DIFFERENTIAL INVOLVEMENT OF S AND L ISOFORMS OF THE CENTROSOMAL MARK4 KINASE IN GLIOMA INITIATION AND PROGRESSION

MARK4 (MAP/Microtubule Affinity-Regulating Kinase 4) belongs to a family of serine-threonine kinases that are able to phosphorylate the Microtubule Associated Proteins, causing their detachment from the microtubules and thus increasing microtubule dynamics. MARK4 gene is ubiquitously expressed and encodes at least two alternatively spliced isoforms, L and S, which differ in the C-terminal region and are differentially regulated in human tissues, especially in the Central Nervous System (CNS). MARK4S predominance in normal brain has been related to a putative role in neuronal differentiation; MARK4L has been found up-regulated in hepatocarcinoma cells and highly expressed in gliomas, suggesting an involvement of the kinase in cycling cells. Gliomas are the most common tumors of the CNS and are characterized by elevated cellular heterogeneity, which has been imputed to the presence of different cells (mature cells / progenitors / stem cells) from which each glioma originates. To further investigate MARK4 up-regulation in glial tumors, we analyzed a panel of 35 glioma tissue samples and 21 glioma cell lines (both low and high malignancy grade) and 6 glioblastoma-derived cancer stem cell populations (GBM CSC; glioblastoma is a IV grade glioma). Human neural progenitors, total human normal brain and mouse neural stem cells (NSC, isolated from the sub-ventricular zone) were also included in the study. A quantitative approach (Real-time PCR; q-PCR) was applied for mRNA analyses whereas MARK4 protein levels and localization were tested by Immunoblotting (IB), Immunohistochemistry (IHC) and Immunofluorescence (IF). We first carried out mutational analysis of the main MARK4 domains, but it didn\u2019t reveal any genomic alteration, as did not the previously performed array-CGH analysis. Integrated approaches of q-PCR, IB and IHC studies show that, although MARK4S and L have a heterogeneous expression within and across different glioma subtypes (consistent with the intrinsic cell heterogeneity of these brain tumors), MARK4L is the prevalent isoform in near all the glioma samples. Conversely, MARK4S mRNA levels display a significant decrease inversely correlating with malignancy grade and are also hardly detectable in both neural and GBM-derived cancer stem cell populations. Therefore, a higher MARK4L prevalence in parallel to low levels of MARK4S characterizes highly undifferentiated cells, such as NSC, and highly malignant cells, such as GBM CSC and glioblastomas, favouring the hypothesis that the ratio between the two MARK4 isoforms is strictly regulated along neural differentiation and may be subverted in gliomagenesis. These findings, together with the observation that in normal brain only the L isoform localizes in the embryonic ventricular zone (VZ) and adult sub-ventricular zone (SVZ), where stem cells reside, suggest that some GBM of our panel originated from the SVZ. In contrast, the concomitant expression of both MARK4 isoforms in the main substrates of glioma transformation, mature glial cells and progenitors, could explain the origin of the Oligodendrogliomas, Astrocytomas and of some GBM here studied. Furthermore, both MARK4 isoforms are expressed in neurons, extending published data of MARK4S as a neuron-specific marker in mouse CNS. By Immunofluorescence we found that the two MARK4 isoforms localize at centrosomes and midbody of normal and glioma cell lines, showing that MARK4 association with these cell compartments is neither isoform- nor tumor-specific. It has been reported that both normal and cancer stem cells can display centrosome amplification, genetic instability and loss of asymmetry with switch to a prevalent symmetric division, which may converge in malignant transformation and unrestrained growth of stem cells. Interestingly, we previously found MARK4 protein in association with glioma aberrant centrosomes, a hint for a possible role of the kinase in the abnormal mitotic processes of human glioma. Symmetric division, besides promoting the expansion of stem cell numbers, may also be permissive for secondary events leading to aneuploidies, since the machinery that controls asymmetric division also regulates the orientation of mitotic spindles and of centrosomes. Finally, an intriguing finding, delineating MARK4L as a tumor marker through its nucleolar association, comes from the evidence that L isoform, in contrast to MARK4S, is detectable in nucleoli and exclusively in cancer cells. Furthermore, the differential expression of MARK4 isoforms in undifferentiated and glial malignant cells expands the concept of MARK4 splice variants dysregulation in mediating tumor initiation and progression

A systematic review and economic evaluation of subcutaneous and sublingual allergen immunotherapy in adults and children with seasonal allergic rhinitis

© Queen’s Printer and Controller of HMSO 2013Severe allergic rhinitis uncontrolled by conventional medication can substantially affect quality of life. Immunotherapy involves administering increasing doses of a specific allergen, with the aim of reducing sensitivity and symptomatic reactions. Recent meta-analyses have concluded that both subcutaneous immunotherapy (SCIT) and sublingual immunotherapy (SLIT) are more effective than placebo in reducing symptoms. It is uncertain which route of administration is more effective and whether or not treatment is cost-effective.National Institute for Health Research Health Technology Assessment programm

Mitochondrial Content and Hepcidin are Increased in Obese Pregnant Mothers

OBJECTIVE: Maternal obesity is characterized by systemic low-grade inflammation and oxidative stress (OxS) with the contribution of fetal sex dimorphism. We recently described increased mitochondrial content (mtDNA) in placentas of obese pregnancies. Here, we quantify mtDNA and hepcidin as indexes of OxS and systemic inflammation in the obese maternal circulation. METHODS: Forty-one pregnant women were enrolled at elective cesarean section: 16 were normal weight (NW) and 25 were obese (OB). Obese women were further classified according to the presence/absence of maternal gestational diabetes mellitus (GDM); [OB/GDM(-)]: n\u2009=\u200915, [OB/GDM(+)]: n\u2009=\u200910. mtDNA and hepcidin were evaluated in blood (real-time PCR) and plasma (ELISA). RESULTS: mtDNA and hepcidin levels were significantly increased in OB/GDM(-) versus NW, significantly correlating with pregestational BMI. Male/female (M/F) ratio was equal in study groups, and overall F-carrying pregnancies showed significantly higher mtDNA and hepcidin levels than M-carrying pregnancies both in obese and normal weight mothers. CONCLUSIONS: Our results indicate a potential compensatory mechanism to increased obesity-related OxS and inflammation, indicated by the higher hepcidin levels found in obese mothers. Increased placental mitochondrial biogenesis, due to lipotoxic environment, may account for the greater mtDNA amount released in maternal circulation. This increase is namely related to F-carrying pregnancies, suggesting a gender-specific placental response

Effects of Alpha-Lipoic Acid and Myoinositol Supplementation on the Oocyte Enviroment of Obese Infertile Women

INTRODUCTION Obesity is characterized by increased inflammation and oxidative stress, resulting in adverse effects on women reproductive potential. Antioxidant supplementation may exert a positive effect on the obese ovarian environment. Indeed, we preliminarily observed a reduction of mitochondrial (mt) DNA content, a marker of oxidative stress, in granulosa cells of obese infertile women supplemented with Sinopol\uae (Laborest SpA), composed by alpha-lipoic acid (ALA) 800 mg, myoinositol (MYO) 2 g, folic acid (FA) 400 ug. This suggested a potential role of Sinopol\uae in reducing oxidative stress in the obese ovarian environment. Here we analyzed Total Antioxidant Capacity (TAC) in follicular fluid and mtDNA levels in granulosa cells, in a larger population of infertile women undergoing in vitro fertilization (IVF). METHODS 19 normal weight (NW) and 24 obese (OB) infertile women were enrolled in our IVF center. Infertility was investigated and a non-ovarian diagnosis was made. Patients did not present any additional pathology. All women were provided with FA and among them 15 OB (OB-SIN) were also supplemented with ALA and MYO, for 2 months before ovarian stimulation. Follicular fluid (FF) and granulosa cells (GC) were collected after oocyte retrieval. TAC was measured in FF by enzymatic assay, mtDNA levels evaluated in GC by Real-time PCR. Results were compared by ANOVA and correlations assessed by Pearson\u2019s correlation (SPSS; IBM). RESULTS OB groups had similar BMI (OB patients supplemented with only folic acid (OB-F): 30.2 \ub1 0.7; OB-SIN: 32.7 \ub1 1.1 kg/m2). Women age was similar in all groups (NW: 36.7 \ub1 0.6; OB-F: 37.6 \ub1 1.7; OB-SIN: 35.9 \ub1 1.1 years). Among OB women, antioxidant capacity was significantly higher in OB-SIN than in OB-F. mtDNA levels showed an opposite trend, being decreased in OB-SIN and increased in OB-F compared to NW, though not reaching statistical significance. mtDNA levels were significantly and inversely correlated with the number of total oocytes and metaphase II (mature) oocytes. Pregnancy rate was similar in NW (36.8%) and OB-SIN (33.3%) women, while it was lower in OB-F patients (11.1%). CONCLUSION We analyzed molecular markers in granulosa cells and follicular fluid as indicators of oocytes oxidative state. Our results suggest that supplementation with a compound of ALA -a natural antioxidant, cofactor in the mt respiratory chain- and MYO -an insulin-sensitizer- might increase antioxidant defenses and reduce oxidative stress in the obese ovarian environment, possibly contributing at restoring physiological conditions. This might improve IVF pregnancy rates in obese infertile women. Further studies are needed to clarify the synergic action of ALA, MYO and FA on the oocyte oxidative environment. Supported by Laborest Sp

Assessment of Cardiovascular Function in Childhood Leukemia Survivors: The Role of the Right Heart

Childhood acute lymphoblastic leukemia (ALL) survivors who underwent chemotherapy with anthracyclines have an increased cardiovascular risk. The aim of the study was to evaluate left and right cardiac chamber performances and vascular endothelial function in childhood ALL survivors. Fifty-four ALL survivors and 37 healthy controls were enrolled. All patients underwent auxological evaluation, blood pressure measurements, biochemical parameters of endothelial dysfunction, flow-mediated dilatation (FMD) of the brachial artery, mean common carotid intima-media thickness (c-IMT), antero-posterior diameter of the infra-renal abdominal aorta (APAO), and echocardiographic assessment. The ALL subjects had significantly lower FMD (p = 0.0041), higher left (p = 0.0057) and right (p = 0.0021) echocardiographic/Doppler Tei index (the non-invasive index for combined systolic and diastolic ventricular function) as compared to controls. Tricuspid annular plane excursion (TAPSE) was 16.9 +/- 1.2 mm vs. 24.5 +/- 3.7 mm, p < 0.0001. Cumulative anthracycline doses were related to TAPSE (p < 0.001). The ALL survivors treated with anthracyclines demonstrated systo/diastolic alterations of the right ventricle and reduced endothelial function compared with healthy controls. The early recognition of subclinical cardiac and vascular impairment during follow up is of utmost importance for the cardiologist to implement strategies preventing overt cardiovascular disease considering the growing number of young adults cured after childhood ALL

Multiple micronutrients and docosahexaenoic acid supplementation during pregnancy : A randomized controlled study

Maternal dietary intake during pregnancy needs to meet increased nutritional demands to maintain metabolism and to support fetal development. Docosahexaenoic acid (DHA) is essential for fetal neuro-/visual development and in immunomodulation, accumulating rapidly within the developing brain and central nervous system. Levels available to the fetus are governed by the maternal diet. In this multicenter, parallel, randomized controlled trial, we evaluated once-daily supplementation with multiple micronutrients and DHA (i.e., multiple micronutrient supplementation, MMS) on maternal biomarkers and infant anthropometric parameters during the second and third trimesters of pregnancy compared with no supplementation. Primary efficacy endpoint: change in maternal red blood cell (RBC) DHA (wt% total fatty acids) during the study. Secondary variables: other biomarkers of fatty acid and oxidative status, vitamin D, and infant anthropometric parameters at delivery. Supplementation significantly increased RBC DHA levels, the omega-3 index, and vitamin D levels. Subscapular skinfold thickness was significantly greater with MMS in infants. Safety outcomes were comparable between groups. This first randomized controlled trial of supplementation with multiple micronutrients and DHA in pregnant women indicated that MMS significantly improved maternal DHA and vitamin D status in an industrialized setting\u2014an important finding considering the essential roles of DHA and vitamin D

Impact of Obesity and Hyperglycemia on Placental Mitochondria

A lipotoxic placental environment is recognized in maternal obesity, with increased inflammation and oxidative stress. These changes might alter mitochondrial function, with excessive production of reactive oxygen species, in a vicious cycle leading to placental dysfunction and impaired pregnancy outcomes. Here, we hypothesize that maternal pregestational body mass index (BMI) and glycemic levels can alter placental mitochondria. We measured mitochondrial DNA (mtDNA, real-time PCR) and morphology (electron microscopy) in placentas of forty-seven singleton pregnancies at elective cesarean section. Thirty-seven women were normoglycemic: twenty-one normal-weight women, NW, and sixteen obese women, OB/GDM(-). Ten obese women had gestational diabetes mellitus, OB/GDM(+). OB/GDM(-) presented higher mtDNA levels versus NW, suggesting increased mitochondrial biogenesis in the normoglycemic obese group. These mitochondria showed similar morphology to NW. On the contrary, in OB/GDM(+), mtDNA was not significantly increased versus NW. Nevertheless, mitochondria showed morphological abnormalities, indicating impaired functionality. The metabolic response of the placenta to impairment in obese pregnancies can possibly vary depending on several parameters, resulting in opposite strains acting when insulin resistance of GDM occurs in the obese environment, characterized by inflammation and oxidative stress. Therefore, mitochondrial alterations represent a feature of obese pregnancies with changes in placental energetics that possibly can affect pregnancy outcomes

Placental ERRγ-CYP19 expressions and circulating 17-Beta Estradiol in IUGR pregnancies

Introduction: Sex steroids are regulating factors for intrauterine growth. 17- f Estradiol (E2) is particularly critical to a physiological pregnancy, as increased maternal E2 was correlated to lower birth weight. The placenta itself is a primary source of estrogens, synthetized from cholesterol precursors. Cytochrome P450 aromatase (encoded by CYP19 gene) is a rate-limiting enzyme for E2 biosynthesis. CYP19 transcription is supported by Estrogen Related-Receptor Gamma (ERR\u3b3), thus having an indirect role in placental steroidogenesis. Here we investigated maternal E2 levels and placental CYP19 and ERR expressions in pregnancies with intrauterine growth restriction (IUGR). Methods: Singleton pregnancies were studied. E2 was measured in maternal plasma by electrochemiluminescence in 16 term controls and 11 IUGR (classified by umbilical artery doppler Pulsatility Index) at elective cesarean section, and also in 13 controls during pregnancy at a gestational age comparable to IUGR. CYP19 and ERR\u3b3 expressions were analyzed in placental tissue. Maternal/fetal characteristics, placental and molecular data were compared among study groups and tested for correlations. Results: Maternal E2 plasma concentrations were significantly decreased in IUGR compared to controls at delivery. When analyzing normal pregnancies at the same IUGR gestational age, E2 levels were not different to IUGR. However, E2 levels at delivery positively correlated with placental efficiency. Placental CYP19 was significantly higher in IUGR placental tissue versus controls, with levels specifically increased in female IUGR placentas. ERR\u3b3 expression was not different among groups. Discussion: We report a positive correlation between 17- f Estradiol levels and placental efficiency, that might indicate a disrupted steroidogenesis in IUGR pregnancies. Moreover, we showed alterations of CYP19 in IUGR placentas, possibly indicating a compensatory effect to the adverse IUGR intrauterine environment, also depending on fetal sex. Further studies are needed to deeper investigate IUGR alterations in the complex interaction among molecules involved in placental steroidogenesis

Mitochondrial DNA content and methylation in fetal cord blood of pregnancies with placental insufficiency

Introduction: Intrauterine growth restriction (IUGR) and preeclampsia (PE) are pregnancy disorders characterized by placental insufficiency with oxygen/nutrient restriction and oxidative stress, all influencing mitochondria functionality and number. Moreover, IUGR and PE fetuses are predisposed to diseases later in life, and this might occur through epigenetic alterations. Here we analyze content and methylation of mitochondrial DNA (mtDNA), for the first time in IUGR and PE singleton fetuses, to identify possible alterations in mtDNA levels and/or epigenetic control of mitochondrial loci relevant to replication (D-loop) and functionality (mt-TF/RNR1: protein synthesis, mt-CO1: respiratory chain complex). Methods: We studied 35 term and 8 preterm control, 31 IUGR, 17 PE/IUGR and 17 PE human singleton pregnancies with elective cesarean delivery. Fetal cord blood was collected and evaluated for biochemical parameters. Extracted DNA was subjected to Real-time PCR to assess mtDNA content and analyzed for D-loop, mt-TF/RNR1 and mt-CO1 methylation by bisulfite conversion and pyrosequencing. Results: mtDNA levels were increased in all pathologic groups compared to controls. Mitochondrial loci showed very low methylation levels in all samples; D-loop methylation was further decreased in the most severe cases and associated to umbilical vein pO2. mt-CO1 methylation levels inversely correlated to mtDNA content. Discussion: Increased mtDNA levels in IUGR, PE/IUGR and PE cord blood may denote a fetal response to placental insufficiency. Hypomethylation of D-loop, mt-TF/RNR1 and mt-CO1 loci confirms their relevance in pregnancy