Low oxygen concentrations inhibit trophoblast cell invasion from early gestation placental explants via alterations in levels of the urokinase plasminogen activator system

Extravillous trophoblast cell (EVT) invasion in early pregnancy occurs in a relatively low-oxygen environment. The role of oxygen in regulation of EVT invasion remains controversial. We hypothesized that 1) culture in 3% oxygen inhibits EVT invasion compared with culture at 8% or 20% oxygen and 2) inhibition of invasion is due to alterations in levels of components of the urokinase plasminogen activator (PLAU, uPA) system rather than through increased apoptosis and/or decreased proliferation. Placental samples (8-10, 12-14, and 16-20 wk gestation) were obtained from women undergoing elective surgical termination of pregnancy or after cesarean section delivery (term) at the Royal Victoria Infirmary, Newcastle upon Tyne, U.K. EVT invasion from placental explants cultured at 3%, 8%, or 20% oxygen was assessed using Matrigel invasion assays. Invasion was assessed on Day 6, explants were harvested for analysis of apoptosis and proliferation, and medium was stored for analysis of PLAU system components by ELISA and casein zymography. Culture at 3% oxygen inhibited EVT invasion. PLAU receptor and plasminogen activator inhibitor-2 protein levels were increased and PLAU activity decreased in these cultures. There was no difference in the proliferation in explants cultured at the three different oxygen concentrations. Apoptosis, assessed by M30 immunostaining, was increased in EVT at both 3% and 8% oxygen. The reduction in the invasive capacity of EVT cultured at 3% oxygen appears to be mediated both by a general inhibition of the PLAU system and a decrease in the number of cells available to invade.     

Immunohistochemical identification of the extravillous trophoblast during the placentation of the degu (Octodon degus)

Recent data indicate that placentation in Octodon degus is similar to that in humans, making it a potential animal model for studies in human placental pathologies related to alterations in the migration of the extravillous trophoblast (EVT). Our objective was to immunohistochemically identify degu EVT during placentation by using cytoskeletal protein markers to establish the normal migratory pattern of the EVT. Fifteen O.degus were divided into three equal groups: day 27, 60, and 84 of gestation. The placentas were immunostained for cytokeratin (CK) and alpha smooth muscle actin (SMA). At day 27, the migrating EVT immunostained for SMA but not for CK. Once the EVT was incorporated in the maternal vessels (day 60) it was positive for CK but negative for SMA. The smooth muscle cells of the mesometrial arteries that remained after EVT invasion were positive for SMA. At day 84, the media muscular layer had partially regenerated but some EVT was still present. Furthermore, at day 27 cyclooxygenase-1 (COX-1) was detected in the endothelium of the maternal decidual vessels. Our results suggest that during the early stages of placentation, the cytoskeletal organization of the actin network of the migrating EVT corresponds to that of a cell with motile behavior. Once the EVT invaded the spiral arteries, the cytoskeleton reorganized, adopting the structure of an epithelial-like cell, expressing CK intermediate filaments. The media muscle layer regenerated near the end of gestation but some EVT remained. During EVT formation the endothelium of the maternal decidual vessels immunostained for COX-1.     

Possible roles for folic acid in the regulation of trophoblast invasion and placental development in normal early human pregnancy

In addition to its role in the prevention of neural tube defects, folic acid has many other physiological functions, including cell proliferation, DNA replication, and antioxidant protection. The aim of this study was to determine the role that folic acid has in regulating placental trophoblast development. Placental explants from placentae at gestational age 7 wk (n = 3) were cultured in folic acid at concentrations of 10(-6) M, 10(-8) M, and 10(-10) M. Extravillous trophoblast (EVT) invasion was assessed following 6-day culture, and explants were used for immunohistochemical evaluation of proliferation (MKI67) and apoptosis (active caspase 3). In addition, an array was performed on cell culture supernatants to examine a range of matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs). Folic acid increased the invasion of EVT cells in this explant model by between 83% and 19% (P = 0.005), and this was associated with increased MKI67 positivity and decreased active caspase 3 positivity; this effect was concentration dependent and showed a biphasic response. In addition, culture in folic acid increased vascular density, as determined by anti-CD31 immunostaining (P = 0.05). The increase in EVT invasion correlated with increased placental explant secretion of MMP2 (P = 0.01), MMP3 (P = 0.01), and MMP9 (P = 0.02). This study demonstrates that folic acid is potentially important in a number of crucial early stages of placental development, including EVT invasion, angiogenesis, and secretion of MMPs, and highlights the need for further studies to address the benefit of longer-term folic acid supplementation throughout pregnancy to prevent pregnancy disorders associated with deficient placental development, including preeclampsia.     

Decidual NK cells alter in vitro first trimester extravillous cytotrophoblast migration: a role for IFN-gamma

Abnormal placentation results in either inadequate (consequences: recurrent miscarriage, intrauterine growth restriction, and preeclampsia) or overzealous (consequences: placenta accreta, increta, and percreta) placentation. NK cells dominate in first trimester decidua and probably control extravillous cytotrophoblast (EVT) invasion. We examined this interaction in a novel way, using NK cells and villous explants from concordant first trimester pregnancies cocultured using a new collagen (two-dimensional) model of placentation. Decidual NK (dNK) cells exerted contact-independent inhibition of normal cytotrophoblast migration, associated with changes in the cytotrophoblast expression of metalloproteases-2 and -9, and plasminogen activator inhibitor-1. dNK cells did not affect EVT proliferation and apoptosis, and cell column formation. dNK cell effects were partially reversed by neutralizing Abs against IFN-gamma. We provide ex vivo human evidence of a direct role for dNK in modulating EVT differentiation as they form columns and then migrate from anchoring villi.     

Decidual-secreted factors alter invasive trophoblast membrane and secreted proteins implying a role for decidual cell regulation of placentation

Inadequate or inappropriate implantation and placentation during the establishment of human pregnancy is thought to lead to first trimester miscarriage, placental insufficiency and other obstetric complications. To create the placental blood supply, specialized cells, the ‘extravillous trophoblast’ (EVT) invade through the differentiated uterine endometrium (the decidua) to engraft and remodel uterine spiral arteries. We hypothesized that decidual factors would regulate EVT function by altering the production of EVT membrane and secreted factors. We used a proteomics approach to identify EVT membrane and secreted proteins regulated by decidual cell factors. Human endometrial stromal cells were decidualized in vitro by treatment with estradiol (10⁻⁸ M), medroxyprogesterone acetate (10⁻⁷ M) and cAMP (0.5 mM) for 14 days. Conditioned media (CM) was collected on day 2 (non-decidualized CM) and 14 (decidualized CM) of treatment. Isolated primary EVT cultured on Matrigel™ were treated with media control, non-decidualized or decidualized CM for 16 h. EVT CM was fractionated for proteins <30 kDa using size-exclusion affinity nanoparticles (SEAN) before trypsin digestion and HPLC-MS/MS. 43 proteins produced by EVT were identified; 14 not previously known to be expressed in the placenta and 12 which had previously been associated with diseases of pregnancy including preeclampsia. Profilin 1, lysosome associated membrane glycoprotein 1 (LAMP1), dipeptidyl peptidase 1 (DPP1/cathepsin C) and annexin A2 expression by interstitial EVT in vivo was validated by immunhistochemistry. Decidual CM regulation in vitro was validated by western blotting: decidualized CM upregulated profilin 1 in EVT CM and non-decidualized CM upregulated annexin A2 in EVT CM and pro-DPP1 in EVT cell lysate. Here, non-decidualized factors induced protease expression by EVT suggesting that non-decidualized factors may induce a pro-inflammatory cascade. Preeclampsia is a pro-inflammatory condition. Overall, we have demonstrated the potential of a proteomics approach to identify novel proteins expressed by EVT and to uncover the mechanisms leading to disease states.     

Advanced Maternal Age‐associated SIRT1 Deficiency Compromises Trophoblast Epithelial−Mesenchymal Transition through an Increase in Vimentin Acetylation

Advanced maternal age (AMA) pregnancies are rapidly increasing and are associated with aberrant trophoblast cell function, poor placentation, and unfavorable pregnancy outcomes, presumably due to premature placental senescence. SIRT1 is an NAD⁺‐dependent deacetylase with well‐known antiaging effects, but its connection with placental senescence is unreported. In this study, human term placentas and first‐trimester villi were collected from AMA and normal pregnancies, and a mouse AMA model was established by cross breeding young and aged male and female C57 mice. SIRT1 expression and activity in HTR8/SVneo cells were genetically or pharmacologically manipulated. Trophoblast‐specific Sirt1‐knockout (KO) mouse placentas were generated by mating Elf5‐Cre and Sirt1 ^fl/fl mice. Trophoblast cell mobility was assessed with transwell invasion and wound‐healing assays. SIRT1‐binding proteins in HTR8/SVneo cells and human placental tissue were identified by mass spectrometry. We identified SIRT1 as the only differentially expressed sirtuin between AMA and normal placentas. It is downregulated in AMA placentas early in the placental life cycle and is barely impacted by paternal age. SIRT1 loss upregulates P53 acetylation and P21 expression and impairs trophoblast invasion and migration. Sirt1‐KO mouse placentas exhibit senescence markers and morphological disruption, along with decreased fetal weight. In trophoblasts, SIRT1 interacts with vimentin, regulating its acetylation. In conclusion, SIRT1 promotes trophoblast epithelial−mesenchymal transition (EMT) to enhance invasiveness by modulating vimentin acetylation. AMA placentas are associated with premature senescence during placentation due to SIRT1 loss. Therefore, SIRT1 may be an antiaging therapeutic target for improving placental development and perinatal outcomes in AMA pregnancies.     

Genetic,Maternal and Placental Factors in the Association between Birth Weight and Physical Fitness: A Longitudinal Twin Study

Background

Adult cardiorespiratory fitness and muscle strength are related to all-cause and cardiovascular mortality. Both are possibly related to birth weight, but it is unclear what the importance is of genetic, maternal and placental factors in these associations.

Design

Peak oxygen uptake and measures of strength, flexibility and balance were obtained yearly during adolescence (10–18 years) in 114 twin pairs in the Leuven Longitudinal Twin Study. Their birth weights had been collected prospectively within the East Flanders Prospective Twin Survey.

Results

We identified linear associations between birth weight and adolescent vertical jump (b = 1.96 cm per kg birth weight, P = 0.02), arm pull (b = 1.85 kg per kg birth weight P = 0.03) and flamingo balance (b = −1.82 attempts to stand one minute per kg birth weight, P = 0.03). Maximum oxygen uptake appeared to have a U-shaped association with birth weight (the smallest and largest children had the lowest uptake, P = 0.01), but this association was no longer significant after adjustment for parental BMI. Using the individual twin’s deviation from his own twin pair’s average birth weight, we found positive associations between birth weight and adolescent vertical jump (b = 3.49, P = 0.0007) and arm pull (b = 3.44, P = 0.02). Δ scores were calculated within the twin pairs as first born twin minus second born twin. Δ birth weight was associated with Δ vertical jump within MZ twin pairs only (b = 2.63, P = 0.009), which indicates importance of placental factors.

Conclusions

We found evidence for an association between adolescent physical performance (strength, balance and possibly peak oxygen uptake) and birth weight. The associations with vertical jump and arm pull were likely based on individual, more specifically placental (in the case of vertical jump) factors. Our results should be viewed as hypothesis-generating and need confirmation, but potentially support preventive strategies to optimize birth weight, for example via placental function, to target later fitness and health.     

MUC4 and MUC1 Expression in Adenocarcinoma of the Stomach Correlates with Vessel Invasion and Lymph Node Metastasis: An Immunohistochemical Study of Early Gastric Cancer

We have previously reported that MUC4 expression is a poor prognostic factor in various carcinomas. Our previous study also showed that MUC1 expression in gastric cancers, including the early and advanced stages is a poor prognostic factor. In the present study, the expression profiles of MUC4 and MUC1 were examined by immunohistochemistry (IHC) using two anti-MUC4 monoclonal antibodies (MAbs), 8G7 and 1G8, and anti-MUC1 MAb DF3 in 104 gastrectomy specimens of early gastric adenocarcinoma with submucosal invasion (pT1b2), including 197 histological subtype lesions. Before the IHC study of the human specimens, we evaluated the specificity of the two MAbs by Western blotting and IHC of two MUC4 mRNA expressing gastric cancer cell lines. MAb 8G7 reacted clearly, whereas MAb 1G8 did not show any reactivity, in either Western blotting or IHC. In the IHC of the gastric cancers, the expression rates of MUC4/8G7 detected by MAb 8G7, MUC4/1G8 detected by MAb 1G8 and MUC1/DF3 detected by MAb DF3 in well differentiated types (70%, 38/54; 67%, 36/54; 52%, 28/54) were significantly higher than those in poorly differentiated types (18%, 10/55; 36%, 20/55; 13%, 7/55) (P<0.0001; P = 0.0021; P<0.0001), respectively. The MUC4/8G7 expression was related with lymphatic invasion (r = 0.304, P = 0.033). On the other hand, the MUC4/1G8 expression was related with lymphatic invasion (r = 0.395, P = 0.001) and lymph node metastasis (r = 0.296, P = 0.045). The MUC1/DF3 expression was related with lymphatic invasion (r = 0.357, P = 0.032) and venous invasion (r = 0.377, P = 0.024). In conclusion, the expression of MUC4 as well as MUC1 in early gastric cancers is a useful marker to predict poor prognostic factors related with vessel invasion.     

Oxygen Tension Modulates Differentiation and Primary Macrophage Functions in the Human Monocytic THP-1 Cell Line

The human THP-1 cell line is widely used as an in vitro model system for studying macrophage differentiation and function. Conventional culture conditions for these cells consist of ambient oxygen pressure (∼20% v/v) and medium supplemented with the thiol 2-mercaptoethanol (2-ME) and serum. In consideration of the redox activities of O₂ and 2-ME, and the extensive experimental evidence supporting a role for reactive oxygen species (ROS) in the differentiation and function of macrophages, we addressed the question of whether culturing THP-1 cells under a more physiologically relevant oxygen tension (5% O₂) in the absence of 2-ME and serum would alter THP-1 cell physiology. Comparisons of cultures maintained in 18% O₂ versus 5% O₂ indicated that reducing oxygen tension had no effect on the proliferation of undifferentiated THP-1 cells. However, decreasing the oxygen tension to 5% O₂ significantly increased the rate of phorbol ester-induced differentiation of THP-1 cells into macrophage-like cells as well as the metabolic activity of both undifferentiated and PMA-differentiated THP-1 cells. Removal of both 2-ME and serum from the medium decreased the proliferation of undifferentiated THP-1 cells but increased metabolic activity and the rate of differentiation under either oxygen tension. In differentiated THP-1 cells, lowering the oxygen tension to 5% O₂ decreased phagocytic activity, the constitutive release of β-hexosaminidase and LPS-induced NF-κB activation but enhanced LPS-stimulated release of cytokines. Collectively, these data demonstrate that oxygen tension influences THP-1 cell differentiation and primary macrophage functions, and suggest that culturing these cells under tightly regulated oxygen tension in the absence of exogenous reducing agent and serum is likely to provide a physiologically relevant baseline from which to study the role of the local redox environment in regulating THP-1 cell physiology.     

Prediction of Nodal Involvement in Primary Rectal Carcinoma without Invasion to Pelvic Structures: Accuracy of Preoperative CT,MR, and DWIBS Assessments Relative to Histopathologic Findings

Objective

To investigate the accuracy of preoperative computed tomography (CT), magnetic resonance (MR) imaging and diffusion-weighted imaging with background body signal suppression (DWIBS) in the prediction of nodal involvement in primary rectal carcinoma patients in the absence of tumor invasion into pelvic structures.

Methods and Materials

Fifty-two subjects with primary rectal cancer were preoperatively assessed by CT and MRI at 1.5 T with a phased-array coil. Preoperative lymph node staging with imaging modalities (CT, MRI, and DWIBS) were compared with the final histological findings.

Results

The accuracy of CT, MRI, and DWIBS were 57.7%, 63.5%, and 40.4%. The accuracy of DWIBS with higher sensitivity and negative predictive value for evaluating primary rectal cancer patients was lower than that of CT and MRI. Nodal staging agreement between imaging and pathology was fairly strong for CT and MRI (Kappa value = 0.331 and 0.348, P<0.01) but was relatively weaker for DWIBS (Kappa value = 0.174, P<0.05). The accuracy was 57.7% and 59.6%, respectively, for CT and MRI when the lymph node border information was used as the criteria, and was 57.7% and 61.5%, respectively, for enhanced CT and MRI when the lymph node enhancement pattern was used as the criteria.

Conclusion

MRI is more accurate than CT in predicting nodal involvement in primary rectal carcinoma patients in the absence of tumor invasion into pelvic structures. DWIBS has a great diagnostic value in differentiating small malignant from benign lymph nodes.     

Human Cytomegalovirus-Induces Cytokine Changes in the Placenta with Implications for Adverse Pregnancy Outcomes

Human cytomegalovirus (CMV) infection of the developing fetus can result in adverse pregnancy outcomes including death in utero. Fetal injury results from direct viral cytopathic damage to the CMV-infected fetus, although evidence suggests CMV placental infection may indirectly cause injury to the fetus, possibly via immune dysregulation with placental dysfunction. This study investigated the effects of CMV infection on expression of the chemokine MCP-1 (CCL2) and cytokine TNF-α in placentae from naturally infected stillborn babies, and compared these changes with those found in placental villous explant histocultures acutely infected with CMV ex vivo. Tissue cytokine protein levels were assessed using quantitative immunohistochemistry. CMV-infected placentae from stillborn babies had significantly elevated MCP-1 and TNF-α levels compared with uninfected placentae (p = 0.001 and p = 0.007), which was not observed in placentae infected with other microorganisms (p = 0.62 and p = 0.71) (n = 7 per group). Modelling acute clinical infection using ex vivo placental explant histocultures showed infection with CMV laboratory strain AD169 (0.2 pfu/ml) caused significantly elevated expression of MCP-1 and TNF-α compared with uninfected explants (p = 0.0003 and p<0.0001) (n = 25 per group). Explant infection with wild-type Merlin at a tenfold lower multiplicity of infection (0.02 pfu/ml), caused a significant positive correlation between increased explant infection and upregulation of MCP-1 and TNF-α expression (p = 0.0001 and p = 0.017). Cytokine dysregulation has been associated with adverse outcomes of pregnancy, and can negatively affect placental development and function. These novel findings demonstrate CMV infection modulates the placental immune environment in vivo and in a multicellular ex vivo model, suggesting CMV-induced cytokine modulation as a potential initiator and/or exacerbator of placental and fetal injury.     

Mesenchymal stroma/stem‐like cells of GARP knockdown inhibits cell proliferation and invasion of mouse colon cancer cells (MC38) through exosomes

Mesenchymal stroma/stem‐like cells (MSCs) have antitumour activity, and MSC‐derived exosomes play a role in the growth, metastasis and invasion of tumour cells. Additionally, glycoprotein A repetition predominant (GARP) promotes oncogenesis in breast cancer. Therefore, GARP is speculated to be a target gene for cancer therapy. We aimed to explore the therapy role of MSC‐derived exosomes targeting GARP in mouse colon cancer cell MC38. We successfully established a GARP knockdown system using three kinds of siRNA‐GARP in MSC cells. Exosomes were isolated from MSC and siGARP‐MSC cells, and verified by the exosome surface protein markers CD9, CD63 and CD81. GARP expression was significantly decreased in siGARP‐MSC exosomes compared with that of MSC exosomes. We found that siGARP‐MSC exosomes inhibited cell proliferation, migration and invasion of MC38 cells, using CCK‐8, colony formation, wound‐healing and Transwell invasion assays. Furthermore, siGARP‐MSC exosomes impeded IL‐6 secretion and partly inactivated JAK1/STAT3 pathway, measured using ELISA and RT‐qPCR. In conclusion, MSC‐derived exosomes targeting GARP are a potential strategy for cancer therapy.     

Sphingosine-1-Phosphate Promotes Extravillous Trophoblast Cell Invasion by Activating MEK/ERK/MMP-2 Signaling Pathways via S1P/S1PR1 Axis Activation

Successful placentation depends on the proper invasion of extravillous trophoblast (EVT) cells into maternal tissues. Previous reports demonstrated that S1P receptors are expressed in the EVT cells and S1P could regulate migration and function of trophoblast cells via S1P receptors. However, little is known about roles of S1P in the invasion of EVT cells. Our study was performed to investigate S1P effect on the invasion of EVT cells. We used the extravillous trophoblast cell line HTR8/SVneo cells to evaluate the effect. In vitro invasion assay was employed to determine the invasion of HTR8/SVneo cells induced by S1P. MMP-2 enzyme activity and relative level in the supernatants of HTR8/SVneo was assessed by gelatin zymography and western blot. Based on the above, siRNA and specific inhibitors were used for the intervention and study of potential signal pathways, and Real-time qPCR and western blot were used to test the mRNA and protein level of potential signal targets. We found that S1P could promote HTR8/SVneo cell invasion and upregulates activity and level of MMP-2. The promotion requires activation of MEK-ERK and is dependent on the axis of S1P/S1PR1. Our investigation of S1P may provide new insights into the molecular mechanisms of EVT invasion.     

Epigenetic Inactivation of EFEMP1 Is Associated with Tumor Suppressive Function in Endometrial Carcinoma

Objective

EFEMP1, the epidermal growth factor–containing fibulin-like extracellular matrix protein 1, functions as an oncogene or a tumor suppressor depending on the cancer types. In this study, we aim to determine whether EFEMP1 affects the tumorigenesis and progression of endometrial carcinoma.

Methods

The expression of EFEMP1 was investigated using immunohistochemistry in a panel of normal endometrium (n = 40), atypical hyperplasia (n = 10) and endometrial carcinoma tissues (n = 84). Methylation status of the EFEMP1 promoter was detected by methylation-specific PCR (MSP) and bisulphite genomic sequencing. Up- or down-regulation of EFEMP1 were achieved by stable or transient transfection with pCMV6/GFP/Neo-EFEMP1 or pGPU6/GFP/Neo-shEFEMP1 respectively. Effects of EFEMP1 on tumor proliferation, invasion and migration were evaluated by MTT, plate colony formation, Transwell and wound healing assay. The nude mouse tumor xenograft assay was used to investigate function of EFEMP1 in vivo.

Results

Compared with normal endometrium (32/40) and atypical hyperplasia (7/10), EFEMP1 expression was much lower in endometrial carcinoma tissues (16/84) (P<0.001 and P = 0.02). EFEMP1 promoter was hypermethylated in endometrial carcinoma tissues (67%) as compared to normal tissue (10%) and down-regulation of EFEMP1 was associated with promoter hypermethylation. Treatment with 5-aza-2′-deoxycytidine (5-aza-dC) and/or trichostatin A (TSA) altered EFEMP1 methylation status, and restored EFEMP1 expression. Moreover, EFEMP1 decreased secretion of MMPs and inhibited tumor cell proliferation, metastasis and invasion in vitro and suppressed tumorigenesis in nude mice. Besides, EFEMP1 increased expression of E-cadherin and suppressed expression of vimentin in endometrial carcinoma.

Conclusion

EFEMP1 is a new candidate tumor suppressor gene in endometrial carcinoma, and is frequently silenced by promoter hypermethylation. It could inhibit tumor growth and invasion both in vitro and in vivo. Our findings propose that targeting EFEMP1 might offer future clinical utility in endometrial carcinoma.     

α-tocopherol prevents oxidative stress-induced proliferative dysfunction in first-trimester human placental (HTR-8/SVneo) cells

Extravillous trophoblasts (EVTs) are the main participants in the process of placentation, an early process critical for placental growth and function involving an adequate invasion and complete remodelling of the maternal spiral arteries during early pregnancy. An increase in oxidative stress during pregnancy is associated with the onset and progression of several pregnancy disorders, including preeclampsia and gestational diabetes mellitus and it also occurs due to exposure of pregnant women to some xenobiotics (eg. alcohol). This study aimed to investigate how oxidative stress affects EVTs, and the ability of several distinct antioxidant agents to prevent these changes. For this, we exposed HTR8/SVneo cells to tert-butylhydroperoxide (0.5 μM; 24 h), which was able to increase lipid peroxidation and protein carbonyl levels. Under these conditions, there was a decrease in proliferation rates, culture growth, migratory and angiogenic capacities and an increase in the apoptosis rates. The antiproliferative effect of TBH was supressed by simultaneous treatment of the cells with α-tocopherol, but other antioxidants (vitamin C, allopurinol, apocynin, N-acetylcysteine, quercetin and resveratrol) were ineffective. α-tocopherol was also able to abolish the effect of TBH on lipid peroxidation and protein carbonyl levels. Overall, our results show that oxidative stress interferes with EVT characteristics essential for the placentation process, which may contribute to the association between oxidative stress and pregnancy disorders. Our results also show that the nature of the in vitro model of oxidative stress-induction is an important determinant of the cellular consequences of oxidative stress and, therefore, of the efficacy of antioxidants.     

Quantification of Cell-Free DNA in Normal and Complicated Pregnancies: Overcoming Biological and Technical Issues

The characterization of cell-free DNA (cfDNA) originating from placental trophoblast in maternal plasma provides a powerful tool for non-invasive diagnosis of fetal and obstetrical complications. Due to its placental origin, the specific epigenetic features of this DNA (commonly known as cell-free fetal DNA) can be utilized in creating universal ‘fetal’ markers in maternal plasma, thus overcoming the limitations of gender- or rhesus-specific ones. The goal of this study was to compare the performance of relevant approaches and assays evaluating the amount of cfDNA in maternal plasma throughout gestation (7.2–39.5 weeks). Two fetal- or placental- specific duplex assays (RPP30/SRY and RASSF1A/β-Actin) were applied using two technologies, real-time quantitative PCR (qPCR) and droplet digital PCR (ddPCR). Both methods revealed similar performance parameters within the studied dynamic range. Data obtained using qPCR and ddPCR for these assays were positively correlated (total cfDNA (RPP30): R = 0.57, p = 0.001/placental cfDNA (SRY): R = 0.85, p<0.0001; placental cfDNA (RASSF1A): R = 0.75, p<0.0001). There was a significant correlation in SRY and RASSF1A results measured with qPCR (R = 0.68, p = 0.013) and ddPCR (R = 0.56, p = 0.039). Different approaches also gave comparable results with regard to the correlation of the placental cfDNA concentration with gestational age and pathological outcome. We conclude that ddPCR is a practical approach, adaptable to existing qPCR assays and well suited for analysis of cell-free DNA in plasma. However, it may need further optimization to surpass the performance of qPCR.     

A Meta-Analysis of P2X7 Gene-762T/C Polymorphism and Pulmonary Tuberculosis Susceptibility

AimWe performed a comprehensive meta-analysis to determine the association between P2X7 -762T/C polymorphism and pulmonary tuberculosis susceptibility.MethodologyBased on comprehensive searches of the PubMed, SCI, Elsevier, China National Knowledge Infrastructure (CNKI) and Wanfang Database, we identified eligible studies about the association between P2X7 -762T/C polymorphism and pulmonary tuberculosis risk. Pooled odds ratio (ORs) and 95% confidence intervals (95%CIs) were calculated in random-effects model.ResultsA total of 2207 tuberculosis cases and 2220 controls in 8 case-control studies were included in this meta-analysis. Allele model (C vs. T: p = 0.15; OR = 0.83, 95% CI = 0.65–1.07), homozygous model (CC vs. TT: p = 0.23; OR = 0.73, 95% CI = 0.44 to 1.22), and heterozygous model (CT vs. TT: p = 0.57; OR = 0.92, 95% CI = 0.68 to 1.24) did not show increased risk of developing pulmonary tuberculosis. Similarly, dominant model (CC+CT vs. TT: p = 0.32; OR = 0.84, 95% CI = 0.59 to 1.19) and recessive model (CC vs. CT+TT: p = 0.08; OR = 0.77, 95% CI = 0.57 to 1.04) failed to show increased risk of developing pulmonary tuberculosis. Subgroup analysis by ethnicity did not detect any significant association between P2X7–762T/C polymorphism and pulmonary tuberculosis susceptibility.ConclusionsP2X7 -762T/C gene polymorphism is not associated with pulmonary tuberculosis susceptibility.     

Diversin Is Overexpressed in Breast Cancer and Accelerates Cell Proliferation and Invasion

Diversin was recently reported to play roles in Wnt and JNK pathways. However, the expression pattern and biological roles of diversin in human breast cancer have not been reported. In the present study, we found that diversin was overexpressed in breast cancer specimens by immunohistochemistry and western blot. Significant association was observed between diversin overexpression and TNM stage (p = 0.0036), nodal metastasis (p = 0.0033), negative estrogen receptor expression (p = 0.0012) and triple-negative status (p = 0.0017). Furthermore, colony formation assay and matrigel invasion assay showed that knockdown of diversin expression in MDA-MB-231 cell line with high endogenous expression decreased cell proliferation and cell invasion. Transfection of diversin plasmid in MCF-7 cell line increased cell proliferation and invasion. Further analysis showed that diversin depletion downregulated JNK phosphorylation while its overexpression upregulated JNK phosphorylation. In conclusion, our study demonstrated that diversin was overexpressed in human breast cancers. Diversin could contribute to breast cancer cell proliferation and invasion.