Rosemary, heather and heterofloral honeys protect towards cytotoxicity of acrylamide in human hepatoma cells

In April 2002 the Swedish Agency for Food Safety alerts of the presence of acrylamide (AA) in carbohydrate-rich foodstuffs subjected to elevated frying temperatures (150 - 180°C) such as fried potatoes, biscuits or breakfast cereals. The International Agency on Research on Cancer (IARC) classified acrylamide as a probable carcinogen, Group 2A. In consequence, one of the priority objectives of Food Safety is to reduce the presence of this substance in food. Numerous studies show the inhibitory effect of antioxidants present in fruit and vegetable extracts against the formation of acrylamide. Honey is also a good source of antioxidants since it contains a great variety of phenolic compounds. Therefore the main objective of this work was to evaluate the protective effect of three Madrid honeys of different floral origin against the AA-induced cytotoxicity in human hepatoma cells (HepG2). The results showed that the acrylamide in concentrations 1.4 and 2.8 mg/ml and in a 24-hour incubation period significantly reduced the percentage of cell viability (67 and 24 %, respectively). In simultaneous treatment of acrylamide (2.8 mg/ml) and the corresponding honeys we noted that rosemary honey in all concentrations tested increased the percentage of cell survival in 40-49 %, while heather honey and heterofloral honey increased cell viability by 54 and 66% respectively. The artificial honey did not mitigate the AA-induced cytotoxic effect. As a result, the protective effect of the evaluated honeys can be attributed to its polyphenolic content and not the sugar constituents.En abril del 2002 la Agencia Sueca de Seguridad Alimentaria alerta de la presencia de acrilamida (AA) en alimentos ricos en carbohidratos sometidos a temperaturas de fritura elevadas (150-180oC) como las patatas fritas, las galletas o los cereales de desayuno. La Agencia Internacional para la investigación del Cáncer (IARC) la clasifica como un agente carcinógeno probable, Grupo 2A. En consecuencia, uno de los objetivos prioritarios de la Seguridad Alimentaria ha consistido en reducir la presencia de esta sustancia en los alimentos. Numerosos estudios muestran el efecto inhibidor de los antioxidantes naturales presentes en extractos de frutas y vegetales en la formación de la acrilamida. La miel es también una buena fuente de antioxidantes ya que contiene una gran variedad de compuestos fenólicos. Por ello, el principal objetivo de este trabajo consistió en evaluar el efecto protector de tres mieles de Madrid de distinto origen floral frente a la citotoxicidad de la acrilamida, en células de hepatoma humano (HepG2). Los resultados obtenidos mostraron que la acrilamida a las concentraciones de 1,4 y de 2,8 mg/ml y un tiempo de incubación de 24 horas redujeron significativamente el porcentaje de viabilidad celular (67 y 24%, respectivamente). En los tratamientos simultáneos de acrilamida (2,8 mg/ml) y las correspondientes mieles observamos que la miel de romero a todas las concentraciones evaluadas incrementaba el porcentaje de viabilidad celular en un 40-49%. Mientras que, la miel de brezo y la miel multifloral lo hicieron en un 54 y 66% respectivamente. La miel artificial no atenúo el efecto citotóxico de la acrilamida. Por tanto, el efecto protector de las mieles evaluadas puede atribuirse a su contenido en polifenoles y no a su contenido en azúcares

Gene Expression Profiling of Muscle Stem Cells Identifies Novel Regulators of Postnatal Myogenesis

International audienceSkeletal muscle growth and regeneration require a population of muscle stem cells, the satellite cells, located in close contact to the myofiber. These cells are specified during fetal and early postnatal development in mice from a Pax3/7 population of embryonic progenitor cells. As little is known about the genetic control of their formation and maintenance, we performed a genome-wide chronological expression profile identifying the dynamic transcriptomic changes involved in establishment of muscle stem cells through life, and acquisition of muscle stem cell properties. We have identified multiple genes and pathways associated with satellite cell formation, including set of genes specifically induced (EphA1, EphA2, EfnA1, EphB1, Zbtb4, Zbtb20) or inhibited (EphA3, EphA4, EphA7, EfnA2, EfnA3, EfnA4, EfnA5, EphB2, EphB3, EphB4, EfnBs, Zfp354c, Zcchc5, Hmga2) in adult stem cells. Ephrin receptors and ephrins ligands have been implicated in cell migration and guidance in many tissues including skeletal muscle. Here we show that Ephrin receptors and ephrins ligands are also involved in regulating the adult myogenic program. Strikingly, impairment of EPHB1 function in satellite cells leads to increased differentiation at the expense of self-renewal in isolated myofiber cultures. In addition, we identified new transcription factors, including several zinc finger proteins. ZFP354C and ZCCHC5 decreased self-renewal capacity when overexpressed, whereas ZBTB4 increased it, and ZBTB20 induced myogenic progression. The architectural and transcriptional regulator HMGA2 was involved in satellite cell activation. Together, our study shows that transcriptome profiling coupled with myofiber culture analysis, provides an efficient system to identify and validate candidate genes implicated in establishment/maintenance of muscle stem cells. Furthermore, tour de force transcriptomic profiling provides a wealth of data to inform for future stem cell-based muscle therapies

Prospective Analysis of Factors Influencing the Antibody Response to Hepatitis B Vaccine in Hemodialysis Patients

Abstract Background. Patients on maintenance hemodialysis typically show a suboptimal immune response to hepatitis B (HB) virus vaccination compared to the non-uremic population. The aim or our study was the identification of factors implicated in the vaccine response of our hemodialysis patients. Methods. We studied prospectively 63 hemodialysis patients who were seronegative for HB (37 males, 26 females). Their mean age was 62.2±11.28 years (range 35-80) and hemodialysis. Duration 55.96±50.1 months (range 6-225) fourteen of them (22.2%) were diabetics. Our patients followed a four-dose vaccination schedule (0, 1, 2 and 6 months) with 40 μg of a recombinant DNA HB vaccine. The antibody response was determined 1 month after the fourth dose of vaccination by assessing the titre of antiHBs antibodies (ab). Immune response was defined as sufficient when the antiHBs ab level was ≥ 12 mIU/ml. During the 6-month vaccination period we also monitored monthly and time-averaged Kt/V, residual renal function (RRF), BMI, serum creatinine, albumin, transferrin, ferritin, CRP, iPTH and the dose of erythropoietin and Vitamin D that they received. Results. An optimal immune response was achieved in 34 patients (54%, responders) (antiHBs: 295.95±341.67 mIU/ml), whereas 29 patients (46%, non-responders) showed a suboptimal response (antiHBs: 1.98±2.92 mIU/ml) (p=1.75x10 -5 ). There was a statistically significant negative correlation between the antiHBs ab titre and BMI (r=-0.28, p=0.024). A significant difference was also found between the BMI of responders and non-responders as groups (24.8±5.5 vs. 27.2±4.5, p=0.02). Grouping our patients according to the existence of diabetes, age (cut off 60 years), and hemodialysis efficiency (Kt/V≥1.2) we found a statistically significant difference in the antiHBs ab titre between diabetics and non-diabetics (8.43±12.3 vs. 200.2±317.7 mIU/ml, p=0.03), younger and older patients (262±365.09 vs. 84.36±189.1 mIU/ml, p=0.0145) and patients with efficient and inefficient hemodialysis (234.71±337.1 vs. 79.14±200.99 mIU/ml, p=0.032). Treatment with vitamin D analogues, RRF and hypoalbuminemia were not found to be implicated in the immune response of our patients

Eine molekulare und zelluläre Analyse

Tightly controlled growth arrest coordinates the equilibrium between cell proliferation and cell differentiation during embryonic tissue formation as well as in adulthood during stem cell-mediated tissue regeneration. Myogenic differentiation requires a coordinated course of tissue-specific gene expression and irreversible cell cycle exit. However, I contributed to showing -using genetic manipulation in the mouse embryo- that these processes can be uncoupled. During development, growth arrest in myogenic cells is mediated by the cyclin-dependent kinase inhibitors (CDKIs) p21 and p57, which act redundantly to promote cell cycle exit. We demonstrated that skeletal muscle progenitors require a direct interaction with the differentiating myoblasts via the Notch signaling pathway to maintain their pool. We also identified a muscle-specific regulatory element of p57 that directly receives the input of Myogenic Regulatory Factors (MRFs) and Notch downstream targets. During my Ph.D. I examined whether this regulatory mechanism is also involved in postnatal myogenesis. Adult skeletal muscle has a remarkable regenerative capacity, involving a stem cell population, called satellite cells (SCs), located on close contact to the myofibers under the basal lamina. At the transition from juvenile to adult state (around 3-4 week during postnatal growth in mice) they enter a non-cycling, quiescent state. Upon injury the SCs rapidly get activated, expand by proliferation and provide differentiated progeny for muscle repair, while a subpopulation self-renews and re-enters quiescence, allowing the support of additional rounds of muscle damage. To understand the mechanisms regulating acquisition of quiescence, we explored the role of the aforementioned CDKIs in adult muscle. Although absent from quiescent SCs, they become rapidly expressed upon activation (even in proliferating myoblasts) and their levels remain high in the differentiating muscle cells. Strikingly, during the course of differentiation p57 translocated from the cytoplasm of activated myoblasts to the nuclei of differentiating cells. Since p57 deficient mice die at birth, we generated a conditional knock-out allele to perform functional studies at the postnatal stages. This new allele, in which the coding region can be removed by the loxP/Cre recombination system, also contains a β- galactosidase reporter allowing the identification of p57-expressing cells. We generated a complete loss of function allele using a ubiquitously expressed Cre, and observed developmental and perinatal phenotypes similar to previously described germline knock-outs. Furthermore, we showed that the reporter inserted in the p57 conditional allele faithfully recapitulates p57 expression profile at embryonic and adult tissues. Conditional ablation of p57 in adult SCs resulted in reduced myogenic differentiation in primary myoblast culture. Similarly, p21-null myoblasts exhibited proliferation and differentiation defects in single myofiber cultures. In vivo regeneration studies with p21 mutants showed an early impact on the SC pool, while both SCs and muscle structure were re- established by the end of the regeneration process. My Ph.D. work suggests that p21 and p57 are at play during adult myogenesis and cell cycle exit, although via different mechanisms compared to the developmental scenario. They both work at the early steps of satellite cell activation but do not compensate for each other’s loss. Future studies will elucidate whether they lie genetically downstream of the MRFs and Notch targets during postnatal myogenesis.Das Gleichgewicht zwischen Zellproliferation und Zelldifferenzierung wird während der embryonalen Gewebebildung sowie während der stammzellvermittelten Geweberegeneration im Adultstadium durch einen streng kontrollierten Wachstumsarrest koordiniert. Die myogene Differenzierung erfordert sowohl eine koordinierte Abfolge von gewebespezifischer Genexpression als auch einen irreversiblen Zellzyklusaustritt. Jedoch konnte ich durch genetische Manipulation von Mausembryos dazu beitragen, aufzuzeigen, dass diese beiden Prozesse voneinander entkoppelt werden können. Ein Wachstumsarrest myogener Zellen wird während der Entwicklung durch die Cyclin-abhängigen Kinaseinhibitoren (CDKIs) p21 und p57 vermittelt. Letztere wirken dabei redundant, um einen Zellzyklusaustritt zu fördern. Wir konnten bereits nachweisen, dass skelettale Muskelvorläuferzellen eine direkte Interaktion mit differenzierenden Myoblasten über den Notch-Signalweg benötigen, um ihren Pool aufrecht zu erhalten. Des Weiteren haben wir ein muskelspezifisches regulatorisches Element von p57 identifiziert, das direkten Input von myogenen Regulationsfaktoren (MRFs) und Notch Stromabwärts-Zielen erhält. Im Rahmen meiner Doktorarbeit habe ich untersucht, ob dieser regulatorische Mechanismus auch in der postnatalen Myogenese involviert ist. Der adulte skelettale Muskel hat eine bemerkenswerte regenerative Kapazität, die eine Stammzellpopulation, sogennante Satellitenzellen (SCs, engl. für "satellite cells") involviert. Diese befinden sich zwischen der Basallamina und der Muskelfaser. Beim Übergang zwischen juvenilen und adultem Stadium (zwischen 3-4 Wochen während des postnatalen Wachstums in Mäusen), gehen die Satellitenzellen in einen nicht-zyklischen Ruhezustand über. Satellitenzellen werden nach einer Verletzung schnell aktiviert, expandieren und stellen differenzierte Abkömmlinge für die Muskelreparatur zur Verfügung. Eine Subpopulation der Satellitenzellen erneuern sich selbst und gehen zurück in den Ruhezustand, so dass bei erneuter Muskelverletzung Unterstützung gewährleistet ist. Um die Mechanismen verstehen zu können, die den Übergang in den Ruhezustand regulieren, haben wir die Rolle der zuvor genannten CDKIs im adulten Muskel untersucht. Obwohl CDKIs in ruhenden Satellitenzellen abwesend sind, werden sie nach Aktivierung der Satellitenzellen schnell exprimiert, und ihr Expressionslevel bleibt in differenzierenden Muskelzellen aufrecht erhalten. Erstaunlicherweise transloziert p57 während des Differenzierungsprozesses vom Zytosplasma in den Zellkern. Da p57-defiziente Mäuse bei Geburt sterben, haben wir eine konditionelle Mausmutante generiert, um funktionale Studien im postnatalen Stadium durchführen zu können. Dieses neue Mausmodell hat ein modifiziertes p57-Allel, in dem die codierende Region von p57 durch loxP/Cre-Rekombination entfernt werden kann. Des Weiteren beinhaltet es auch das Reportgen β-Galactosidase, um p57-exprimierende Zellen identifizieren zu können. Wir haben durch eine ubiquitär exprimierte Cre- Rekombinase vollständige Knockout-Mäuse generiert und dabei entwicklungsorientierte und perinatale Phänotypen, ähnlich wie bei bereits beschriebenen Knockout- Mäusen, beobachtet. Des Weiteren konnten wir zeigen, dass der in das konditionelle p57-Allel eingefügte Reporter dem Expressionsprofil von p57 im embryonalen und adulten Gewebe entspricht. Konditionelle Ablation von p57 in Satellitenzellen resultierte in einer reduzierten myogenen Differenzierung von primären Myoblastenkulturen. p21 -defiziente-Myoblasten wiesen ähnliche Proliferation- und Differenzierungsdefekte in einzelnen Muskelfaser-Kulturen auf. In-vivo- Regenerationsstudien mit p21-Mutanten haben eine initiale Reduktion der Satellitenzellenanzahl gezeigt. Zum Ende des Regenerationsprozesses waren die Anzahl der Satellitenzellen sowie die Muskelstruktur jedoch re-etabliert. Meine Doktorarbeit lässt darauf schließen, dass p21 und p57 während der adulten Myogenese und des Zellzyklusaustritts eine Rolle spielen, jedoch unterscheidet sich der Mechanismus im Vergleich zum pränatalem Stadium. Beide fungieren bei frühzeitigen Schritten der Satellitenzellenaktivierung, aber kompensieren sich nicht für den gegenseitigen Verlust. Künftige Studien werden zeigen, ob p21 und p57 von MRFs und Notch während der postnatalen Myogenese reguliert werden

Analyse moléculaire et cellulaire de l'interaction entre sortie du cycle et myogenèse dans les cellules souches du muscle du squelette

Ce travail de thèse a porté sur l'étude de l'équilibre entre la prolifération et la différenciation dans le cadre de la myogenèse embryonaire et postnatale. Chez l'embryon, le sortie du cycle cellulaire est contrôlé par p57 et p21 pendant la myogenèse. Nous avons montré que la voie de signalisation Notch ainsi que les facteurs de régulation myogéniques (MRFs) régulent l'expression de p57 dans les cellules progénitrices et les myoblastes en différentiation. Chez l'adulte, p21 et p57 ne sont pas exriés dans la population quiescente de cellules souches du muscle (cellules satellites - SCs). p21 et p57 sont rapidement induits après activation et durant la différentiation des SCs. Ex vivo, les myoblastes déficients pour le gène p21 présentent des défauts de prolifération et de différenciation. In vivo, l'étude de la régénération musculaire chez les mutants p21 a montré une réduction précoce des SCs, avec un retard de reconstitution du tissu musculaire. Afin de pouvoir étudier le rôle de p57 après la naissance (les mutants p57 meurent à la naissance) nous avons généré un modèle murin permettant de muter le gène p57 de manière spatio-temporelle avec le système de recombinaison Cre/LoxP. Nous avons combiner notre allèle p57 conditionnel avec une Cre exprimée de manière ubiquitaire, et observé des phénotypes identiques aux phénotypes décrits précédemment chez les souris présentant une perte du p57. L'ablation conditionnelle de p57 dans les SCs adultes, a conduit à une diminution de la différenciation myogénique in vitro. Notre travail suggère que p21 et p57 jouent un rôle important dans la régulation de la différenciation et le cycle cellulaire dans le muscle adulte.This thesis focuses on the coordination of proliferation and differentiation in embryonic and adult myogenesis. During development, we demonstrated that skeletal muscle progenitors interact with the differentiating myoblasts via the Notch pathway to maintain their pool. It has previously been established that p57 and p21 redundantly promote cell cycle exit in developing muscle and we showed that Notch and Myogenic Regulatory Factors act through muscle-specific regulation of p57. We then examined p21 and p57 in adult skeletal muscle stem cells, called satellite cells (SCs). Although absent from quiescent SCs, p21 and p57 are expressed upon activation (including proliferating myoblasts) and differentiation. p21-null myoblasts exhibited proliferation and differentiation defects in myofiber cultures, implicating p21 at the early activation phase. In vivo muscle regeneration studies with p21 mutants showed an early impact on the SC pool, while SCs and muscle structure were re-established by the end of regeneration. Since p57-deficient mice die at birth, we generated a conditional knock-out (KO) allele for postnatal studies using the loxP/Cre recombination system. With a ubiquitous Cre we observed developmental and perinatal phenotypes similar to previously described KO embryos. The new p57 allele also includes a β-galactosidase reporter and we showed that it recapitulates p57 expression profile in embryonic and adult tissues. Conditional ablation of p57 in adult SCs reduced myogenic differentiation in primary myoblast culture. Our work suggests that p21 and p57 are involved in adult myogenesis and cell cycle exit, working at the early steps of satellite cell activation

Analyse moléculaire et cellulaire de l'interaction entre sortie du cycle et myogenèse dans les cellules souches du muscle du squelette

This thesis focuses on the coordination of proliferation and differentiation in embryonic and adult myogenesis. During development, we demonstrated that skeletal muscle progenitors interact with the differentiating myoblasts via the Notch pathway to maintain their pool. It has previously been established that p57 and p21 redundantly promote cell cycle exit in developing muscle and we showed that Notch and Myogenic Regulatory Factors act through muscle-specific regulation of p57. We then examined p21 and p57 in adult skeletal muscle stem cells, called satellite cells (SCs). Although absent from quiescent SCs, p21 and p57 are expressed upon activation (including proliferating myoblasts) and differentiation. p21-null myoblasts exhibited proliferation and differentiation defects in myofiber cultures, implicating p21 at the early activation phase. In vivo muscle regeneration studies with p21 mutants showed an early impact on the SC pool, while SCs and muscle structure were re-established by the end of regeneration. Since p57-deficient mice die at birth, we generated a conditional knock-out (KO) allele for postnatal studies using the loxP/Cre recombination system. With a ubiquitous Cre we observed developmental and perinatal phenotypes similar to previously described KO embryos. The new p57 allele also includes a β-galactosidase reporter and we showed that it recapitulates p57 expression profile in embryonic and adult tissues. Conditional ablation of p57 in adult SCs reduced myogenic differentiation in primary myoblast culture. Our work suggests that p21 and p57 are involved in adult myogenesis and cell cycle exit, working at the early steps of satellite cell activation.Ce travail de thèse a porté sur l'étude de l'équilibre entre la prolifération et la différenciation dans le cadre de la myogenèse embryonaire et postnatale. Chez l'embryon, le sortie du cycle cellulaire est contrôlé par p57 et p21 pendant la myogenèse. Nous avons montré que la voie de signalisation Notch ainsi que les facteurs de régulation myogéniques (MRFs) régulent l'expression de p57 dans les cellules progénitrices et les myoblastes en différentiation. Chez l'adulte, p21 et p57 ne sont pas exriés dans la population quiescente de cellules souches du muscle (cellules satellites - SCs). p21 et p57 sont rapidement induits après activation et durant la différentiation des SCs. Ex vivo, les myoblastes déficients pour le gène p21 présentent des défauts de prolifération et de différenciation. In vivo, l'étude de la régénération musculaire chez les mutants p21 a montré une réduction précoce des SCs, avec un retard de reconstitution du tissu musculaire. Afin de pouvoir étudier le rôle de p57 après la naissance (les mutants p57 meurent à la naissance) nous avons généré un modèle murin permettant de muter le gène p57 de manière spatio-temporelle avec le système de recombinaison Cre/LoxP. Nous avons combiner notre allèle p57 conditionnel avec une Cre exprimée de manière ubiquitaire, et observé des phénotypes identiques aux phénotypes décrits précédemment chez les souris présentant une perte du p57. L'ablation conditionnelle de p57 dans les SCs adultes, a conduit à une diminution de la différenciation myogénique in vitro. Notre travail suggère que p21 et p57 jouent un rôle important dans la régulation de la différenciation et le cycle cellulaire dans le muscle adulte

From cyclins to CDKIs: Cell cycle regulation of skeletal muscle stem cell quiescence and activation

International audienc

Bu-M-P-ing Iron: How BMP Signaling Regulates Muscle Growth and Regeneration

International audienceThe bone morphogenetic protein (BMP) pathway is best known for its role in promoting bone formation, however it has been shown to play important roles in both development and regeneration of many different tissues. Recent work has shown that the BMP proteins have a number of functions in skeletal muscle, from embryonic to postnatal development. Furthermore, complementary studies have recently demonstrated that specific components of the pathway are required for efficient muscle regeneration

Pax7 haploinsufficiency impairs muscle stem cell function in Cre-recombinase mice and underscores the importance of appropriate controls

International audienceAbstract Ever since its introduction as a genetic tool, the Cre-lox system has been widely used for molecular genetic studies in vivo in the context of health and disease, as it allows time- and cell-specific gene modifications. However, insertion of the Cre-recombinase cassette in the gene of interest can alter transcription, protein expression, or function, either directly, by modifying the landscape of the locus, or indirectly, due to the lack of genetic compensation or by indirect impairment of the non-targeted allele. This is sometimes the case when Cre-lox is used for muscle stem cell studies. Muscle stem cells are required for skeletal muscle growth, regeneration and to delay muscle disease progression, hence providing an attractive model for stem cell research. Since the transcription factor Pax7 is specifically expressed in all muscle stem cells, tamoxifen-inducible Cre cassettes (CreERT2) have been inserted into this locus by different groups to allow targeted gene recombination. Here we compare the two Pax7-CreERT2 mouse lines that are mainly used to evaluate muscle regeneration and development of pathological features upon deletion of specific factors or pathways. We applied diverse commonly used tamoxifen schemes of CreERT2 activation, and we analyzed muscle repair after cardiotoxin-induced injury. We show that consistently the Pax7-CreERT2 allele targeted into the Pax7 coding sequence (knock-in/knock-out allele) produces an inherent defect in regeneration, manifested as delayed post-injury repair and reduction in muscle stem cell numbers. In genetic ablation studies lacking proper controls, this inherent defect could be misinterpreted as being provoked by the deletion of the factor of interest. Instead, using an alternative Pax7-CreERT2 allele that maintains bi-allelic Pax7 expression or including appropriate controls can prevent misinterpretation of experimental data. The findings presented here can guide researchers establish appropriate experimental design for muscle stem cell genetic studies

SOXF factors regulate murine satellite cell self-renewal and function through inhibition of β-catenin activity

International audienceMuscle satellite cells are the primary source of stem cells for postnatal skeletal muscle growth and regeneration. Understanding genetic control of satellite cell formation, maintenance, and acquisition of their stem cell properties is on-going, and we have identified SOXF (SOX7, SOX17, SOX18) transcriptional factors as being induced during satellite cell specification. We demonstrate that SOXF factors regulate satellite cell quiescence, self-renewal and differentiation. Moreover, ablation of Sox17 in the muscle lineage impairs postnatal muscle growth and regeneration. We further determine that activities of SOX7, SOX17 and SOX18 overlap during muscle regeneration, with SOXF transcriptional activity requisite. Finally, we show that SOXF factors also control satellite cell expansion and renewal by directly inhibiting the output of β-catenin activity, including inhibition of Ccnd1 and Axin2. Together, our findings identify a key regulatory function of SoxF genes in muscle stem cells via direct transcriptional control and interaction with canonical Wnt/β-catenin signalin