatic β cells of hyperglycaemic NOD mice. Elevated miR-203a expression inhibited the proliferation and promoted the apoptosis of MIN6 cells. IRS2/Irs2 is a novel target gene directly regulated by miR-203a and miR-203a overexpression downregulated the expression of IRS2. Irs2 silencing reduced cell proliferation and increased apoptosis. Irs2 overexpression could abolish the pro-apoptotic and anti-proliferative effects of miR-203a on MIN6 cells. Hyperglycemia in newly hyperglycemic NOD mice was under control after treatment with miR-203a inhibitor. Our study suggests that miR-203a regulates pancreatic β cell proliferation and apoptosis by targeting IRS2, treatment with miR-203a inhibitors and IRS2 might provide a new therapeutic strategy for T1DM.

BackgroundMapping of allele-specific DNA methylation (ASM) can be a post-GWAS strategy for localizing regulatory sequence polymorphisms (rSNPs). However, the advantages of this approach, and the mechanisms underlying ASM in normal and neoplastic cells, remain to be clarified.ResultsWe performed whole genome methyl-seq on diverse normal cells and tissues and three types of cancers (multiple myeloma, lymphoma, glioblastoma multiforme). After excluding imprinting, the data pinpointed 15,114 high-confidence ASM differentially methylated regions (DMRs), of which 1,842 contained SNPs in strong linkage disequilibrium or coinciding with GWAS peaks. ASM frequencies were increased 5 to 9-fold in cancers vs. matched normal tissues, due to widespread allele-specific hypomethylation and focal allele-specific hypermethylation in poised chromatin. Cancers showed increased allele switching at ASM loci, but disruptive SNPs in specific classes of CTCF and transcription factor (TF) binding motifs were similarly correlated with ASM in cancer and non-cancer. Rare somatic mutations affecting these same motif classes tracked with de novo ASM in the cancers. Allele-specific TF binding from ChIP-seq was enriched among ASM loci, but most ASM DMRs lacked such annotations, and some were found in otherwise uninformative “chromatin deserts”.ConclusionsASM is increased in cancers but occurs by a shared mechanism involving disruptive SNPs in CTCF and TF binding sites in both normal and neoplastic cells. Dense ASM mapping in normal plus cancer samples reveals candidate rSNPs that are difficult to find by other approaches. Together with GWAS data, these rSNPs can nominate specific transcriptional pathways in susceptibility to autoimmune, neuropsychiatric, and neoplastic diseases. Custom genome browser tracks with annotated ASM loci can be viewed at a UCSC browser session hosted by our laboratory (https://bit.ly/tycko-asm)

Differentiation of first trimester human placental cytotrophoblast (CTB) from an anchorage-dependent epithelial phenotype into the mesenchymal-like invasive extravillous trophoblast (EVT) is crucial in the development of the maternal-fetal interface. We showed previously that differentiation of first trimester CTB to EVT involves an epithelial-mesenchymal transition (EMT). Here we compare the epithelial-mesenchymal characteristics of CTB and EVT derived from normal third trimester placenta or placenta previa versus abnormally invasive placenta (AIP). CTB and EVT were isolated from normal term placenta or placenta previa following Caesarean section and EVT from AIP following Caesarean hysterectomy. Cell identity was validated by measurement of cytokeratin-7 and HLA-G. Comparing normal term CTB with EVT from normal term placenta or placenta previa for differential expression analysis of genes associated with the EMT showed changes in >70% of the genes probed. While demonstrating a mesenchymal phenotype relative to CTB, many of the gene expression changes in third trimester EVT were reduced relative to the first trimester EVT. We suggest that third trimester EVT are in a more constrained, metastable state compared to first trimester equivalents. By contrast, EVT from AIP demonstrate characteristics that are more mesenchymal than normal third trimester EVT, placing them closer to first trimester EVT on the EMT spectrum, consistent with a more invasive phenotype.

Workshops are an important part of the IFPA annual meeting as they allow for discussion of specialised topics. At IFPA meeting 2016 there were twelve themed workshops, four of which are summarized in this report. These workshops related to various aspects of placental biology but collectively covered areas of decidual-trophoblast interaction, regulation of trophoblast invasion, immune cells at the maternal-fetal interface, and placental inflammation.

PURPOSE Atopic dermatitis (AD) is a common inflammatory disease that can affect the eye, resulting in ocular pathologies, including blepharitis, keratitis, and uveitis; however, the pathogenic mechanisms underlying the ocular manifestations of AD are not well understood. METHODS In the present study, we characterized the ocular pathologies that develop in the Stat6VT mouse model of AD. We examined the cytokine profile of the eyelid lesions, measured the behavioral response, and documented the treatment response to topical steroids. RESULTS Our results show that Stat6VT mice spontaneously developed blepharitis, keratitis, and uveitis similar to that observed in patients with AD. Histologic findings of allergic inflammation in affected eyelids in this model include the presence of a lymphocyte-predominant infiltrate and tissue eosinophilia in the dermis. Gene expression analysis of affected eyelid tissue by quantitative PCR revealed increased amounts of mRNAs for the Th2 cytokines IL-4, IL-5, and IL-13. In addition, increased eyelid scratching was seen in Stat6VT mice with blepharitis. Topical treatment with the corticosteroid clobetasol reduced eyelid inflammation, tissue eosinophilia, and Th2 cytokine expression. CONCLUSIONS The development of AD-like ocular pathologies in this model supports the idea that in humans, AD-associated disease of the eye may be driven by Th2-mediated inflammation and demonstrates that the Stat6VT mouse may be a useful system in which to further investigate pathogenesis of and treatment strategies for blepharitis and other ocular diseases that develop in association with AD.

Platelet activating factor (PAF) has long been associated with acute edema and inflammatory responses. PAF acts by binding to a specific G-protein coupled receptor (PAF-R, Ptafr). However, the role of chronic PAF-R activation on sustained inflammatory responses has been largely ignored. We recently demonstrated that mice lacking the PAF-R (Ptafr-/- mice) exhibit increased cutaneous tumorigenesis in response to a two-stage chemical carcinogenesis protocol. Ptafr-/- mice also exhibited increased chronic inflammation in response to phorbol ester application. In this present study, we demonstrate that topical application of the non-hydrolysable PAF mimetic (carbamoyl-PAF (CPAF)), exerts a potent, dose-dependent, and short-lived edema response in WT mice, but not Ptafr -/- mice or mice deficient in c-Kit (c-Kit W-sh/W-sh mice). Using an ear inflammation model, co-administration of topical CPAF treatment resulted in a paradoxical decrease in both acute ear thickness changes associated with a single PMA application, as well as the sustained inflammation associated with chronic repetitive PMA applications. Moreover, mice treated topically with CPAF also exhibited a significant reduction in chemical carcinogenesis. The ability of CPAF to suppress acute and chronic inflammatory changes in response to PMA application(s) was PAF-R dependent, as CPAF had no effect on basal or PMA-induced inflammation in Ptafr-/- mice. Moreover, c-Kit appears to be necessary for the anti-inflammatory effects of CPAF, as CPAF had no observable effect in c-Kit W-sh/W-sh mice. These data provide additional evidence that PAF-R activation exerts complex immunomodulatory effects in a model of chronic inflammation that is relevant to neoplastic development.

Calcipotriene is a topical vitamin D3 analogue approved for the treatment of plaque and scalp psoriasis. We report the case of a 2-year-old boy whose atopic dermatitis (AD) flared in response to application of calcipotriene 0.005% cream and solution for a mistaken diagnosis of plaque and scalp psoriasis. We investigated whether the patient's eruption was secondary to an allergic contact dermatitis. In the Stat6VT mouse model of AD we tested whether calcipotriene could induce the otherwise-spontaneous AD-like phenotype. Closed patch testing was done on the patient with calcipotriene solution and cream, moisturizing cream, and 51% isopropanol. Stat6VT and wild-type (WT) mice were treated for 7 days with calcipotriene solution or vehicle (isopropanol) applied to the right and left upper back skin, respectively, after which mice were followed longitudinally for 10 weeks. Biopsy specimens from prior treatment sites were then collected for histology and RNA isolation. RNA was analyzed for interleukin (IL-4) expression using quantitative polymerase chain reaction. Patch testing was negative. Stat6VT mice, in contrast to WT mice, developed a persistent eczematous dermatitis at sites of calcipotriene application. Clinical and histologic features and high IL-4 transcript levels were consistent with the spontaneous AD-like phenotype seen in Stat6VT mice. At sites of active disease, calcipotriene can worsen a flare of AD. In Stat6VT mice, calcipotriene can induce the AD-like phenotype.

Field cancerization refers to areas of grossly normal epithelium that exhibit increased risk for tumor occurrence. Unfortunately, elucidation of the locoregional changes that contribute to increased tumor risk is difficult due to the inability to visualize the field. In this study, we use a noninvasive optical-based imaging approach to detail spatiotemporal changes in subclinical hyperemia that occur during experimental cutaneous carcinogenesis. After acute inflammation from 10 weeks of UVB irradiation subsides, small areas of focal hyperemia form and were seen to persist and expand long after cessation of UVB irradiation. We show that these persistent early hyperemic foci reliably predict sites of angiogenesis and overlying tumor formation. More than 96% of the tumors (57 of 59) that developed following UVB or 7,12-dimethylbenz(a)anthracene/phorbol 12-myristate 13-acetate (DMBA/PMA) treatment developed in sites of preexisting hyperemic foci. Hyperemic foci were multifocal and heterogeneously distributed and represented a minor fraction of the carcinogen-treated skin surface (10.3% of the imaging area in vehicle-treated animals). Finally, we also assessed the ability of the anti-inflammatory agent, celecoxib, to suppress hyperemia formation during photocarcinogenesis. The chemopreventive activity of celecoxib was shown to correlate with its ability to reduce the area of skin that exhibit these hyperemic foci, reducing the area of imaged skin containing hyperemic foci by 49.1%. Thus, we propose that a hyperemic switch can be exploited to visualize the cancerization field very early in the course of cutaneous carcinogenesis and provides insight into the chemopreventive activity of the anti-inflammatory agent celecoxib. Cancer Res; 73(1); 150–9. ©2012 AACR.

Objectives: One aspect of our studies of abnormally invasive placenta (AIP, placenta accreta) involves investigation of cytotrophoblast (CTB) differentiation into invasive extravillous trophoblast (EVT) in normal and AIP pregnancies. Differentiation involves a process with similarities to the epithelial-mesenchymal transition (EMT), observed in gastrulation, wound-healing and cancer metastasis. We asked whether the CTB to EVT change was an EMT process and whether it was further advanced in AIP. Methods: We compared CTB and EVT from normal, term pregnancies (n1⁄44,8) and EVT from placenta previa (EVT-PP; n1⁄44) and AIP pregnancies (EVT-AIP; n1⁄48) obtained in the mid-third trimester. All AIP pregnancies were placenta percreta as determined by histopathology. After EVT purification by Percoll gradient and immunomagnetic separation targeted to HLA-G, cellular RNA was extracted and analysed on targeted PCR arrays containing EMT-related genes (Qiagen). Results: The EVT/EVT-PP comparison showed no significant differences and EVT-PP results were included in the EVT group thereafter. CTB/EVT comparison showed alterations in 48 of 84 genes (p

Introduction: Placenta accreta-related pathologies are characterized by excessive trophoblast invasion of the uterus. Evidence suggests that the trophoblast-decidual interaction is abnormal in these pathologies. We decided to isolate extravillous trophoblast (EVT) from term placenta as a precursor to obtaining and investigating these cells in accreta-related pathologies. Methods: Basal plate tissue (2-3 mm) from normal term placenta was digested using trypsin, collagenase or dispase, individually or in combination. After digestion cells were spun on a 30%:60% discontinuous Percoll gradient and the interface layer was collected. Cells were incubated with a mAb to HLA-G (clone MEM/G9) coupled to phycoerythrin (PE; Abcam) for 30min, washed, then incubated for a further 15minwith anti-PEmagnetic microbeads. Positive selection of HLA-G-labeled cells was performed on an AutoMACS magnetic cell separator (Miltenyi). Cell fractions were analyzed by flow cytometry using PE and the dead cell stain, SYTOX Green, as markers. Results: Use of collagenase or dispase in DMEM/F12 for digestion produced a significant degree of cell death whereas there was minimal cell death with these agents when digested in HBSS. A combination of 0.25% trypsin, 0.1% dispase, 0.1% collagenase IV in HBSS (3 x 20min) gave the best digestion as assessed by number of cells released and percentage of dead cells (1.6 0.4%; mean SEM, n1⁄43). Optimal anti-HLA-G dilutionwas 1:500. HLA-G-labeled cells comprised 0.9 0.3% of total after digestion, 23.7 5.7% after gradient separation and 94.3 2.5% following magnetic bead separation. Of the non-PE (HLA-G) labeled cells remaining in final fraction, 5.9 1.0% were SYTOX Green-stained suggesting that the primary contaminating component is a dead cell population. Conclusions: The method outlined here selects for a fraction highly enriched in the EVT-specific marker, HLA-G. This technique appears to isolate EVT cells from third trimester placenta and will be of value in the investigation of accreta-related pathologies.