Effect of Nicotine on CYP2B1 Expression in a Glioma Animal Model and Analysis of CYP2B6 Expression in Pediatric Gliomas.

Cyclophosphamide (CPA) is a pro-drug commonly used in the chemotherapeutic schemes for glioma treatment but has high toxicity and the side effects include brain damage and even death. Since CPA is activated mainly by CY2B6, over-expression of the enzyme in the tumor cells has been proposed to enhance CPA activation. In this study, we explored the induction of the Cyp2b1 (homologous to CYP2B6) by nicotine in an animal rat model with glioma. Gene expression and protein levels were analyzed by RT-PCR and Western blot. Nicotine treatment increased CYP2B1 protein levels in the healthy animals' brain tissue. In the brain tissue of animals with glioma, the CYP2B1 showed a high expression, even before nicotine treatment. Nicotine did not increase significantly the CYP2B1 protein expression in the tumor, but increased its expression in the tumor vicinity, especially around blood vessels in the cortex. We also explored CY2B6 expression in glioma samples derived from pediatric patients. Tumor tissue showed a variable expression of the enzyme, which could depend on the tumor malignancy grade. Induction of the CYP2B6 in pediatric gliomas with lower expression of the enzyme, could be an alternative to improve the antitumoral effect of CPA treatment.

Decidualization Mediated by Steroid Hormones Modulates the Innate Immunity in Response to Group B Streptococcal Infection in vitro.

BACKGROUND: Decidual cells play a role in the modulation of the innate immune response to protect pregnancy against infection. Steroid hormones regulate the innate immune response in different tissues, and they are involved in several biological processes like decidualization. The aim of this study was to assess if steroid hormones modulate the innate immunity in endometrial stromal cells (ESCs) and decidual stromal cells (DSCs) in response to group B streptococcus (GBS) infection in vitro. METHODS: Primary cultures of ESC were differentiated into DSC using 36 nM estradiol + 300 nM progesterone, and both were infected with GBS overnight. Concentrations of pro- and anti-inflammatory mediators (interleukin [IL]-1ß, IL-6, tumor necrosis factor [TNF]-α, IL-10, and TGF-ß), chemokines (IL-8 and GCP-2), and human ß-defensins (HBD-1, HBD-2, and HBD-3) were measured in the culture supernatants. RESULTS: DSCs showed a significant increase in IL-6 (p < 0.05), TNF-α (p < 0.05), IL-10 (p < 0.01), and TGF-ß (p < 0.05) secretion after GBS infection, while these changes were not observed in infected ESCs. IL-8 and GCP-2 increased after GBS infection, regardless of decidualization. ß-Defensins 1-3 decreased (p < 0.05) in ESCs after GBS infection, and hormone decidualization preserved the secretion of these antimicrobial peptides. CONCLUSIONS: Decidualization mediated by steroid hormones balance the pro- and anti-inflammatory response at the maternal-fetal interface under infection conditions.

Isolation of Primary Human Decidual Cells from the Fetal Membranes of Term Placentae.

The maternal decidua is a transient and dynamic tissue that functions as an immunoprivileged matrix related to nutritional and endocrine processes. The function of decidual cells is key to the success of embryo implantation and the maintenance of pregnancy with a positive maternal-fetal outcome. Therefore, establishing a method to optimize the isolation of primary decidual cells is essential. Our protocol described here provides a good yield of decidual cells in an optimized time.

Resistin Modulates Low-Density Lipoprotein Cholesterol Uptake in Human Placental Explants via PCSK9.

Maternal metabolic status influences pregnancy and, consequently, the perinatal outcome. Resistin is a pro-inflammatory adipokine predominantly expressed and secreted by mononuclear cells, adipose tissue, and placental trophoblastic cells during pregnancy. Recently, we reported an inverse association between maternal resistin levels and fetal low-density lipoprotein cholesterol (LDL-C). Then, in this work, we used a human placental explant model and the trophoblast cell line JEG-3 to evaluate whether resistin affects placental LDL-C uptake. Resistin exposure induced the transcription factor SREBP-2, LDLR, and PCSK9 mRNA expression, and changes at the protein level were confirmed by immunohistochemistry and Western blot. However, for LDLR, the changes were not consistent between mRNA and protein levels. Using a labeled LDL-cholesterol (BODIPY FL LDL), uptake assay demonstrated that the LDL-C was significantly decreased in placental explants exposed to a high dose of resistin and a lesser extent in JEG-3 cells. In summary, resistin induces PCSK9 expression in placental explants and JEG-3 cells, which could be related to negative regulation of the LDLR by lysosomal degradation. These findings suggest that resistin may significantly regulate the LDL-C uptake and transport from the maternal circulation to the fetus, affecting its growth and lipid profile.

Persistence of decidual NK cells and KIR genotypes in healthy pregnant and preeclamptic women: a case-control study in the third trimester of gestation.

BACKGROUND: Natural Killer (NK) cells are the most abundant lymphocytes in the decidua during early gestation. The interactions of NK cells with the extravillous cytotrophoblast have been associated with a normal spiral artery remodeling process, an essential event for a successful pregnancy. Recent data indicate that alterations in the amount of decidual NK (dNK) cells contribute to the development of preeclampsia (PE). Moreover, genetic studies suggest that Killer Immunoglobulin-like Receptors (KIR) expressed in dNK cells influence the susceptibility to PE. Although dNK cells have been well characterized during early pregnancy, they have been scarcely studied in the third trimester of gestation. The aim of this work was to characterize dNK cells at the last trimester of gestation and to analyze the KIR genotype of healthy and PE women. METHODS: Decidual samples were obtained during Caesarean section from control (n = 10) and PE (n = 9) women. Flow cytometric analysis of CD3, CD56, CD16 and CD9 was used to characterize and quantify dNK cells in both groups. Cell surface markers from decidual leukocytes were compared with PBMC from healthy donors.KIR genotyping was performed in genomic DNA (control, n = 86; PE, n = 90) using PCR-SSP. RESULTS: The results indicate that dNK cells persist throughout pregnancy. They represented 20% of total leukocytes in control and PE groups, and they expressed the same cell surface markers (CD3-, CD56+, CD16- and CD9+) as dNK in the first trimester of gestation. There were no significant differences in the percentage of dNK cells between control and PE groups. The analysis of KIR gene frequencies and genotypes was not statistically different between control and PE groups. The ratio of activating to inhibitory genes indicated that the overall inhibitory balance (0.2-0.5) was more frequent in the PE group (control, 31.3% vs PE, 45.5%), and the activating balance (0.6-1.1) was more frequent in the control group (control, 68.6% vs PE, 54.4%). However this difference was not significant. CONCLUSION: We demonstrated the persistence of dNK cells in PE and control women at the third trimester of pregnancy; these dNK cells had a similar phenotype to those found during early pregnancy. The predominance of a KIR inhibitory balance in the PE group could be associated to the physiopathology of PE.

Mitochondrial DNA Copy Number Adaptation as a Biological Response Derived from an Earthquake at Intrauterine Stage.

An altered mitochondrial DNA copy number (mtDNAcn) at birth can be a marker of increased disease susceptibility later in life. Gestational exposure to acute stress, such as that derived from the earthquake experienced on 19 September 2017 in Mexico City, could be associated with changes in mtDNAcn at birth. Our study used data from the OBESO (Biochemical and Epigenetic Origins of Overweight and Obesity) perinatal cohort in Mexico City. We compared the mtDNAcn in the umbilical cord blood of 22 infants born before the earthquake, 24 infants whose mothers were pregnant at the time of the earthquake (exposed), and 37 who were conceived after the earthquake (post-earthquake). We quantified mtDNAcn by quantitative real-time polymerase chain reaction normalized with a nuclear gene. We used a linear model adjusted by maternal age, body mass index, socioeconomic status, perceived stress, and pregnancy comorbidities. Compared to non-exposed newborns (mean ± SD mtDNAcn: 0.740 ± 0.161), exposed and post-earthquake newborns (mtDNAcn: 0.899 ± 0.156 and 0.995 ± 0.169, respectively) had increased mtDNAcn, p = 0.001. The findings of this study point at mtDNAcn as a potential biological marker of acute stress and suggest that experiencing an earthquake during pregnancy or before gestation can have programing effects in the unborn child. Long-term follow-up of newborns to women who experience stress prenatally, particularly that derived from a natural disaster, is warranted.

Gestational Weight Gain Influences the Adipokine-Oxidative Stress Association during Pregnancy.

INTRODUCTION AND OBJECTIVE: The weight gained during pregnancy could determine the immediate and future health of the mother-child dyad. Excessive gestational weight gain (EGWG) due to abnormal adipose tissue (AT) accumulation is strongly associated with adverse perinatal outcomes as gestational diabetes, macrosomia, obesity, and hypertension further in life. Dysregulation of adipokine, AT dysfunction, and an imbalance in the prooxidant-antioxidant systems are critical features in altered AT accumulation. This study was aimed to investigate the association between adipokines and oxidative stress markers in pregnant women and the influence of the GWG on this association. METHODS: Maternal blood samples were obtained in the third trimester of pregnancy (n = 74) and serum adipokines (adiponectin, leptin, and resistin), oxidative damage markers: 8-oxo-2'-deoxyguanosine (8-oxodG), lipohydroperoxides (LOOH), malondialdehyde (MDA), and carbonylated proteins (CP), and glucose a metabolic marker were measured. RESULTS: Women with EGWG had low adiponectin levels than women with adequate weight gain (AWG) or insufficient weight gain (IWG). Multiple linear regression models revealed a positive association between adiponectin and 8-oxodG in women with AWG (B = 1.09, 95% CI: 164-222, p = 0.027) and IWG (B = 0.860, 95% CI: 0.199-1.52, p = 0.013) but not in women with EGWG. In women with EGWG, leptin was positively associated with LOOH (p = 0.018), MDA (p = 0.005), and CP (p = 0.010) oxidative markers. CONCLUSION: Our findings suggest that concurrent mechanisms regulate adipokine production and oxidative stress in pregnant women and that this regulation is influenced by GWG, probably due to an excessive AT accumulation.

[The two leading hypothesis regarding the molecular mechanisms and etiology of preeclampsia, and the Mexican experience in the world context]. / Estado actual de la preeclampsia en México: de lo epidemiológico a sus mecanismos moleculares.

Preeclampsia (PE) is one of the most severe complications of pregnancy. PE is responsible for the highest rates of morbidity and mortality for both pregnant women and the neonate. In this review, we first address general aspects of PE and its diagnosis, along with some epidemiological aspects of this disease in the mexican population, in particular the experience from the Instituto Mexicano del Seguro Social. Even though over the last 20 years a great deal of evidence has accumulated regarding PE's pathophysiology, an exact mechanism to explain its etiology has not been established. This review aims to cover the status of two of the most important hypotheses in the etiology of PE: the immunological and the placental ischemia hypotheses. Recent data suggest that Natural Killer cells (NK) play a major role in the decidual spiral arteriole remodeling and in normal placental development. In genetic studies, KIR receptors present in NK cells have been involved in the susceptibility for the disease. In this review, we discuss data of our group regarding the presence of NK cells in the decidua, at the end of pregnancy and the genotypes of KIR receptors in normal and preeclamptic Mexican population. PE is characterized by abnormal placentation and hypoxia with an increase of anti-angiogenic factors; the Hypoxia-inducible factor 1-alfa (HIF1-alfa) is over expressed in PE. In this review, we also included some of our results concerning the polymorphisms and regulation of HIF in preeclamptic women.

High Plasmatic Levels of Advanced Glycation End Products are Associated with Metabolic Alterations and Insulin Resistance in Preeclamptic Women.

AIMS: The purpose of this study was to investigate the association between plasmatic levels of advanced end glycation products (AGEs) and the metabolic profile in subjects diagnosed with preeclampsia, due to the known relation of these molecules with oxidative stress and inflammation, which in turn are related with PE pathogenesis. BACKGROUND: It has been reported that increased levels of AGEs are observed in patients with preeclampsia as compared with healthy pregnant subjects, which was mainly associated with oxidative stress and inflammation. Besides, in women with preeclampsia, there are metabolic changes such as hyperinsulinemia, glucose intolerance, dyslipidemia, among others, that are associated with an exacerbated insulin resistance. Additionally, some parameters indicate the alteration of hepatic function, such as increased levels of liver enzymes. However, the relationship of levels of AGEs with altered lipidic, hepatic, and glucose metabolism parameters in preeclampsia has not been evaluated. OBJECTIVE: To investigate the association between plasmatic levels of AGEs and hepatic, lipid, and metabolic profiles in women diagnosed with preeclampsia. METHODS: Plasma levels of AGEs were determined by a competitive enzyme-linked immunosorbent assay (ELISA) in 15 patients diagnosed with preeclampsia and 28 normoevolutive pregnant subjects (control group). Hepatic (serum creatinine, gammaglutamyl transpeptidase, aspartate transaminase, alanine transaminase, uric acid, and lactate dehydrogenase), lipid (apolipoprotein A, apolipoprotein B, total cholesterol, triglycerides, low-density lipoproteins, and high-density lipoproteins), and metabolic variables (glucose, insulin, and insulin resistance) were assessed. RESULTS: Plasmatic levels of AGEs were significantly higher in patients with preeclampsia as compared with the control. A positive correlation between circulating levels of AGEs and gamma-glutamyl transpeptidase, uric acid, glucose, insulin, and HOMA-IR levels was found in patients with preeclampsia. In conclusion, circulating levels of AGEs were higher in patients with preeclampsia than those observed in healthy pregnant subjects. Besides, variables of hepatic and metabolic profile, particularly those related to insulin resistance, were higher in preeclampsia as compared with healthy pregnant subjects. Interestingly, there is a positive correlation between AGEs levels and insulin resistance. CONCLUSIONS: Circulating levels of AGEs were higher in patients with preeclampsia than those observed in healthy pregnant subjects. Besides, hepatic and metabolic profiles, particularly those related to insulin resistance, were higher in preeclampsia as compared with healthy pregnant subjects. Interestingly, there is a positive correlation between AGEs levels and insulin resistance, suggesting that excessive glycation and an impaired metabolic profile contribute to the physiopathology of preeclampsia.

Von Hippel-Lindau disease germline mutations in Mexican patients with cerebellar hemangioblastoma.

OBJECT: Central nervous system (CNS) hemangioblastomas are benign vascular tumors arising either sporadically or as a manifestation of von Hippel-Lindau (VHL) disease, a hereditary cancer syndrome. The authors studied a series of patients with CNS hemangioblastomas and their families to identify germline mutations in the VHL tumor suppressor gene and to establish a predictive testing and screening protocol. METHODS: Patients admitted between 2002 and 2004 to the Instituto Nacional de Neurología y Neurocirugía for hemangioblastoma were prospectively enrolled, together with their at-risk family members. The authors performed the molecular analysis of the VHL gene by using polymerase chain reaction and direct genetic sequencing. All asymptomatic mutation carriers underwent genetic counseling and tumor surveillance. Ninety-eight individuals were tested for VHL mutations--23 symptomatic and 75 asymptomatic individuals belonging to 16 families. Seven of the families had definite clinical criteria of VHL disease, five had sporadic hemangioblastoma, and four had CNS hemangioblastoma combined with minor visceral signs. Molecular genetic testing identified five germline mutations in six of the definite VHL families (sensitivity 85%), but none in the possible VHL and sporadic hemangioblastoma cases; four of these mutations had been previously described and one is a novel mutation present in two unrelated families. After patients carrying the mutation were identified, they underwent clinical screening and asymptomatic VHL-related lesions were identified in 43%. CONCLUSIONS: Genetic testing for mutations in the VHL gene is crucial in patients with CNS hemangioblastoma. The prompt identification of patients carrying the genetic mutation allows for a multidisciplinary screening protocol to decrease morbidity and mortality rates in these patients, while avoiding costly and invasive procedures for noncarriers.

Maternal and Fetal Lipid and Adipokine Profiles and Their Association with Obesity.

Background. Maternal metabolic changes impact fetal metabolism resulting in a higher risk for developing chronic diseases later in life. The aim of this study was to assess the association between maternal and fetal adipokine and lipid profiles, as well as the influence of maternal weight on this association. Methods. Healthy pregnant women at term who delivered by C-section were enrolled. Maternal and fetal glucose, lipid profile, adiponectin, leptin, and resistin levels were analyzed by obesity and maternal weight gain. Statistics included descriptives, correlations, and mean differences (SPSS v20.0). Results. Adiponectin and resistin concentrations were higher in fetal blood, while leptin was lower (p < 0.05). A significant inverse association between maternal resistin and fetal LDL-cholesterol (LDL-C) (r = -0.327; p = 0.022) was observed. A positive correlation was found between maternal and fetal resistin (r = 0.358; p = 0.013). Women with excessive weight gain had higher leptin levels and their fetuses showed higher LDL-C levels (p < 0.05). Conclusions. Maternal resistin showed an inverse association with fetal LDL-C, suggesting that maternal adiposity status may play an active role in the regulation of fetal lipid profile and consequently, in fetal programming. Excessive maternal weight gain during pregnancy may exert an effect over metabolic mediators in both mother and newborn.

Matrix Metalloproteinase-3 (MMP-3) Is an Endogenous Activator of the MMP-9 Secreted by Placental Leukocytes: Implication in Human Labor.

BACKGROUND: The activity of matrix degrading enzymes plays a leading role in the rupture of the fetal membranes under normal and pathological human labor, and matrix metalloproteinase-9 (MMP-9) it is considered a biomarker of this event. To gain further insight into local MMP-9 origin and activation, in this study we analyzed the contribution of human placental leukocytes to MMP-9 secretion and explored the local mechanisms of the pro-enzyme activation. METHODS: Placental blood leukocytes were obtained from women at term gestation without labor and maintained in culture up to 72 h. MMP-9 activity in the culture supernatants was determined by zymography and using a specific substrate. The presence of a potential pro-MMP-9 activator in the culture supernatants was monitored using a recombinant biotin-labeled human pro-MMP-9. To characterize the endogenous pro-MMP-9 activator, MMP-1, -3, -7 and -9 were measured by multiplex assay in the supernatants, and an inhibition assay of MMP-9 activation was performed using an anti-human MMP-3 and a specific MMP-3 inhibitor. Finally, production of MMP-9 and MMP-3 in placental leukocytes obtained from term pregnancies with and without labor was assessed by immunofluorescence. RESULTS: Placental leukocytes spontaneously secreted pro-MMP-9 after 24 h of culture, increasing significantly at 48 h (P≤0.05), when the active form of MMP-9 was detected. Culture supernatants activated the recombinant pro-MMP-9 showing that placental leukocytes secrete the activator. A significant increase in MMP-3 secretion by placental leukocytes was observed since 48 h in culture (P≤0.05) and up to 72 h (P≤0.001), when concentration reached its maximum value. Specific activity of MMP-9 decreased significantly (P≤0.005) when an anti-MMP-3 antibody or a specific MMP-3 inhibitor were added to the culture media. Placental leukocytes from term labor produced more MMP-9 and MMP-3 compared to term non-labor cells. CONCLUSIONS: In this work we confirm that placental leukocytes from human term pregnancies are able to secrete large amounts of MMP-9, and that the production of the enzyme it is enhanced by labor. We also demonstrate for the first time that endogenous MMP-3 plays a major role in MMP-9 activation process. These findings support the contribution of placental leukocytes to create the collagenolytic microenvironment that induces the rupture of the fetal membranes during human labor.

Polymorphisms in the hypoxia-inducible factor 1 alpha gene in Mexican patients with preeclampsia: A case-control study.

BACKGROUND: Although the etiology of preeclampsia is still unclear, recent work suggests that changes in circulating angiogenic factors play a key role in its pathogenesis. In the trophoblast of women with preeclampsia, hypoxia-inducible factor 1 alpha (HIF-1α) is over-expressed, and induces the expression of non-angiogenic factors and inhibitors of trophoblast differentiation. This observation prompted the study of HIF-1α and its relation to preeclampsia. It has been described that the C1772T (P582S) and G1790A (A588T) polymorphisms of the HIF1A gene have significantly greater transcriptional activity, correlated with an increased expression of their proteins, than the wild-type sequence. In this work, we studied whether either or both HIF1A variants contribute to preeclampsia susceptibility. RESULTS: Genomic DNA was isolated from 150 preeclamptic and 105 healthy pregnant women. Exon 12 of the HIF1A gene was amplified by PCR, and the genotypes of HIF1A were determined by DNA sequencing.In preeclamptic women and controls, the frequencies of the T allele for C1772T were 4.3 vs. 4.8%, and the frequencies of the A allele for G1790A were 0.0 vs. 0.5%, respectively. No significant differences were found between groups. CONCLUSION: The frequency of the C1772T and G1790A polymorphisms of the HIF1A gene is very low, and neither polymorphism is associated with the development of preeclampsia in the Mexican population.