Endocannabinoid regulation in the cervix during pregnancy: insights into molecular mechanisms of premature labor.

In brief: The cervix plays a crucial role not only in the maintenance of pregnancy but also during delivery, when it undergoes extensive changes. This study highlights the involvement of the endocannabinoidome in cervical remodeling, emphasizing its relevance in the shift from a nonpregnant to pregnant state and its potential contribution to preterm delivery in inflammatory contexts. Abstract: During pregnancy, the main role of the cervix is to isolate the fetus from outside pathogens and maintain the relatively closed system of uterine gestation. Conversely, toward the end of pregnancy, the cervix must be remodeled to increase flexibility and allow the delivery. This process is called cervical remodeling and dysregulation of the process plays a role in premature delivery. The endocannabinoidome plays an important role in several reproductive events; however, its function on cervical tissue throughout pregnancy is poorly understood. The goal of this study was to evaluate the presence and participation of the endocannabinoidome in lipopolysaccharide (LPS)-induced cervical changes. Therefore, we evaluated key components of the endocannabinoidome in cervical tissue from nonpregnant mice and pregnant mice with and without LPS treatment. Using mass spectrometric analysis, we found an increase in anandamide and 2-arachidonoylglycerol in the cervix of pregnant mice when compared to nonpregnant mice. We have also found a reduction in FAAH protein expression in these tissues. Furthermore, when treated with LPS, we observed a reduction in the cervical immunostaining with anti-CB1 and anti-CB2 antibodies. Likewise, using cervix explants from pregnant mice, we found that LPS significantly increased cervical metalloprotease activity and cyclooxygenase 2, which were subsequently modulated by cannabinoid receptor antagonists. Collectively, our findings suggest that an LPS-induced imbalance of cervix endocannabinoidome likely contributes to premature cervical remodeling, which is part of the key components that contribute to premature delivery.

CB1 receptor expression and signaling are required for dexamethasone-induced aversive memory consolidation.

The molecular processes that underlie long-term memory formation involve signaling pathway activation by neurotransmitter release, which induces the expression of immediate early genes, such as Zif268, having a key role in memory formation. In this work, we show that the cannabinoid CB1 receptor signaling is necessary for the effects of dexamethasone on the behavioral response in an inhibitory avoidance task, on dexamethasone-induced ERK phosphorylation, and on dexamethasone-dependent Zif268 expression. Furthermore, we provide primary evidence for the mechanism responsible for this crosstalk between cannabinoid and glucocorticoid-mediated signaling pathways, showing that dexamethasone regulates endocannabinoid metabolism by inhibiting the activity of the Fatty acid amide hydrolase (FAAH), an integral membrane enzyme that hydrolyzes endocannabinoids and related amidated signaling lipids. Our results provide novel evidence regarding the role of the endocannabinoid system, and in particular of the CB1 receptor, as a mediator of the effects of glucocorticoids on the consolidation of aversive memories.

Maternal obesity reverses the resistance to LPS-induced adverse pregnancy outcome and increases female offspring metabolic alterations in cannabinoid receptor 1 knockout mice.

Maternal overnutrition negatively impacts the offspring's health leading to an increased risk of developing chronic diseases or metabolic syndrome in adulthood. What we eat affects the endocannabinoid system (eCS) activity, which in turn modulates lipogenesis and fatty acids utilization in hepatic, muscle, and adipose tissues. This study aimed to evaluate the transgenerational effect of maternal obesity on cannabinoid receptor 1 knock-out (CB1 KO) animals in combination with a postnatal obesogenic diet on the development of metabolic disturbances on their offspring. CB1 KO mice were fed a control diet (CD) or a high-fat diet (HFD; 33% more energy from fat) for 3 months. Offspring born to control and obese mothers were also fed with CD or HFD. We observed that pups born to an HFD-fed mother presented higher postnatal weight, lower hepatic fatty acid amide hydrolase activity, and increased blood cholesterol levels when compared to the offspring born to CD-fed mothers. When female mice born to HFD-fed CB1 KO mothers were exposed to an HFD, they gained more weight, presented elevated blood cholesterol levels, and more abdominal adipose tissue accumulation than control-fed adult offspring. The eCS is involved in several reproductive physiological processes. Interestingly, we showed that CB1 KO mice in gestational day 15 presented resistance to LPS-induced deleterious effects on pregnancy outcome, which was overcome when these mice were obese. Our results suggest that an HFD in CB1 receptor-deficient mice contributes to a "nutritional programming" of the offspring resulting in increased susceptibility to metabolic challenges both perinatally and during adulthood.

Maternal environmental enrichment modulates the immune response against an inflammatory challenge during gestation and protects the offspring.

The production of pro-inflammatory cytokines during inflammatory processes has been associated with preterm birth (PTB) and fetal injury in humans and mice. We previously demonstrated that exposition to an enriched environment (EE), defined as a noninvasive and biological significant stimulus of the sensory pathway combined with voluntary physical activity, prevented PTB and perinatal death induced by the systemic administration of bacterial lipopolysaccharide (LPS) in mice. This work aimed to analyze whether EE modulates the immune response to the inflammatory process induced by LPS in peripheral blood and the amniotic fluid (AF). We observed that EE modulated maternal white blood cell count and its response to LPS. Furthermore, we found higher levels of IL-10 and a higher percentage of B cells in AF from EE exposed mothers compared to controls. Albeit LPS significantly increased IL-6 levels in AF from both groups, it was 3.6 times higher in control environment (CE) exposed group when compared to EE. Similarly, levels of IL-22 were significantly increased by LPS in both groups, but it was 6.7 times higher in EE group. Interestingly, levels of PGE2 in AF were only increased in the EE-LPS treated group, and a positive correlation between IL-22 and PGE2 levels was observed. During lactation, EE prevented LPS-induced delay in physical landmarks analyzed to assess offspring development. Our results suggest that EE modulates the immune response to systemic LPS-administration protecting the offspring. We propose that an EE-like protocol could be designed for pregnant women aiming at preventing the sequelae present in premature children.

The enrichment of maternal environment prevents pre-term birth in a mice model.

Maternal lifestyle affects both mother health and pregnancy outcome in humans. Several studies have demonstrated that interventions oriented toward reducing stress and anxiety have positive effects on pregnancy complications such as preeclampsia, excessive gestational weight, gestational diabetes and preterm birth. In this work, we showed that the environmental enrichment (EE), defined as a noninvasive and biologically significant stimulus of the sensory pathway combined with voluntary physical activity, prevented preterm birth (PTB) rate by 40% in an inflammatory mouse model induced by the systemic administration of bacterial lipopolysaccharide (LPS). Furthermore, we found that EE modulates maternal metabolism and produces an anti-inflammatory environment that contributes to pregnancy maintenance. In pregnant mice uterus, EE reduces the expression of TLR4 and CD14 (the LPS receptor and its coactivator protein), preventing the LPS-induced increase in PGE2 and PGF2α release and nitric oxide synthase (NOS) activity. In cervical tissue, EE inhibits cervical ripening events, such as PGE2 release, matrix metalloproteinase (MMP)-9 increased activity and neutrophil recruitment, therefore conserving cervical function. It seems that EE exposure could mimic the stress and anxiety-reducing techniques mentioned above, explaining, at least partially, the beneficial effects of having a healthy lifestyle before and during gestation. Furthermore, we propose that designing an EE protocol for humans could be a noninvasive and preventive therapy for pregnancy complications, averting pre-term birth occurrence and dreaded sequelae that are present in the offspring born too soon.

Biomarcadores y parto pretérmino / Biomarkers and preterm birth

INTRODUCCIÓN El nacimiento pretérmino es la principal causa de muerte en menores de 5 años a nivel mundial. La identificación temprana durante el embarazo de las mujeres en riesgo de parto pretérmino (PP) espontáneo es importante para que puedan beneficiarse de potenciales estrategias preventivas. Sin embargo, pocas estrategias han demostrado ser efectivas en la práctica y hasta el momento ninguna prueba biológica ha demostrado ser exacta para predecir el PP. Por ende, a pesar de los avances del conocimiento mencionados, la capacidad para predecir quién experimentará un PP es notablemente limitada. De hecho, la Organización Mundial de la Salud no recomienda el uso del cribado biológico para estimar riesgo de PP, ya que ninguna prueba ha demostrado hasta el momento ser suficientemente beneficiosa. Existe entonces, una clara necesidad de identificar en forma precisa y confiable a las mujeres con mayor riesgo de PP, idealmente antes de la aparición de los síntomas. OBJETIVO GENERAL Evaluar la asociación de nuevos biomarcadores sanguíneos con el parto pretérmino. Como objetivos primarios se planteó explorar asociación entre los niveles sanguíneos de Progesterona (P4), de Receptores nucleares de P4 (PR A y PR B), de la enzima que degrada P4, de Endocanabinoides y de la expresión proteica de FAAH, y el PP. Como objetivos secundarios se planteó medir la tasa de reclutamiento al estudio y la tasa de seguimiento, y evaluar los procedimientos para la recolección, el transporte, el procesamiento y el almacenamiento de las muestras biológicas. MÉTODO Se llevó a cabo un estudio observacional prospectivo de tipo caso-cohorte, en el cual las mujeres fueron enroladas entre la semana 13 y 19 de gestación. Se hizo un seguimiento de las participantes que incluyó la toma de dos muestras sanguíneas durante el segundo trimestre de embarazo, la recolección de datos referidos a los antecedentes clínicos obstétricos, y los datos del parto. RESULTADOS Hasta el momento pudieron contestarse los objetivos de factibilidad. En las instituciones participantes se obtuvo una tasa de elegibilidad baja, del 32%, y una tasa de reclutamiento de más del 95%. En este grupo de mujeres se produjeron 36 partos de los cuales cinco fueron pretérmino. DISCUSIÓN Sin bien no hemos podido hasta el momento responder al objetivo principal del estudio, hemos logrado analizar la factibilidad de la realización de un estudio de esta complejidad y describir a las mujeres enroladas en cuanto a las variables de interés vinculadas al parto pretérmino. Hemos enfrentado problemas de reclutamiento y falta de fondos para poder realizar el análisis bioquímico de las muestras. Consideramos que los resultados de factibilidad que arrojó este estudio facilitan la comprensión de los componentes claves que entran en juego durante la implementación de un estudio tan complejo. Hemos demostrado que el estudio es factible de implementar si se logra una adecuada elección de los hospitales/centros participantes; suficiente tiempo y recursos para la etapa inicial en que todos los actores y procesos deben funcionar de manera articulada; adecuado tiempo para enrolamiento y seguimiento al parto de las mujeres embarazadas elegibles, y un presupuesto mayor para llevar adelante un estudio que requiere personal interdisciplinario, e insumos y reactivos importados

Peripheral Blood Mononuclear Cells Infiltration Downregulates Decidual FAAH Activity in an LPS-Induced Embryo Resorption Model.

Maternal infections with gram-negative bacteria are associated with miscarriage and are one of the most common complications during pregnancy. Previous studies from our group have shown that lipopolysaccharide (LPS)-activated infiltrating peripheral blood mononuclear cells (PBMC) into decidual tissue plays an important role in the establishment of a local inflammatory process that results in embryo cytotoxicity and early embryo resorption. Moreover, we have also shown that an increased endocannabinoid tone mediates LPS-induced deleterious effects during early pregnancy loss. Here, we sought to investigate whether the infiltrating PBMC modulates the decidual endocannabinoid tone and the molecular mechanisms involved. PBMC isolated from 7-day pregnant mice subjected to different treatments were co-cultured in a transwell system with decidual tissue from control 7-day pregnant mice. Decidual fatty acid amide hydrolase (FAAH) activity was measured by radioconvertion, total decidual protein nitration by Western blot (WB), and decidual FAAH nitration by immunoprecipitation followed by WB. We found that co-culture of PBMC obtained from LPS-treated mice increased the level of nitration of decidual FAAH, which resulted in a negative modulation of decidual FAAH activity. Interestingly, co-treatment with progesterone or aminoguanidine prevented this effect. We found that LPS-treated PBMC release high amounts of nitric oxide (NO) which causes tyrosine nitration of decidual FAAH, diminishing its enzymatic activity. Inactivation of FAAH, the main degrading enzyme of anandamide and similar endocannabinoids, could lead to an increased decidual endocannabinoid tone with embryotoxic effects. J. Cell. Physiol. 232: 1441-1447, 2017. © 2016 Wiley Periodicals, Inc.

Endocannabinoid system and pregnancy.

The endocannabinoid system (eCS), is a complex system, comprising the main endogenous ligands anandamide and 2-arachidonoyl glycerol, the cannabinoid receptors CB1 and CB2 and the biosynthetic and degrading enzymes. Cumulative evidence shows that the eCS plays an important role in reproduction, from egg fertilization to parturition. Therefore, alterations in this system, either by recreation/therapeutic use of cannabis or deregulation of the endogenous cannabinoids, might lead to adverse pregnancy outcomes, including retardation in embryo development, poor blastocyst implantation, inhibition of decidualization, miscarriage and compromised placentation. Nevertheless, the molecular mechanisms by which the eCS participates in different stages of pregnancy remain poorly understood. In this review, we will examine the evidence from animal and human studies to support the role of the eCS in implantation, early-to-late pregnancy and placentation as well as the difficulties of targeting this system for treatment of female infertility.

A role for the endocannabinoid system in premature luteal regression and progesterone withdrawal in lipopolysaccharide-induced early pregnancy loss model.

STUDY QUESTION: What is the role of the endocannabinoid system (eCS) in the alterations of the endocrine system in a murine model of lipopolysaccharide (LPS)-induced miscarriage? SUMMARY ANSWER: In 7-days pregnant wild type, but not cannabinoid receptor type 1 knockout (CB1-KO) mice, LPS increased COX-2 expression and prostaglandin F2α (PGF2α) production in the uterus leading to lower expression of prolactin receptor in the ovary and a marked regression of corpora lutea (CL), suggesting that the eCS mediates the deleterious effects of LPS on reproductive events. WHAT IS KNOWN ALREADY: Appropriate systemic progesterone levels are critical for a successful pregnancy outcome. Precocious loss of luteal progesterone (P4) secretion leads to miscarriage in rodents. We have previously shown that LPS administration to pregnant mice induces embryonic resorption accompanied by a dramatic decrease in systemic progesterone levels in a murine model of inflammatory miscarriage, with the eCS mediating these LPS-induced deleterious effects. STUDY DESIGN SAMPLES/MATERIALS, METHODS: CD1 wild-type (WT) and CB1-KO mice were randomly allocated to Vehicle (saline; i.p.) or LPS (0.5 µg/g body weight; i.p.) treated groups: (WT-Vehicle; WT-LPS; CB1-KO-Vehicle and CB1-KO-LPS). A single injection was given on day 7 of pregnancy and tissues (blood, ovary, uterus) were collected 6, 12, 24 and 48 h later. P4 and PGF2α plasma levels were determined by radioimmunoassay. Cyclooxygenase-2 (COX-2) mRNA (RT-PCR) and protein (Western blot) content in uterus was assayed. COX-2 and prolactin receptor (PrlR) mRNA levels in the ovary were assayed by RT-PCR. Tissue morphology of the CL was assessed by haematoxylin-eosin staining. MAIN RESULTS AND THE ROLE OF CHANCE: Treatment of 7-day pregnant WT mice with LPS induced a P4 withdrawal (p < 0.05), increased in uterine COX-2 mRNA and protein expression (p < 0.05) as well as an increase in uterine PGF2α production (p < 0.05). These changes were absent in LPS-treated 7-day pregnant CB1-KO mice. In ovarian tissues, LPS treatment to 7-day pregnant WT mice induced a downregulation of PrlR mRNA expression (p < 0.05) together with an increase in COX-2 mRNA expression (p < 0.05) and PGF2α content (p < 0.05). These effects were absent in the CB1-KO mice. Collectively, our results suggest a role for the eCS mediating LPS-induced deleterious effects on reproductive tissues. LIMITATIONS, REASONS FOR CAUTION: An important caveat of this study is the endocrine differences between mice and humans during pregnancy (e.g. P4 is produced by the CL throughout pregnancy in mice, whereas this is not the case in humans), which limits the extrapolation of the results presented here. WIDER IMPLICATIONS OF THE FINDINGS: Our findings provide new insights in the role of the endocannabinoid system in the physiopathology of reproduction as well as the role of this endogenous system as a mediator of LPS deleterious effects on reproductive tissues. LARGE SCALE DATA: None. STUDY FUNDING AND COMPETING INTERESTS: Dr Ana María Franchi was funded by Agencia Nacional para la Promoción Científica y Tecnológica (PICT 2010/0813 and PICT 2013/0097) and by Consejo Nacional de Investigaciones Científicas y Técnicas (PIP 2012/0061). The authors have no competing interests.

Progesterone modulates the LPS-induced nitric oxide production by a progesterone-receptor independent mechanism.

Genital tract infections caused by Gram-negative bacteria induce miscarriage and are one of the most common complications of human pregnancy. LPS administration to 7-day pregnant mice induces embryo resorption after 24h, with nitric oxide playing a fundamental role in this process. We have previously shown that progesterone exerts protective effects on the embryo by modulating the inflammatory reaction triggered by LPS. Here we sought to investigate whether the in vivo administration of progesterone modulated the LPS-induced nitric oxide production from peripheral blood mononuclear cells from pregnant and non-pregnant mice. We found that progesterone downregulated LPS-induced nitric oxide production by a progesterone receptor-independent mechanism. Moreover, our results suggest a possible participation of glucocorticoid receptors in at least some of the anti-inflammatory effects of progesterone.

Lipopolysaccharide-induced murine embryonic resorption involves changes in endocannabinoid profiling and alters progesterone secretion and inflammatory response by a CB1-mediated fashion.

Genital tract infections are a common complication of human pregnancy that can result in miscarriage. We have previously shown that a lipopolysaccharide (LPS) induces embryonic resorption in a murine model of inflammatory miscarriage. This is accompanied by a dramatic decrease in systemic progesterone levels associated with a robust pro-inflammatory response that results in embryo resorption. Here, we tested the hypothesis that the endogenous cannabinoid system (eCS), through cannabinoid receptor 1 (CB1), plays a role in regulating progesterone levels and, therefore, the pro-inflammatory response. We show that LPS treatment in pregnant mice causes significant changes in the eCS ligands, which are reversed by progesterone treatment. We further show the CB1-KO mice maintain higher plasma progesterone levels after LPS treatment, which is associated with a feebler uterine inflammatory response and a significant drop in embryo resorption. These data suggest that manipulation of CB1 receptors and/or ligands is a potential therapeutic avenue to decrease infection-induced miscarriage.

GAP-43 slows down cell cycle progression via sequences in its 3'UTR.

Growth-associated protein 43 (GAP-43) is a neuronal phosphoprotein associated with initial axonal outgrowth and synaptic remodeling and recent work also suggests its involvement in cell cycle control. The complex expression of GAP-43 features transcriptional and posttranscriptional components. However, in some conditions, GAP-43 gene expression is controlled primarily by the interaction of stabilizing or destabilizing RNA-binding proteins (RBPs) with adenine and uridine (AU)-rich instability elements (AREs) in its 3'UTR. Like GAP-43, many proteins involved in cell proliferation are encoded by ARE-containing mRNAs, some of which codify cell-cycle-regulating proteins including cyclin D1. Considering that GAP-43 and cyclin D1 mRNA stabilization may depend on similar RBPs, this study evaluated the participation of GAP-43 in cell cycle control and its underlying mechanisms, particularly the possible role of its 3'UTR, using GAP-43-transfected NIH-3T3 fibroblasts. Our results show an arrest in cell cycle progression in the G0/G1 phase. This arrest may be mediated by the competition of GAP-43 3'UTR with cyclin D1 3'UTR for the binding of Hu proteins such as HuR, which may lead to a decrease in cyclin D1 expression. These results might lead to therapeutic applications involving the use of sequences in the B region of GAP-43 3'UTR to slow down cell cycle progression.

Progesterone reverts LPS-reduced FAAH activity in murine peripheral blood mononuclear cells by a receptor-mediated fashion.

Increased anandamide concentrations are associated with pregnancy failure. Anandamide levels are regulated by the fatty acid amide hydrolase (FAAH). The aim of the study was to investigate the role of progesterone (P) on FAAH modulation in murine peripheral blood mononuclear cells (PBMC) under septic conditions. We observed that in vivo administration of LPS to non-pregnant (NP) mice decreased FAAH activity of PBMC while in pregnant mice no changes in FAAH activity were observed. NP animals administered with P had a similar response to LPS as the pregnant animals. Also, NP mice injected with P antagonist and P showed that the effect of P on LPS-reduced FAAH activity was impaired. Furthermore, LPS produced a decrease in the ratio of PR-B/PR-A in NP animals. Our results showed that, in our model the endotoxin decreased PBMC's FAAH activity and this condition was reverted by P in a receptor-mediated fashion.

Progesterone is essential for protecting against LPS-induced pregnancy loss. LIF as a potential mediator of the anti-inflammatory effect of progesterone.

Lipopolysaccharide (LPS) administration to mice on day 7 of gestation led to 100% embryonic resorption after 24 h. In this model, nitric oxide is fundamental for the resorption process. Progesterone may be responsible, at least in part, for a Th2 switch in the feto-maternal interface, inducing active immune tolerance against fetal antigens. Th2 cells promote the development of T cells, producing leukemia inhibitory factor (LIF), which seems to be important due to its immunomodulatory action during early pregnancy. Our aim was to evaluate the involvement of progesterone in the mechanism of LPS-induced embryonic resorption, and whether LIF can mediate hormonal action. Using in vivo and in vitro models, we provide evidence that circulating progesterone is an important component of the process by which infection causes embryonic resorption in mice. Also, LIF seems to be a mediator of the progesterone effect under inflammatory conditions. We found that serum progesterone fell to very low levels after 24 h of LPS exposure. Moreover, progesterone supplementation prevented embryonic resorption and LPS-induced increase of uterine nitric oxide levels in vivo. Results show that LPS diminished the expression of the nuclear progesterone receptor in the uterus after 6 and 12 h of treatment. We investigated the expression of LIF in uterine tissue from pregnant mice and found that progesterone up-regulates LIF mRNA expression in vitro. We observed that LIF was able to modulate the levels of nitric oxide induced by LPS in vitro, suggesting that it could be a potential mediator of the inflammatory action of progesterone. Our observations support the view that progesterone plays a critical role in a successful pregnancy as an anti-inflammatory agent, and that it could have possible therapeutic applications in the prevention of early reproductive failure associated with inflammatory disorders.

Opposite effects of methanandamide on lipopolysaccharide-induced prostaglandin E2 and F2α synthesis in uterine explants from pregnant mice.

Prostaglandins (PG) are effective abortifacients and are important mediators of lipopolisaccharide (LPS)-induced embryonic resorption (ER). Besides, anandamide (AEA) has been described as one of the major endocannabinoids present in the uterus suggesting that it might play a role in reproduction. It has been reported that high levels of AEA are associated with pregnancy failure and that LPS increases AEA production. Also, it has been observed that AEA modulates PG production in different tissues. In this sense, we studied whether LPS-induced PG production is modulated by AEA and we also assessed the effect of this endocannabinoid on PG metabolism in an in vitro model. Uterine explants from BALB/c implantation sites were cultured in the presence of LPS plus cannabinoid receptor (CB) specific antagonists and PG production was assessed. Then, we studied the effect of exogenous AEA on different steps of PG metabolic pathway. We showed that AEA is involved in LPS-induced PG biosynthesis. Also, we observed that AEA exerts opposite effects on PGE(2) and PGF(2α) biosynthesis, by inhibiting PGE(2) production and increasing PGF(2α) levels. We suggest that AEA could be involved in the mechanisms implicated in LPS-induced ER. A better understanding of how AEA could be affecting ER could help developing specific interventions to prevent this pathology.

Lipopolysaccharide-induced murine embryonic resorption involves nitric oxide-mediated inhibition of the NAD+-dependent 15-hydroxyprostaglandin dehydrogenase.

The initial inactivation of prostaglandins (PGs) is mediated by 15-hydroxyprostaglandin dehydrogenase (15-PGDH). PGs are potent mediators of several biological processes, including inflammation and reproduction. In uterus, PGs play a key role in infection-induced pregnancy loss, in which concentration of this mediator increased. This process is accompanied with the induction of nitric oxide synthase expression and a marked increase in uterine levels of nitric oxide. There is no information concerning nitric oxide contribution to potential changes in PG catabolism, but experimental evidence suggests that nitric oxide modulates PG pathways. The specific objectives of the study were to evaluate the protein expression of HPGD (15-PGDH) and to characterize the nitric oxide-dependent regulation of this enzyme in a model of lipopolysaccharide (LPS)-induced embryonic resorption. Results show that LPS decreased HPGD protein expression and augmented PGE synthase activity; therefore, PGE2 levels increased in uterus in this inflammatory condition. Just as LPS, the treatment with a nitric oxide donor diminished HPGD protein expression in uterine tissue. In contrast, the inhibition of nitric oxide synthesis both in control and in LPS-treated mice increased 15-PGDH levels. Also, we have found that this enzyme and PGE2 levels are not modulated by peroxynitrite, an oxidant agent derived from nitric oxide. This study suggests that LPS and nitric oxide promote a decrease in the ability of the uterus for PG catabolism during bacterially triggered pregnancy loss in mice.

Inflammatory agents involved in septic miscarriage.

Even though the understanding of the cause of early pregnancy loss due to chromosomal abnormalities has improved, there is a dearth of knowledge of the causes of loss in euploid conceptuses. Maternal infections are a cause of abort in humans, but the mechanisms are not clear, so we have developed a murine model to study the mechanism of septic abortion by inducing embryonic resorption (ER) with lipopolysaccharide (LPS). We demonstrated that augmented production of nitric oxide (NO) and prostaglandins (PG) is involved in ER, and that inhibitors of their synthesis could prevent ER. Also, we observed an increase in the oxidative damage, evidenced by nitration of tyrosine proteins, due to the peroxynitrite anion. Since an association between chronic marijuana smoking and early miscarriage has been shown in women, we studied the participation of anandamide (AEA), the principal endocannabinoid, on the mechanism of action of LPS. We showed that LPS-induced NO synthesis and tissue damage were mediated by AEA, and that this endotoxin inhibited AEA degradation and increased its synthesis. These results suggest that several inflammatory molecules participate in the mechanism of early pregnancy loss and that their modulation could be useful tools to prevent it.

Curcumin induces cell-arrest and apoptosis in association with the inhibition of constitutively active NF-kappaB and STAT3 pathways in Hodgkin's lymphoma cells.

Although treatment of Hodgkin's lymphoma (HL) with a multi-drug approach has been very successful, its toxicity becomes evident after several years as secondary malignancies and cardiovascular disease. Therefore, the current goal in HL treatment is to find new therapies that specifically target the deregulated signaling cascades, such as NF-kappaB and STAT3, which cause Hodgkin and Reed-Sternberg (H-RS) cell proliferation and resistance of apoptosis. Based on the above information, we investigated the capacity of curcumin to inhibit NF-kappaB and STAT3 in H-RS cells, characterizing the functional consequences. Curcumin is incorporated into H-RS cells and acts inhibiting both NF-kappaB and STAT3 activation, leading to a decreased expression of proteins involved in cell proliferation and apoptosis, e.g. Bcl-2, Bcl-xL, cFLIP, XIAP, c-IAP1, survivin, c-myc and cyclin D1. Interestingly, curcumin caused cell cycle arrest in G2-M and a significant reduction (80-97%) in H-RS cell viability. Furthermore, curcumin triggered cell death by apoptosis, as evidenced by the activation of caspase-3 and caspase-9, changes in nuclear morphology and phosphatidylserine translocation. The above findings provide a mechanistic rationale for the potential use of curcumin as a therapeutic agent for patients with HL.