SPTBN1 Mediates the Cytoplasmic Constraint of PTTG1, Impairing Its Oncogenic Activity in Human Seminoma.

Seminoma is the most common testicular cancer. Pituitary tumor-transforming gene 1 (PTTG1) is a securin showing oncogenic activity in several tumors. We previously demonstrated that nuclear PTTG1 promotes seminoma tumor invasion through its transcriptional activity on matrix metalloproteinase 2 (MMP-2) and E-cadherin (CDH1). We wondered if specific interactors could affect its subcellular distribution. To this aim, we investigated the PTTG1 interactome in seminoma cell lines showing different PTTG1 nuclear levels correlated with invasive properties. A proteomic approach upon PTTG1 immunoprecipitation uncovered new specific securin interactors. Western blot, confocal microscopy, cytoplasmic/nuclear fractionation, sphere-forming assay, and Atlas database interrogation were performed to validate the proteomic results and to investigate the interplay between PTTG1 and newly uncovered partners. We observed that spectrin beta-chain (SPTBN1) and PTTG1 were cofactors, with SPTBN1 anchoring the securin in the cytoplasm. SPTBN1 downregulation determined PTTG1 nuclear translocation, promoting its invasive capability. Moreover, a PTTG1 deletion mutant lacking SPTBN1 binding was strongly localized in the nucleus. The Atlas database revealed that seminomas that contained higher nuclear PTTG1 levels showed significantly lower SPTBN1 levels in comparison to non-seminomas. In human seminoma specimens, we found a strong PTTG1/SPTBN1 colocalization that decreases in areas with nuclear PTTG1 distribution. Overall, these results suggest that SPTBN1, along with PTTG1, is a potential prognostic factor useful in the clinical management of seminoma.

Short-Chain Fatty Acids Modulate Sperm Migration through Olfactory Receptor 51E2 Activity.

The non-orthotopic expression of olfactory receptors (ORs) includes the male reproductive system, and in particular spermatozoa; their active ligands could be essential to sperm chemotaxis and chemical sperm-oocyte communication. OR51E2 expression has been previously reported on sperm cells' surface. It has been demonstrated in different cellular models that olfactory receptor 51E2 (OR51E2) binds volatile short-chain fatty acids (SCFAs) as specific ligands. In the present research, we make use of Western blot, confocal microscopy colocalization analysis, and the calcium-release assay to demonstrate the activation of sperm cells through OR51E2 upon SCFAs stimulus. Moreover, we perform a novel modified swim-up assay to study the involvement of OR51E2/SCFAs in sperm migration. Taking advantage of computer-assisted sperm analysis (CASA system), we determine the kinematics parameters of sperm cells migrating towards SCFAs-enriched medium, revealing that these ligands are able to promote a more linear sperm-cell orientation. Finally, we obtain SCFAs by mass spectrometry in cervico-vaginal mucus and show for the first time that a direct incubation between cervical mucus and sperm cells could promote their activation. This study can shed light on the possible function of chemosensory receptors in successful reproduction activity, laying the foundation for the development of new strategies for the treatment of infertile individuals.

Alpha-Lipoic Acid Plays a Role in Endometriosis: New Evidence on Inflammasome-Mediated Interleukin Production, Cellular Adhesion and Invasion.

Endometriosis is an estrogen-linked gynecological disease defined by the presence of endometrial tissue on extrauterine sites where it forms invasive lesions. Alterations in estrogen-mediated cellular signaling seems to have an essential role in the pathogenesis of endometriosis. Higher estrogen receptor (ER)-ß levels and enhanced ER-ß activity were detected in endometriotic tissues. It is well known that ER-ß interacts with components of the cytoplasmic inflammasome-3 (NALP-3), the NALP-3 activation increases interleukin (IL)-1ß and IL-18, enhancing cellular adhesion and proliferation. Otherwise, the inhibition of ER-ß activity suppresses the ectopic lesions growth. The present study aims to investigate the potential effect of α-lipoic acid (ALA) on NALP-3 and ER-ß expression using a western blot analysis, NALP-3-induced cytokines production by ELISA, migration and invasion of immortalized epithelial (12Z) and stromal endometriotic cells (22B) using a 3D culture invasion assay, and matrix-metalloprotease (MMPs) activity using gelatin zymography. ALA significantly reduces ER-ß, NALP-3 protein expression/activity and the secretion of IL-1ß and IL-18 in both 12Z and 22B cells. ALA treatment reduces cellular adhesion and invasion via a lower expression of adhesion molecules and MMPs activities. These results provide convincing evidence that ALA might inhibit endometriosis progression.

Helicobacter pylori infection contributes to placental impairment in preeclampsia: basic and clinical evidences.

BACKGROUND: Preeclampsia (PE) is a major cause of maternal and neonatal morbidity and mortality. Epidemiological association between Helicobacter pylori (Hp) infection and PE onset has been widely shown. The aim of this study was to analyze a possible correlation between Hp infection and the severity of clinical presentation of PE and to identify an immunologic mechanism triggered by Hp infection potentially contributing to the pathogenesis of PE. MATERIALS AND METHODS: Sera from 93 preeclamptic women and 87 healthy pregnant women were tested for Hp infection by immunoassay and for anti-CagA antibodies by Western blot assay. The serologic results were correlated with the clinical features of PE. The functional effect of serum IgG fractions, positive or negative for Hp, from preeclamptic women or controls were tested on trophoblast and endothelial cell cultures and in a murine model of angiogenesis. RESULTS: Preeclamptic women showed higher seroprevalence of Hp infection (57.0%) compared to controls (33.3%) (P<.001). The seropositivity for CagA-positive strains of Hp was 45.2% in preeclamptic women vs 13.7% in controls (P<.001). In PE women, Hp infection was associated with abnormality of uterine arteries Doppler (P<.001). Hp+ IgG fractions from preeclamptic women bound to trophoblast and endometrial endothelial cell cultures, reducing in vitro invasiveness and angiogenesis, respectively, and inhibited angiogenesis in mice. CONCLUSIONS: Our data show, for the first time, an association between Hp infection and PE with abnormal uterine arteries Doppler velocimetry, suggesting a role for Hp infection in impairing placental development and increasing the risk to develop PE. This study opens the new perspective of a potential screening and treatment for Hp infection in pregnancy.

Potential new mechanisms of placental damage in celiac disease: anti-transglutaminase antibodies impair human endometrial angiogenesis.

Celiac disease (CD) is an autoimmune enteropathy triggered by gluten ingestion and characterized by circulating anti-transglutaminase type 2 (anti-TG2) autoantibodies. An epidemiological link between maternal CD and increased risk of pregnancy failure has been established; however, the mechanism underlying this association is still poorly understood. Because proper endometrial angiogenesis and decidualization are prerequisites for placental development, we investigated the effect of anti-TG2 antibodies on the process of endometrial angiogenesis. Binding of anti-TG2 antibodies to human endometrial endothelial cells (HEECs) was evaluated by ELISA. Angiogenesis was studied in vitro on HEECs and in vivo in a murine model. In particular, we investigated the effect of anti-TG2 antibodies on HEEC matrix metalloprotease-2 (MMP-2) activity by gelatin zymography, cytoskeletal organization and membrane properties by confocal microscopy, and activation of extracellular signal-regulated kinases (ERKs) and focal adhesion kinase (FAK) by Western blot analysis. Anti-TG2 antibodies bound to HEECs and decreased newly formed vessels both in vitro and in vivo. Anti-TG2 antibodies impaired angiogenesis by inhibiting the activation of MMP-2, disarranging cytoskeleton fibers, changing the physical and mechanical properties of cell membranes, and inhibiting the intracellular phosphorylation of FAK and ERK. Anti-TG2 antibodies inhibit endometrial angiogenesis affecting the TG2-dependent migration of HEECs and extracellular matrix degradation, which are necessary to form new vessels. Our results identify pathogenic mechanisms of placental damage in CD.

Antibodies anti-CagA cross-react with trophoblast cells: a risk factor for pre-eclampsia?

BACKGROUND: Previous studies reported an epidemiological association between CagA-positive H. pylori strains and pre-eclampsia. As antibodies anti-CagA cross-react with endothelial cells and trophoblast cells show an endothelial phenotypic profile, we hypothesized that anti-CagA antibodies may recognize antigens of cytotrophoblast cells, thus impairing their function. MATERIALS AND METHODS: Placenta samples were obtained from healthy women. Cytotrophoblast cells were cultured in a medium containing increasing concentration of polyclonal anti-CagA antibodies. Binding of anti-CagA antibodies to cytotrophoblast cells was evaluated by cell ELISA and immunofluorescence assay. Invasive potential of those cells was assessed by an invasion culture system and by measuring of MMP-2. Protein sequencing was performed on antigens precipitated by anti-CagA antibodies. Measurement of phosphorylated ERK expression and NF-kB DNA-binding activity in trophoblast cells incubated with anti-CagA or irrelevant antibodies was also performed. RESULTS: Anti-CagA antibodies recognized ß-actin of cytotrophoblast cells, showing a dose-dependent binding. Incubation of cytotrophoblast cells with increasing doses of anti-CagA antibodies significantly reduced their invasiveness and determined a significant decrease in phosphorylated ERK expression and a reduced NF-kB translocation activity. CONCLUSIONS: This study shows that anti-CagA antibodies recognize ß-actin of cytotrophoblast cells, reducing their invasiveness ability, possibly giving a biological explanation for the epidemiological association.

PTTG1/ZEB1 Axis Regulates E-Cadherin Expression in Human Seminoma.

(1) Background: PTTG1 sustains the EMT process and the invasiveness of several neoplasms. We previously showed the role of nuclear PTTG1 in promoting invasiveness, through its transcriptional target MMP2, in seminoma in vitro models. Here, we investigated the key players involved in PTTG1-mediated EMT in human seminoma. (2) Methods: Two seminoma cell lines and four human seminoma tumor specimens were used. E-Cadherin gene regulation was investigated using Western blot, real-time PCR, and luciferase assay. Immunoprecipitation, ChIP, RE-ChIP, and confocal microscopy analysis were performed to evaluate the interplay between PTTG1 and ZEB1. Matrigel invasion and spheroid formation assays were applied to functionally investigate PTTG1 involvement in the EMT of seminoma cell lines. RNA depletion and overexpression experiments were performed to verify the role of PTTG1/ZEB1 in E-Cadherin repression and seminoma invasiveness. E-Cadherin and ZEB1 levels were analyzed in human testicular tumors from the Atlas database. (3) Results: PTTG1 transcriptionally represses E-Cadherin in seminoma cell lines through ZEB1. The cooperation of PTTG1 with ZEB1 has a significant impact on cell growth/invasion properties involving the EMT process. Analysis of the Atlas database of testicular tumors showed significantly lower E-Cadherin levels in seminoma, where PTTG1 showed nuclear staining. Finally, PTTG1 and ZEB1 strongly localize together in the periphery of the tumors. (4) Conclusions: These results strengthen the evidence for a role of PTTG1 in the EMT process in human seminomas through its cooperation with the transcriptional repressor ZEB1 on the E-Cadherin gene. Our data enrich the molecular characterization of seminoma, suggesting that PTTG1 is a prognostic factor in seminoma clinical management.

α-Lipoic Acid and its Role on Female Reproduction.

α-lipoic Acid (ALA), also known as thioctic acid, is a biological thiol present in all types of prokaryotic and eukaryotic cells. It has been shown that ALA or its reduced form, DHLA, has several positive effects on human health, acting as a biological antioxidant, metal chelator and detoxifying agent. It is able to reduce the oxidation of several antioxidant agents like glutathione, vitamins C and E, and modulate insulin and NF-kB signaling pathways. ALA's pharmacological effects are not only related to its antioxidant properties but it shows an anti-inflammatory action. In particular, ALA is able to reduce inflammasome activity, the pro-inflammatory cytokine levels, such as TNF-α, IL-1ß, IL-6, IL-18 and IL-17, interferon (INF)-γ as well as the production of Vascular and Intercellular cell adhesion protein (VCAM-1 and ICAM-1). In recent papers, ALA has been indicated as a possible therapeutic approach to several endocrine or inflammatory disorders affecting female reproduction. Aim of the current review was to assess whether ALA has an evidence- based beneficial role on gynecological and obstetrical diseases such as polycystic ovary syndrome (PCOS), endometriosis, and miscarriage.

Nuclear Localization of PTTG1 Promotes Migration and Invasion of Seminoma Tumor through Activation of MMP-2.

(1) Background: PTTG1 sustains the invasiveness of several cancer types. We previously reported that in seminomas, PTTG1 was detected in the peripheral area of the tumor and in the leading infiltrative edge. Here, we investigate the PTTG1 role on the invasive properties of seminoma. (2) Methods: three seminoma cell lines were used as in vitro model. PTTG1 levels and localization were investigated by biochemical and immunofluorescence analyses. Wound-healing, Matrigel invasion assays, and zymography were applied to study migratory and invasive capability of the cell lines. RNA interference and overexpression experiments were performed to address the PTTG1 role in seminoma invasiveness. PTTG1 and its target MMP-2 were analyzed in human testicular tumors using the Atlas database. (3) Results: PTTG1 was highly and differentially expressed in the seminoma cell lines. Nuclear PTTG1 was positively correlated to the aggressive phenotype. Its modulation confirms these results. Atlas database analysis revealed that PTTG1 was localized in the nucleus in seminoma compared with non-seminoma tumors, and that MMP-2 levels were significantly higher in seminomas. (4) Conclusions: nuclear PTTG1 promotes invasiveness of seminoma cell lines. Atlas database supported these results. These data lead to the hypothesis that nuclear PTTG1 is an eligible prognostic factor in seminomas.

Anti-tissue transglutaminase antibodies from celiac patients are responsible for trophoblast damage via apoptosis in vitro.

OBJECTIVES: The association between maternal celiac disease (CD) and both reduced fertility and increased risk of adverse pregnancy-related events has been long documented. However, no evidences are available regarding the pathogenic mechanisms of this link. The aim of this study was to determine whether anti-tissue transglutaminase (anti-tTG) antibodies are involved in the damage of trophoblastic cells in vitro. METHODS: Human primary trophoblastic cells, isolated from term placenta, were exposed to anti-tTG immunoglobulin G (IgG) antibodies, both commercially available and separated from sera of three untreated celiac women. The ability of anti-tTG antibodies to bind to trophoblastic cells, invasiveness of placental cells through a layer of extracellular matrix, and the activity of cellular matrix metalloprotease (MMP) and cellular apoptosis were evaluated, as indicators of trophoblast damage, by TdT-mediated dUTP digoxigenin nick end labeling (TUNEL) and annexin V expression. RESULTS: Anti-tTG IgG showed a specific dose- and time-dependent binding to human trophoblast. In addition, trophoblastic cells, after being exposed to anti-tTG IgG antibodies, both commercially available and separated from sera of celiac women, showed an impaired invasiveness, a decreased activity of cellular MMP, and a greater percentage of TUNEL positivity and annexin V positivity. CONCLUSIONS: We showed that the binding of anti-tTG antibodies to trophoblast might represent a key mechanism by which the embryo implantation and pregnancy outcome are impaired in untreated celiac pregnant women. Because healthy trophoblast development is essential for placental and fetal development, these data provide a novel mechanism for CD-induced infertility, early pregnancy loss, and intrauterine growth retardation.

Antiphospholipid antibodies affect human endometrial angiogenesis.

Antiphospholipid antibodies (aPL) represent an important risk factor for thrombosis and recurrent miscarriage in patients with antiphospholipid syndrome (APS). The mechanisms of aPL-mediated pregnancy failure have been researched. Previous studies demonstrated that aPL bind trophoblast cells, reducing proliferation, human chorionic gonadotrophin release, and in vitro invasiveness. Recent data suggest that aPL are also able to react with human decidual cells, inducing a proinflammatory phenotype. Decidua, a newly formed tissue on the maternal side of the human placenta, is characterized by active angiogenesis and structural modifications of the spiral arteries in early pregnancy. Since angiogenesis is a critical component of normal placentation, the purpose of our study was to evaluate the role of aPL on human endometrial angiogenesis. For this reason, we investigated the effect of aPL on in vitro endometrial endothelial cell (HEEC) angiogenesis, VEGF secretion by ELISA, matrix metalloproteinases (MMPs) activity by gelatin zymography, and DNA binding activity of NFKB by a sensitive multiwell colorimetric assay. Furthermore, we performed experiments to study whether aPL affects in vivo angiogenesis in a murine model. We found that aPL significantly decrease the number and the total length of the tubules formed by HEEC on in vitro Matrigel assay and reduce newly formed vessels in aPL-inoculated mice. Moreover, aPL reduce significantly both VEGF and MMPs production and, at the nuclear level, NFKB DNA binding activity. From our results, it appears that aPL are associated with an inhibition of angiogenesis, suggesting further additional mechanisms to explain the defective placentation in the APS.

Expression of Pinopodes in the Endometrium from Recurrent Pregnancy Loss Women. Role of Thrombomodulin and Ezrin.

BACKGROUND: Pinopode expression has been suggested as a marker of endometrial receptivity. METHODS: We set up an experimental study comparing endometrial tissue from recurrent pregnancy loss (RPL, n = 30) and fertile control (CTR, n = 20) women in terms of pinopode expression/morphology; expression of thrombomodulin (TM) and ezrin; cytoskeletal organization. Endometrial samples were collected during implantation window and evaluated by scanning electron microscopy, western blot, and immunofluorescence. RESULTS: We found that RPL endometrial tissue showed: (i) increased pinopodes density (* p < 0.05); (ii) a reduced diameter of pinopodes (* p < 0.05); (iii) a decreased TM and ezrin expression (p < 0.05). Additionally, confocal images showed a significantly reduced expression of phosphorylated (p)-ezrin, confirming the results obtained through immunoblot analysis. Immunofluorescence staining showed that in CTR samples, junctions between cells are intact and clearly visible, whereas actin filaments appear completely lost in RPL endometrial samples; this suggests that, due to the impaired expression and activity of TM and ezrin, actin does not bind to plasma membrane in order to orchestrate the cytoskeletal actin filaments. CONCLUSIONS: Our findings suggest that an impaired expression of TM and expression/activation of ezrin may affect the connection between the TM and actin cytoskeleton, impairing the organization of cytoskeleton and, eventually, the adequate pinopode development.

Synthetic PreImplantation Factor (sPIF) reduces inflammation and prevents preterm birth.

Preterm birth (PTB) is the leading cause of neonatal morbidity and mortality and spontaneous PTB is a major contributor. The preceding inflammation/infection contributes not only to spontaneous PTB but is associated with neonatal morbidities including impaired brain development. Therefore, control of exaggerated immune response during pregnancy is an attractive strategy. A potential candidate is synthetic PreImplantation Factor (sPIF) as sPIF prevents inflammatory induced fetal loss and has neuroprotective properties. Here, we tested maternal sPIF prophylaxis in pregnant mice subjected to a lipopolysaccharides (LPS) insult, which results in PTB. Additionally, we evaluated sPIF effects in placental and microglial cell lines. Maternal sPIF application reduced the LPS induced PTB rate significantly. Consequently, sPIF reduced microglial activation (Iba-1 positive cells) and preserved neuronal migration (Cux-2 positive cells) in fetal brains. In fetal brain lysates sPIF decreased IL-6 and INFγ concentrations. In-vitro, sPIF reduced Iba1 and TNFα expression in microglial cells and reduced the expression of pro-apoptotic (Bad and Bax) and inflammatory (IL-6 and NLRP4) genes in placental cell lines. Together, maternal sPIF prophylaxis prevents PTB in part by controlling exaggerated immune response. Given the sPIF`FDA Fast Track approval in non-pregnant subjects, we envision sPIF therapy in pregnancy.

Resistin modulates glucose uptake and glucose transporter-1 (GLUT-1) expression in trophoblast cells.

The adipocytokine resistin impairs glucose tolerance and insulin sensitivity. Here, we examine the effect of resistin on glucose uptake in human trophoblast cells and we demonstrate that transplacental glucose transport is mediated by glucose transporter (GLUT)-1. Furthermore, we evaluate the type of signal transduction induced by resistin in GLUT-1 regulation. BeWo choriocarcinoma cells and primary cytotrophoblast cells were cultured with increasing resistin concentrations for 24 hrs. The main outcome measures include glucose transport assay using [(3)H]-2-deoxy glucose, GLUT-1 protein expression by Western blot analysis and GLUT-1 mRNA detection by quantitative real-time RT-PCR. Quantitative determination of phospho(p)-ERK1/2 in cell lysates was performed by an Enzyme Immunometric Assay and Western blot analysis. Our data demonstrate a direct effect of resistin on normal cytotrophoblastic and on BeWo cells: resistin modulates glucose uptake, GLUT-1 messenger ribonucleic acid (mRNA) and protein expression in placental cells. We suggest that ERK1/2 phosphorylation is involved in the GLUT-1 regulation induced by resistin. In conclusion, resistin causes activation of both the ERK1 and 2 pathway in trophoblast cells. ERK1 and 2 activation stimulated GLUT-1 synthesis and resulted in increase of placental glucose uptake. High resistin levels (50-100 ng/ml) seem able to affect glucose-uptake, presumably by decreasing the cell surface glucose transporter.

Effect of alpha-lipoic acid and myoinositol on endometrial inflammasome from recurrent pregnancy loss women.

PROBLEM: A significant increased expression/activation of one of the most well-characterized inflammasomes, the NAcht leucine-rich-repeat protein-3 (NALP-3), in the endometrium from idiopathic recurrent pregnancy loss women (RPL) has been previously found by our research group. We therefore, suggested this event as being one of the molecular mechanisms altering endometrial inflammatory status during early pregnancy. In the present research, we attempt to investigate whether molecules with anti-inflammatory activity, alpha-lipoic acid (ALA), and/or myoinositol affect the endometrial NALP-3 expression and activation. METHOD OF STUDY: Women with a history of idiopathic RPL (n = 30) were included in the study and compared to a control group (n = 15). Endometrial tissues were collected by hysteroscopy during the mid-luteal phase. RPL women underwent a three-month prescription of tablets containing ALA plus myoinositol (Sinopol® ). After treatment, hysteroscopic biopsies were repeated in RPL patients. Inflammasome expression was evaluated by immunohistochemical and Western blot analysis. NALP-3 activation was studied by quantifying the secretion of both caspase-1 and interleukin (IL)-1ß and IL-18 through ELISA. In ex vivo experiments, the effects of each molecule on endometrial inflammasome were studied. RESULTS: Sinopol® significantly reduced the RPL endometrial inflammasome expression and activation. ALA, but not myoinositol, significantly reduced the endometrial inflammasome expression and activity. CONCLUSION: Our data suggest a role for ALA on RPL inflammasome. Understanding the mechanisms involved in RPL and the observation that specific molecules are able to interfere with such complex at the endometrium might provide new rational design approaches to a personalized evaluation of endometrial status and, ultimately, a targeted medicine.

Endometrial microbes and microbiome: Recent insights on the inflammatory and immune "players" of the human endometrium.

In recent years, extended scientific works shed light on the important role played by the endometrium in early pregnancy. This review examines our current knowledge about the delicate balance between microbial and cellular immune agents at endometrial level: All of them might affect endometrial receptivity. In contrast to the classical thinking of human endometrium as a sterile tissue, several recent studies have drawn attention to a resident population of microorganisms, which reaches only a 30% of concordance with those of the cervical-vaginal flora. At present, the understanding of the microbiome in relation to human reproduction is in its infancy and further studies are needed to clarify the activity of endometrial microbiome and the possible effects of a "reproductive tract dysbiosis" on fertility. Moreover, in the human endometrium, there is a complex system works preventing the risk of infection as well as enabling, when pregnancy occurs, the acceptance of the blastocyst. In this way, the endometrium plays a central role in the uterine immune surveillance. A better understanding of the different agents that may affect endometrial receptivity would improve the diagnosis and treatment of obstetric complications related to defective implantation and placentation.

An Emerging Role of Endometrial Inflammasome in Reproduction: New Therapeutic Approaches.

BACKGROUND: One of the common complications of pregnancy is spontaneous pregnancy loss which occurs in an estimated 5- 15% of pregnancies. Of all women 1%-5% suffer from Recurrent Pregnancy Loss (RPL). Despite the fact that RPL has been associated to various anatomic, hormonal, immune, hematologic, and genetic defects, in 30% of the patients, screening tests included in the RPL workup may have negative results. Recently, we demonstrated a significant increased activation of endometrial NALP-3 inflammasome, and a caspase-1 dependent secretion of IL-18 and IL-1ß in the endometrial tissues obtained from RPL women compared with a fertile women group. The inflammasome has emerged as a key player in innate immunity and inflammation. An abnormal inflammasome activation, in absence of detectable infectious causes, might be one of the molecular mechanisms involved in establishing an unreceptive endometrium, potentially leading to early fetal loss. Upon activation, this multiprotein complex makes possible the caspase- 1-mediated proteolytic processing of proinflammatory cytokines generating their respective mature secretory forms. CONCLUSION: The understanding of molecular modulation of inflammasome associated pathways is critical for drug design, development and delivery. To date many promising inhibitors of inflammasome complex activation have been described, such as MCC950, ß-Hydroxybutyrate or Micro RNAs that affect NALP3 expression and activation. Furthermore, several herbal extracts and its bioactive constituents have shown to be effective in inflammatory response mediated by NLRP3 inflammasome activation. Nevertheless all these molecules represent a significant progress toward developing therapies that target IL-18 and IL-1ß secretion in a variety of diseases.

Synthetic PreImplantation Factor (PIF) prevents fetal loss by modulating LPS induced inflammatory response.

Maternal control of inflammation is essential during pregnancy and an exaggerated response is one of the underlying causes of fetal loss. Inflammatory response is mediated by multiple factors and Toll-like receptors (TLRs) are central. Activation of TLRs results in NALP-3 mediated assembly of apoptosis-associated speck-like protein containing a CARD (ASC) and caspase-1 into the inflammasome and production of pro-inflammatory cytokines IL-1ß and IL-18. Given that preventing measures are lacking, we investigated PreImplantation Factor (PIF) as therapeutic option as PIF modulates Inflammation in pregnancy. Additionally, synthetic PIF (PIF analog) protects against multiple immune disorders. We used a LPS induced murine model of fetal loss and synthetic PIF reduced this fetal loss and increased the embryo weight significantly. We detected increased PIF expression in the placentae after LPS insult. The LPS induced serum and placenta cytokines were abolished by synthetic PIF treatment and importantly synthetic PIF modulated key members of inflammasome complex NALP-3, ASC, and caspase-1 as well. In conclusion our results indicate that synthetic PIF protects against LPS induced fetal loss, likely through modulation of inflammatory response especially the inflammasome complex. Given that synthetic PIF is currently tested in autoimmune diseases of non-pregnant subjects (clinicaltrials.gov, NCT02239562), therapeutic approach during pregnancy can be envisioned.

Resistin regulates human choriocarcinoma cell invasive behaviour and endothelial cell angiogenic processes.

Resistin is a novel hormone that is secreted by human adipocytes and mononuclear cells and is probably associated with insulin resistance. Recently, resistin has been postulated to play a role in pregnancy, and resistin gene expression has been observed in placental tissues. However, it is still not known if resistin is able to affect trophoblast functions and development. Therefore, we investigated the hypothesis that resistin might regulate trophoblast production of matrix metalloproteinases (MMPs), the tissue inhibitors of metalloproteinases (TIMPs), trophoblast invasive behavior and the angiogenic processes. In human choriocarcinoma cells (BeWo), resistin (10-100 ng/ml) enhanced both MMP-2 protein and mRNA expression, significantly reduced TIMP-1 and TIMP-2 and increased trophoblast-like cell invasiveness. We analyzed the effect of resistin on an in vitro angiogenesis system for endothelial cells (HUVEC) and we evaluated its ability to modulate the secretion of an angiogenic factor, vascular endothelial growth factor (VEGF). Our data showed that resistin induced VEGF production and we observed that the addition of resistin stimulated endothelial cell tube formation. These findings suggest that resistin might be able to induce BeWo cell invasiveness and to contribute to the control of placental vascular development.

Placental Chemokine Receptor D6 Is Functionally Impaired in Pre-Eclampsia.

BACKGROUND: Pre-eclampsia (PE) is a major cause of maternal and perinatal morbidity and mortality worldwide. It is defined by new onset of hypertension and proteinuria after the 20th week of gestation and characterized by systemic exaggerated inflammatory response. D6 is a chemokines scavenger receptor that binds with high affinity CC chemokines, internalizes and targets the ligands for degradation. It is expressed in trophoblast-derived tissues and prevents excessive placenta leukocyte infiltration.The aim of this study was to investigate the expression and function of D6 in human placentae from pre-eclamptic and healthy pregnant women. METHODS AND RESULTS: Plasma levels of D6-binding CC chemokines (CCL-2, CCL-3, CCL-4, CCL-7, CCL-11) and pro-inflammatory cytokines (IL-6, TNF-α, CRP) were analyzed in 37 healthy pregnant women and 38 patients with PE by multiplex bead assay. Higher circulating levels of CCL7, CCL11, IL-6, (p<0.0001) and CRP (p<0.05) were observed in PE women compared to controls. Levels of circulating CCL4 were decreased in PE (p<0.001), while no significant differences of CCL2, CCL3 or TNF-α levels were detected. Immunofluorescent staining of placental sections showed higher expression of D6 receptor in the PE syncytiotrophoblast. Confocal and Western blot (WB) analyses revealed a prevalent distribution of D6 in trophoblast cells membranes in PE. Increased activation of D6 intracellular pathway was observed by Western blot analyses of p-LIMK and p-cofilin in trophoblast cell lysates. D6 functional assays showed reduced scavenging of CCL2 in PE cells compared to controls. Since actin filaments spatial assembling is essential for D6 intracellular trafficking and scavenging activity, we investigated by confocal microscopy trophoblast cytoskeleton organization and we observed a dramatic disarrangement in PE compared to controls. CONCLUSIONS: our results suggest membrane distribution of D6 receptor on trophoblast cell membranes in PE, together with reduced functionality, probably due to cytoskeleton impairment.