Dissertations / Theses on the topic 'GDF-15'

La circulation extracorporelle compte parmi les progrès techniques majeurs associés à la chirurgie cardiaque. Elle constitue aussi l’une des causes de complications principales car responsable d’une réponse inflammatoire généralisée qui résulte de la conjugaison des effets du stress oxydant et des cytokines libérés, contribuerait à la dysfonction multi-organe aboutissant aux complications myocardiques et rénales survenant au cours des périodes per- et postopératoires. La première partie de notre travail avait pour objectif d’explorer l’évolution des taux circulants du GDF-15, cytokine associée au stress oxydant et à l’inflammation, dans ce contexte de chirurgie cardiaque. Notre étude prospective a démontré pour la première fois que cette procédure est accompagnée de l’augmentation du GDF-15 dont les taux plasmatiques sont associés aux lésions postopératoires cardiaques et rénales.L’évaluation du risque opératoire repose sur un ensemble de scores dont le calcul est basé essentiellement sur des caractéristiques cliniques. Ces scores présentent toutefois un certaines limitations. Chez les patients « médicaux » atteints de pathologies cardiovasculaires la stratification du risque est définie en associant des caractéristiques cliniques à l’évaluation des taux circulants de biomarqueurs. L’objectif de cette seconde partie a donc été de mettre en évidence le pouvoir prédictif du GDF-15 en tant que biomarqueur circulant dans la survenue de complications rénales au cours de la chirurgie cardiaque sous CEC. Nous avons mis en évidence que les patients présentant des taux préopératoires élevés de GDF-15 sont à risque de développer une insuffisance rénale aigue postopératoire
Ischemic cardiac diseases are the most frequent and deleterious pathologies leading to important cardiovascular-related mortality worldwide. One of the alternative therapies consists to treat these patients using cardiac surgery. Cardiopulmonary bypass was developed to greatly improve this surgical procedure. However, some adverse effects can occur during cardiac surgery associated with cardiopulmonary bypass due to the inflammatory response. This phenomenon is the result of various mechanisms including oxidative stress and inflammatory cytokines which lead to multi-organ failure and then to myocardial and renal injuries occurring during the peri- and post-operative periods.The first part of this work was designed to evaluate in the context of cardiac surgery the kinetics of plasma GDF-15 levels, an oxidative stress and inflammation related cytokine. Our prospective study demonstrated for the first time the kinetic increase in plasma GDF-15 levels which were associated to postoperative cardiac and renal injuries.Currently, operative risk evaluation is based on score calculation including clinical criteria. These risk scores present some limitations. Concerning other cardiac patients out of surgical fields, the risk assessment is defined using clinical parameters and biomarkers evaluation (cardiac troponin, BNP, Nt-proBNP). Thus, we aimed to determine whether pre-operative GDF-15 as plasma biomarker could help to identify patients at high risk of renal injuries. We found that patients with the highest pre-operative plasma GDF-15 levels are at risk for post-operative acute kidney injury

Une des difficultés majeures dans le traitement des cancers est l’acquisition de résistance par les cellules tumorales vis-à-vis de la mort induite par les différentes chimiothérapies. Au sein du laboratoire, nous nous intéressons aux propriétés anti-tumorales d’un donneur de monoxyde d’azote (NO), le Glycéryl TriNitrate (GTN), fréquemment utilisé dans le traitement de l’angine de poitrine. Au cours de ce travail, nous avons étudié les mécanismes moléculaires par lesquels le GTN sensibilise les cellules tumorales de plusieurs types de cancer (colique, mammaire, prostatique) à la mort impliquant des voies de signalisation régulées par des cytokines telles que le TNFα, l’IL-6 ou encore le GDF-15.Une meilleure compréhension des mécanismes sous-jacents à l’action anti-tumorale du GTN permettrait de potentialiser son utilisation comme nouvelle thérapie anti-cancéreuse.Modèle colique : le GTN, en présence de la cytokine pro-inflammatoire TNFα, sensibilise les cellules cancéreuses coliques et mammaires à l’apoptose. Du point de vue mécanistique, le GTN induit la S-nitrosylation de cIAP1, inhibant ainsi son activité ubiquitine E3 ligase. Ce qui abroge la voie de signalisation classique NF-кB de survie cellulaire activée par la voie TNFα/TNFR1 en faveur d’une voie de signalisation pro-apoptotique.Modèle mammaire : le GTN intervient au niveau de la migration cellulaire en altérant la voie de signalisation Jak2/STAT3 activée par la cytokine pro-inflammatoire IL-6, dans un modèle de cancer du sein triple négatif. En présence de dérivés du platine (carboplatine) générant de l’IL-6, le GTN freine la migration des cellules en induisant la S-nitrosylation, et probablement l’inactivation, de la kinase Jak-2, indispensable pour l’activation de la voie.Modèle prostatique : le GTN sensibilise à la mort les cellules cancéreuses prostatiques résistantes au docétaxel en modulant le taux de deux marqueurs de résistance à cette chimiothérapie : la clusterine (CLU) et le growth differentiation factor 15 (GDF-15). Au niveau moléculaire, le GTN diminue le taux de l'isoforme cytoprotectrice soluble de la CLU (sCLU) et augmente le taux de l'isoforme cytotoxique nucléaire (nCLU) dans les cellules prostatiques résistantes au docétaxel. Plus particulièrement, en présence de GTN, nous avons établi un lien entre le GDF-15 et la modulation du taux des isoformes de la CLU
One of the main difficulties in the treatment of cancers is the acquisition of resistance by the tumor cells vis-à-vis the death induced by the different chemotherapies. In the laboratory, we are interested in the anti-tumor properties of a nitric oxide (NO) donor, Glyceryl TriNitrate (GTN), frequently used in the treatment of angina pectoris. In this work, we investigated the molecular mechanisms by which GTN sensitizes tumor cells of several types of cancer (colonic, mammary, prostate) to death involving signaling pathways regulated by cytokines such as TNFα, IL-6 or GDF-15.A better understanding of the mechanisms underlying the GTN's anti-tumor action would make it possible to use it as a new anti-cancer therapy.Colon model: GTN, in the presence of the pro-inflammatory cytokine TNFα, sensitizes colon and mammary cancer cells to apoptosis. From a mechanistic point of view, GTN induces S-nitrosylation of cIAP1, thus inhibiting its ubiquitin E3 ligase activity. This abrogates the classical NF-κB signaling pathway of TNFα / TNFR1 activated cell survival in favor of a pro-apoptotic signaling pathway.Mammary model: GTN intervenes at the level of cell migration by altering the Jak2 / STAT3 signaling pathway activated by the pro-inflammatory cytokine IL-6, in a model of triple negative breast cancer. In the presence of platinum (carboplatin) derivatives generating IL-6, GTN inhibits cell migration by inducing S-nitrosylation, and probably inactivation, of Jak-2 kinase, essential for the activation of the way.Prostate model: GTN sensitizes prostatic prostate cancer cells to death by modulating the level of two markers of resistance to this chemotherapy: clusterin (CLU) and growth differentiation factor 15 (GDF-15). At the molecular level, GTN decreases the level of the soluble cytoprotective isoform of CLU (sCLU) and increases the level of nuclear cytotoxic isoform (nCLU) in prostatic cells resistant to docetaxel. More particularly, in the presence of GTN, we have established a link between GDF-15 and the modulation of the isoform rate of the CLU

Memoria para optar al Título Profesional de Médico Veterinario
El factor de crecimiento diferencial 9 (GDF-9) y la proteína morfogénica ósea 15 (BMP-15) regulan en diferentes especies procesos esenciales para la maduración del ovocito, tales como la expansión del cúmulo. Este estudio evaluó en caninos la expresión génica de GDF-9 y BMP-15 en las células del cúmulo (CCs) obtenidas de complejos cúmulos ovocitos (COCs) a través del ciclo estral en relación a la maduración in vitro (IVM) de los ovocitos y la expansión de estas células. Las CCs se obtuvieron de COCs de folículos antrales de anestro, proestro, estro y diestro, previo y después de la IVM, expandidas y no expandidas. Para lograr mayor cantidad de CCs, antes y después de la IVM, estas se cultivaron in vitro por 48 h en medio Dulbecco’s Modified Eagle’s Medium (DMEM) con suero fetal bovino. El desarrollo meiótico luego de la IVM se evaluó mediante microscopía de fluorescencia, clasificándolo en vesícula germinal (VG), reinicio meiótico (GVBD), primera metafase (MI) y segunda metafase (MII). La expresión relativa de GDF-9 y BMP-15 en las CCs se evaluó mediante q-PCR. Estos genes se encontraron en todas las etapas del ciclo antes y después de la IVM, con diferencias significativas entre estas en relación a la expansión y maduración de los ovocitos. De acuerdo a los estados del ciclo estral, en las CCs de COCs no madurados, los niveles de GDF-9 fueron mayores (P < 0,05) durante el estro, seguido por el diestro y los menores (P < 0,05) niveles en proestro y anestro. En BMP-15, los mayores (P < 0,05) niveles de mRNA se observaron en estro y diestro. En las CCs expandidas no hubo diferencias en GDF-9 entre las distintas etapas del ciclo, pero en BMP-15 los menores (P < 0,05) niveles de mRNA se observaron en estro. Luego de la maduración, hubo un incremento (P < 0,05) de los transcriptos de ambos genes en las CCs de anestro, proestro y diestro, excepto los niveles de mRNA de BMP-15 durante el diestro. En estro en cambio, hubo una disminución significativa de la expresión de los dos genes luego de la IVM. No se encontraron diferencias significativas en la capacidad de alcanzar el estado de MII al considerar la etapa del ciclo. Sin embargo, los COCs obtenidos de proestro y diestro que expandieron sus CCs pudieron reiniciar la meiosis en mayor (P < 0,05) porcentaje en comparación con las demás etapas del ciclo y los COCs de estro que expandieron sus CCs lograron mayores (P < 0,05) porcentajes de MII en relación a los que no expandieron el cúmulo. En conclusión, GDF-9 y BMP-15 se expresan en las CCs en todas las etapas del ciclo con un patrón diferente de acuerdo a cada una y estarían involucrados en la expansión del cúmulo de la perra, pudiendo estar relacionado con la maduración meiótica in vitro, al menos en aquellos ovocitos en etapa estral.
Differential growth factor 9 (GDF-9) and bone morphogenic protein 15 (BMP-15) regulate essential processes for oocyte maturation in different species, such as cumulus expansion. This study evaluated gene expression of GDF-9 and BMP-15 in canine cumulus cells (CCs) obtained from cumulus-oocyte complexes (COCs) through the estrous cycle in relation to oocyte in vitro maturation (IVM) and the expansion of CCs. The CCs were obtained from COCs of antral follicles from anestrus, proestrus, estrus, diestrus phases, before and after IVM, expanded and non-expanded. To achieve more CCs, before and after IVM, these were cultured in vitro for 48 h in Dulbecco's Medium Modified Eagle's Medium (DMEM) with fetal calf serum. The meiotic development after IVM was evaluated by fluorescence microscopy, classifying it in germinal vesicle (GV), germinal vesicle breakdown (GVBD), first metaphase (MI) and second metaphase (MII). The relative expression of GDF-9 and BMP-15 in the CCs was evaluated by q-PCR. These genes were found in all stages of the reproductive cycle, before and after IVM, with significant differences between them in relation to the expansion and oocytes maturation. According to the estrous cycle stages, CCs from non-matured COCs expressed higher (P < 0.05) levels of GDF-9 mRNA during estrus in comparison with the other phases and the lowest (P < 0.05) levels at proestrus and anestrus. The highest (P < 0.05) levels of BMP-15 transcripts were observed in estrus and diestrus. There were no differences in GDF-9 gene expression comparing expanded CCs among the different phases of the estrous cycle, regarding BMP- 15 transcripts, the lowest (P < 0.05) levels were registered in estrus. After IVM, the level of both genes increased (P < 0.05) in CCs of anestrus, proestrus and diestrus, except BMP-15 during diestrus. In contrast, in estrus GDF-9 transcripts significantly decreased after IVM. Non expanded CCs exhibited the lowest levels of both genes at anestrus and proestrus, whereas at diestrus the levels of GDF-9 in non-matured CCs were lower (P < 0.05) than that of the expanded cells, the levels of BMP-15 were similar. No differences were found in the oocyte ability to reach MII state when comparing the phases of the estrous cycle. However, COCs obtained from proestrus and diestrus that expanded their CCs showed higher (P < 0.05) percentage of meiosis resumption than to the other phases. At estrus expanded CCs achieved higher (P < 0.05) percentages of MII in comparison to those did not expand. In conclusion, GDF-9 and BMP-15 are expressed in the CCs over the estrous cycle with different patterns according each phase and these genes maybe involved in the cumulus expansion in canines, maybe related to the in vitro maturation, at least in oocytes at estrus phase
Financiamiento: Proyecto Fondecyt 1171670

Memoria para optar al Título Profesional de Médico Veterinario
El ciclo estral de la hembra canina presenta algunas características únicas al ser comparadas con otras especies de mamíferos domésticos. Sin embargo, poco se conoce de las características del desarrollo folicular en relación a otras especies. El Factor de Crecimiento Diferencial 9 (GDF-9) y la Proteína Morfogenética Ósea 15 (BMP-15), son miembros de la superfamilia de Factores de Crecimiento Transformantes Beta (TGF-β), y han demostrado un rol fundamental en el desarrollo folicular en muchas especies. El objetivo de este estudio fue analizar la presencia de ambas proteínas en células foliculares proveniente de folículos antrales del ovario canino mediante citometría de flujo. Se obtuvieron células de la granulosa y del cúmulo a través de aspiración y raspado de folículos antrales de distintos niveles de desarrollo y en distintas etapas del ciclo estral. Las células se fijaron e incubaron con anticuerpo anti GDF-9 humano y anti BMP-15 de ratón (1:100) y anticuerpos secundarios conjugados con FITC y PerCP (1:500), respectivamente. Al análisis por citometría se hizo un Gate discriminatorio por tamaño y complejidad en el Dot plot inicial y adicionalmente se discriminó con marcadores de CD45 para leucocitos y yoduro de propidio (IP) para eritrocitos y debris en los histogramas correspondientes. Los resultados se analizaron con ANOVA y regresión lineal. La expresión de GDF-9 disminuyó (P<0.05) a medida que avanzó el desarrollo folicular en Anestro y Proestro/Estro, pero aumentó (P<0.05) en Diestro. BMP-15 aumentó (P<0.05) su presencia en folículos antrales más desarrollados en la etapa de Anestro, disminuyendo (P<0.05) en Proestro/Estro al alcanzar un mayor nivel de desarrollo. Estas proteínas por tanto, se expresaron en las células foliculares de caninos en distintas etapas del ciclo estral y de manera diferente durante el desarrollo folicular, donde ambas podrían estar relacionadas con las características particulares.
The estrous cycle of the bitch presents unique features when compared to other domestic mammals, however, little is known about the characteristics of the follicular development compared with other species. The Growth Differential Factor 9 (GDF-9) and Bone Morphogenetic Protein 15 (BMP-15) are members of the Transforming Growth Factor (TGF-β) superfamily and have demonstrated an important role during the follicular development of many species. The aim of this study was to analyze the presence of both proteins in the follicular cells from canine ovarian antral follicles by flow cytometry. Granulosa and cumulus cells were obtained by aspiration and scraping of antral follicles from different sizes and stages of the estrous cycle. Cells were fixed and incubated with first antibodies against human GDF-9 and mouse BMP-15 (1:100) and second antibodies conjugated with FITC and PerCP (1:500), respectively. A size and complexity discriminatory gate was used for the cytometryc analysis in the initial dot plot and, additionally, a CD45 marker for leukocyte and Iodum Propide for erythrocyte and debris discrimination in the corresponding histograms. The results were analyzed with ANOVA and lineal regression. GDF-9 expression decreased (P<0,05) during follicular development in Anestrous and Proestrous/Estrous, but increased during Diestrous (P<0,05). BMP-15 expression increased (P<0,05) during follicular development in Anestrous, but decreased in roestrous/Estrous (P<0,05) compared to small and medium sizes. Therefore, these proteins are expressed in canine antral follicle cells during different stages of the estrous cycle and at different levels of develop of the antral follicle, where both may be related to the special features of the bitch.
Financiamiento: Proyecto Fondecyt No. 1140658

Antecedentes: La insuficiencia cardíaca con fracción de eyección del ventrículo izquierdo (FEVI) preservada e intermedia (ICFEp, ICFEmr) es un diagnóstico frecuente y responsable de un gran número de consultas e ingresos. Los mecanismos fisiopatológicos no están claros, lo que dificulta un tratamiento específico. No existen predictores de mortalidad y reingreso robustos que permitan identificar de forma precoz a los pacientes de mayor riesgo. Se han introducido diferentes biomarcadores en la práctica clínica habitual, siendo indiscutible el papel de los péptidos natriuréticos. Objetivo: Evaluar predictores de mortalidad y reingreso en una cohorte de pacientes con ICFEp e ICFEmr en seguimiento durante 5 años en una unidad de insuficiencia cardiaca (UIC). Métodos: Se incluyeron en el estudio pacientes con ICFEp e ICFEmr evaluados por primera vez en la UIC y se obtuvieron muestras de sangre por venopunción periférica para el posterior análisis de diferentes biomarcadores. Resultados: Se incluyeron 311 pacientes: n=221 (70%) con FEVI >50% y n=90 (30%) con FEVI entre 40-49%. Edad media: 68 años (32% mujeres). Etiología más frecuente isquémica (34%), seguida idiopática (24%).   Se evaluaron diferentes biomarcadores (GDF-15, NT-proBNP, TnT us, Galectina-3) sin encontrar diferencias significativas en el valor medio entre ICFEp e ICFEmr. No hubo diferencias tampoco en el seguimiento ni en la tasa de reingreso. La mortalidad en el seguimiento fue: 35% en ICFEp y 22% en ICFEmr (p=0,02).   El análisis multivariado identificó la clase funcional 3-4 (p=0.04); presión arterial sistólica (p=0.01); tamaño de la aurícula izquierda (p<0.03) y edad (p<0.0001) como predictores independientes de mortalidad. Cuando los biomarcadores se agregaron al modelo, sólo GDF15 resultó estadísticamente significativo (p<0,01). El IDI después de la inclusión de GDF15 en el modelo con los factores predictores de mortalidad fue de 0,033, lo que aumenta la capacidad de predecir muerte en un 3,3% (p<0,004). El NRI, que representa la cuantificación de la mejora en la clasificación de eventos (mortalidad), descontando lo que empeora para los no eventos, fue de 0.548 (p=0.000005). Conclusiones: El análisis de GDF-15 mejoró el modelo predictivo de mortalidad y reingreso basado en parámetros clínicos en pacientes con insuficiencia cardíaca con fracción de eyección preservada o intermedia.
Background: Heart failure with preserved and intermediate (HFpEF, HFmrEF) ejection fraction (LVEF) is a frequent diagnosis and responsible of a large number of consultations and admissions.  The pathophysiological mechanisms are not clear, which makes specific treatment difficult. There are no predictors of mortality and re-entry that allow early identification of patients at higher risk. Different biomarkers have been introduced in routine clinical practice, the role of natriuretic peptides is indisputable. Objective: To evaluate predictors of mortality and readmission in a cohort of patients with HFpEF and HFmrEF followed up for 5 years in a Heart Failure Unit (HFU). Methods: Patients with HFpEF and HFmrEF evaluated for the first time in the HFU were included in the study and blood samples were obtained by peripheral venipuncture for the subsequent analysis of different biomarkers. Results: 311 patients were included: n = 221 (70%) with LVEF> 50% and n = 90 (30%) with LVEF between 40-49%. Mean age: 68 years (32% women). Most frequent aetiology was ischemic (34%).   Different biomarkers were evaluated (GDF-15, NT-proBNP, hs TnT, Galectin-3) without finding significant differences in the mean value between HFpEF and HFmrEF. There were no differences either in the follow-up or in the re-entry rate. Mortality at follow-up was: 35% in HFpEF and 22% in HFmrEF (p = 0.02).   The multivariate analysis identified functional class 3-4 (p=0.04); systolic blood pressure (p=0.01); left atrial size (p<0.03) and age (p<0.0001) as independent predictors of mortality. When the biomarkers were added to the model, only GDF-15 was statistically significant (p <0.01). The IDI after the inclusion of GDF-15 in the model with the predictors of mortality was 0.033, which increases the ability to predict death by 3.3% (p <0.004). The NRI, which represents the quantification of the improvement in the classification of events (mortality), discounting what worsens for non-events, was 0.548 (p = 0.000005). Conclusions: The analysis of GDF-15 improved the predictive model of mortality and re-entry based on clinical parameters in patients with heart failure with preserved or intermediate ejection fraction.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico
O objetivo deste trabalho foi investigar os efeitos da artrite encefalite caprina (CAE) sobre a expressÃo de BMP-15, BMPR-IB e GDF-9 em folÃculos ovarianos, bem como os efeitos de fitohemaglutinina (PHA) e EGF na sobrevivÃncia e ativaÃÃo de folÃculos primordiais, e na expressÃo de genes para o sistema de TNF-α no tecido ovariano cultivado caprino. Os nÃveis de BMP-15, BMPR-IB e GDF-9 em folÃculos primordiais/primÃrios e secundÃrios, bem como em CCOs e parede folicular de folÃculos antrais foram avaliados por PCR em tempo real (experimento 1). Para os estudos in vitro, fragmentos de tecido ovariano foram cultivados por seis dias em α-MEM sozinho ou suplementado com EGF (100ug), PHA (10μg/mL) ou ambos (experimento 2). Antes e depois do cultivo, o tecido ovariano foi processado para anÃlise morfolÃgica ou armazenado para avaliar a expressÃo de RNAm para TNF-α e seus receptores. Os resultados mostraram que a expressÃo de BMP-15 e GDF-9 em folÃculos primordiais/primÃrios de cabras infectadas foram significativamente maiores do que em animais saudÃveis, mas a expressÃo de GDF-9 em folÃculos secundÃrios de cabras infectadas foi significativamente menor. AlÃm disso, a expressÃo de RNAm para BMP-15 na parede folicular de folÃculos antrais de cabras infectadas foi significativamente maior do que em cabras saudÃveis. ApÃs o cultivo dos fragmentos ovarianos em todos os meios testados, observou-se a reduÃÃo nos percentuais de folÃculos primordiais, e aumento de folÃculos em desenvolvimento quando comparado ao grupo controle nÃo cultivado. AlÃm disso, houve um aumento significativo no diÃmetro folicular apÃs cultivo em meio suplementado com EGF. ApÃs o cultivo de tecido ovariano de cabras infectadas em meio suplementado com PHA, os folÃculos primordiais apresentavam diÃmetros maiores do que os de animais saudÃveis. AlÃm disso, observou-se um aumento nos nÃveis de RNAm para TNF-α apÃs cultivo de tecido ovariano na presenÃa de ambos EGF e PHA em animais saudÃveis, mas este mesmo tratamento proporcionou uma reduÃÃo de RNAm para TNF-α e aumento dos transcritos de TNFR-II em animais infectados. Pode-se concluir que a CAE influencia a expressÃo de RNAm para BMP-15 e GDF-9 em folÃculos ovarianos caprinos e a PHA e o EGF diferencialmente regulam a expressÃo de TNF-α e TNFR-II em tecidos ovarianos.
This study aims to investigate the effects of caprine arthritis encephalitis (CAE) on the expression of BMP-15, BMPR-IB and GDF-9 in ovarian follicles, as well as the effects of phytohemagglutinin (PHA) and EGF on the survival and activation of primordial follicles, and on expression of mRNA for TNF-α and its receptors in cultured goat ovarian tissue. The levels of BMP-15, BMPR-IB and GDF-9 in primordial/primary and secondary follicles, as well as in COCs and follicular walls from antral follicles were evacuated by real-time PCR (experiment 1). Ovarian tissues were cultured for six days in α-MEM+ alone or supplemented with EGF (100Âg/mL), PHA (10Âg/mL) or both (experiment 2). Before and after culture, ovarian fragments were processed for morphological analysis or stored to evaluate the expression of mRNA for TNFα and its receptors. The results showed that the expression of BMP-15 and GDF-9 in primordial/primary follicles from infected goats was significantly higher than in health animals, but the expression of GDF-9 in secondary follicles from infected goats was significantly lower. Additionally, the expression of mRNA for BMP-15 in follicular wall of antral follicles from infected goats was significantly higher than in healthy goats. After culturing ovarian fragments in all tested media, reduced percentages of primordial follicles, and increased of developing follicles was observed when compared to uncultured control. Furthermore, there was a significant increase in follicular diameter after culture in medium supplemented with EGF. Ovarian tissue from infected goats cultured in medium supplemented with PHA had primordial follicles with higher diameters than those from healthy animals. An increase in the levels of mRNAs for TNF-α was observed after culturing ovarian tissue in presence of both EGF and PHA in healthy animals, but this same treatment promoted a reduction of mRNAs for TNF-α and and increase of TNFR-II transcripts in infected animals. In conclusion, CAE influences the expression of mRNA for BMP-15 and GDF-9 in goat ovarian follicles and PHA and EGF differentially regulate the expression of TNFα and TNFR-II in cultured ovarian tissue.

LOPES, T.A. Influência da Artrite Encefalite Caprina sobre a expressão de RNAm para GDF-9, BMP-15 e BMPR-IB em folículos ovarianos e ativação in vitro de folículos primordiais em meio suplementado com fitohemaglutinina e EGF. 2014. 101 f. Dissertação (MESTRADO EM BIOTECNOLOGIA) - Campus de Sobral, Universidade Federal do Ceará, Sobral, 2014.
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This study aims to investigate the effects of caprine arthritis encephalitis (CAE) on the expression of BMP-15, BMPR-IB and GDF-9 in ovarian follicles, as well as the effects of phytohemagglutinin (PHA) and EGF on the survival and activation of primordial follicles, and on expression of mRNA for TNF-α and its receptors in cultured goat ovarian tissue. The levels of BMP-15, BMPR-IB and GDF-9 in primordial/primary and secondary follicles, as well as in COCs and follicular walls from antral follicles were evacuated by real-time PCR (experiment 1). Ovarian tissues were cultured for six days in α-MEM+ alone or supplemented with EGF (100μg/mL), PHA (10μg/mL) or both (experiment 2). Before and after culture, ovarian fragments were processed for morphological analysis or stored to evaluate the expression of mRNA for TNFα and its receptors. The results showed that the expression of BMP-15 and GDF-9 in primordial/primary follicles from infected goats was significantly higher than in health animals, but the expression of GDF-9 in secondary follicles from infected goats was significantly lower. Additionally, the expression of mRNA for BMP-15 in follicular wall of antral follicles from infected goats was significantly higher than in healthy goats. After culturing ovarian fragments in all tested media, reduced percentages of primordial follicles, and increased of developing follicles was observed when compared to uncultured control. Furthermore, there was a significant increase in follicular diameter after culture in medium supplemented with EGF. Ovarian tissue from infected goats cultured in medium supplemented with PHA had primordial follicles with higher diameters than those from healthy animals. An increase in the levels of mRNAs for TNF-α was observed after culturing ovarian tissue in presence of both EGF and PHA in healthy animals, but this same treatment promoted a reduction of mRNAs for TNF-α and and increase of TNFR-II transcripts in infected animals. In conclusion, CAE influences the expression of mRNA for BMP-15 and GDF-9 in goat ovarian follicles and PHA and EGF differentially regulate the expression of TNFα and TNFR-II in cultured ovarian tissue.
O objetivo deste trabalho foi investigar os efeitos da artrite encefalite caprina (CAE) sobre a expressão de BMP-15, BMPR-IB e GDF-9 em folículos ovarianos, bem como os efeitos de fitohemaglutinina (PHA) e EGF na sobrevivência e ativação de folículos primordiais, e na expressão de genes para o sistema de TNF-α no tecido ovariano cultivado caprino. Os níveis de BMP-15, BMPR-IB e GDF-9 em folículos primordiais/primários e secundários, bem como em CCOs e parede folicular de folículos antrais foram avaliados por PCR em tempo real (experimento 1). Para os estudos in vitro, fragmentos de tecido ovariano foram cultivados por seis dias em α-MEM sozinho ou suplementado com EGF (100ug), PHA (10μg/mL) ou ambos (experimento 2). Antes e depois do cultivo, o tecido ovariano foi processado para análise morfológica ou armazenado para avaliar a expressão de RNAm para TNF-α e seus receptores. Os resultados mostraram que a expressão de BMP-15 e GDF-9 em folículos primordiais/primários de cabras infectadas foram significativamente maiores do que em animais saudáveis, mas a expressão de GDF-9 em folículos secundários de cabras infectadas foi significativamente menor. Além disso, a expressão de RNAm para BMP-15 na parede folicular de folículos antrais de cabras infectadas foi significativamente maior do que em cabras saudáveis. Após o cultivo dos fragmentos ovarianos em todos os meios testados, observou-se a redução nos percentuais de folículos primordiais, e aumento de folículos em desenvolvimento quando comparado ao grupo controle não cultivado. Além disso, houve um aumento significativo no diâmetro folicular após cultivo em meio suplementado com EGF. Após o cultivo de tecido ovariano de cabras infectadas em meio suplementado com PHA, os folículos primordiais apresentavam diâmetros maiores do que os de animais saudáveis. Além disso, observou-se um aumento nos níveis de RNAm para TNF-α após cultivo de tecido ovariano na presença de ambos EGF e PHA em animais saudáveis, mas este mesmo tratamento proporcionou uma redução de RNAm para TNF-α e aumento dos transcritos de TNFR-II em animais infectados. Pode-se concluir que a CAE influencia a expressão de RNAm para BMP-15 e GDF-9 em folículos ovarianos caprinos e a PHA e o EGF diferencialmente regulam a expressão de TNF-α e TNFR-II em tecidos ovarianos.

GDF-15 ist ein atypisches Mitglied der TGF-b-Superfamilie. Unter physiologischen Bedingungen kommt es nur in der Plazenta in größeren Mengen vor, während es in zahlreichen Tumoren überexprimiert gefunden wurde. Die genaue Funktion von GDF-15 im Tumorkontext ist nicht genau geklärt. Aufgrund der häufigen und hohen Expression in Tumoren scheint GDF-15 eine wesentliche Funktion im Tumorprogress auszuüben. Das Ovarialkarzinom (OvCA) nimmt die Stellung als tödlichste gynäkologische Erkrankung ein. Da der Tumor meist erst in fortgeschrittenen Stadien diagnostiziert wird, sind bis heute die Heilungschancen schlecht. Häufig kommt es zum Rezidiv nach zunächst erfolgreicher Chemotherapie und mit 30% ist die 5-Jahres-Überlebenschance gering. Für die chemoresistenten Fälle gibt es bis zum heutigen Zeitpunkt keine effektive Therapie. Dies verdeutlicht die Notwendigkeit, neue innovative Therapiestrategien zu entwickeln. Günstige immunologische Parameter korrelieren mit der Überlebensdauer von OvCA-Patientinnen, was die Immuntherapie beim OvCA in den Fokus der experimentellen klinischen Therapie rückt. Doch um neue immuntherapeutische Strategien entwickeln zu können, müssen zunächst immunologisch relevante Angriffspunkte identifiziert werden. Das in vielen Tumoren exprimierte GDF-15 ist mit einem der stärksten immunsuppressiven Faktoren verwandt, was die Vermutung nahe legt, dass auch GDF-15 eine immunologisch relevante Funktion im Tumorkontext ausüben könnte. Daher wurden die Expression und die mögliche Funktion von GDF-15 als Immunmodulator im Ovarialkarzinom untersucht. Expressionsanalysen von OvCA-Gewebe und primären OvCA-Zellen zeigten, dass GDF-15 das am stärksten überexprimierte Gen der untersuchten TGF-b-Familienmitglieder im OvCA ist. Auch als sezerniertes Protein wird GDF-15 in vivo und in vitro im OvCA detektiert, was auf eine funktionale Rolle von GDF-15 im OvCA hindeutet. Normalerweise eliminiert das Immunsystem entartete körpereigene Zellen. Manchmal gelingt es Tumorzellen jedoch, sich dieser Immunüberwachung zu entziehen und dem Immunsystem zu „entwischen“. Inwieweit GDF-15 bei der Koordination des „immune escape“ des OvCA eine Rolle spielt, sollte im Fokus dieser Arbeit stehen. Das Hauptaugenmerk lag dabei auf der Wirkung von GDF-15 auf NK-Zellen, da diese als frühe Effektoren und wichtige Mediatoren zwischen angeborenem und adaptivem Immunsystem nicht nur eine Schlüsselrolle bei der immunologische Überwachung spielen, sondern sich dadurch auch als ideale Werkzeuge für die Tumorimmuntherapie auszeichnen. Exogenes GDF-15 hemmt in vitro die Lyseaktivität von NK-Zellen gegenüber OvCA-Zellen. Endogene GDF-15-Defizienz der OvCA-Zellen sensitiviert diese für NK-Zell-Lyse und endogene GDF-15-Überexpression mindert die NK-Lyseaktivität. Die Hemmung der NK-Lyseaktivität kann durch verschiedene synergistisch wirkende Mechanismen erfolgen: durch Rezeptormodulation, durch direkte Modulation des Lysemechanismus und durch Apoptoseregulation. Wie TGF-b1 reguliert GDF-15 die Expression des aktivierenden NK-Rezeptors NKG2D von der Zelloberfläche herunter und induziert zusätzlich die Expression des inhibierenden Rezeptors CD305 und die des mit NKG2A- und NKG2C-assoziierten Rezeptors CD94. Daneben greift GDF-15 direkt in den Lysemechanismus der NK-Zellen ein, indem es die Granzym B-Expression beeinflusst. Darüber hinaus sensitiviert GDF-15 Immunzellen für die Apoptose durch die Induktion von Fas/CD95. Signaltransduktionsanalysen zeigen, dass GDF-15 in Immunzellen die SMAD-Proteine zeitverzögert zu TGF-b aktiviert, was auf eine indirekte Wirkung schließen lässt. Zusätzlich kann GDF-15 auch die die p38/MAPK in Immunzellen aktivieren. Die Genregulation von GDF-15 und TGF-b1 in NK-Zellen ist sehr verschieden. Beide Zytokine regulieren überwiegend Gene aus gleichen Funktionalitätsclustern, allerdings sind die einzelnen von TGF-b1 und GDF-15 regulierten Gene verschieden. Nur drei Gene (CD55, Caspase-8 und Apolipoprotein 6) sind durch GDF-15 und TGF-b1 gleich reguliert. Zusammengefasst zeigt sich eine funktionale Analogie von GDF-15 und TGF-b1 in NK-Zellen. TGF-b1 scheint eine stärkere Wirkung zu induzieren, dafür zeigt GDF-15 hier ein breiteres Funktionalitätsspektrum. Durch die Charakterisierung der funktionalen Rolle von GDF-15 als Immunmodulator in Tumoren ist hier ein neuer potentieller Angriffspunkt identifiziert worden, welcher Grundlage für neue Tumortherapiestrategien, nicht nur für das OvCA, sondern auch für andere GDF-15-exprimierende Tumore sein kann
GDF-15 is an atypical member of the TGF-b superfamily. Under physiological conditions it is expressed primarily in the placenta, whereas it is over-expressed in many tumors. So far, the exact function of GDF-15 in tumor context remains unknown. Due to the high and frequent expression in different tumors GDF-15 might play an important role in tumor progression. Ovarian cancer (OvCA) is one of the most deadly gynaecological malignancies. Often OvCA is diagnosed in late stages and therapies frequently fail. Initially, chemotherapy is successful but in many cases tumors re-grow and the 5-year survival rate is less than 30%. Until now, no cure for chemoresistent OvCA exists which demonstrates the need for new experimental strategies. Immunotherapy is one major subject of experimental OvCA therapy, as favorable immunological parameters correlate with the survival of OvCA patients. In order to design new immunotherapeutic strategies against OvCA reliable and immunologically relevant targets have to be identified. GDF-15 is not only highly expressed in different tumors but is also closely related to one of the most immunosuppressive factors known suggesting a potential immunosuppressive role for GDF-15 itself. Thus, in this work GDF-15 expression and potential function as an immune modulator in ovarian cancer is analyzed. Expression analyses of OvCA tissue and primary OvCA cells reveal GDF-15 with the highest over-expression in OvCA of all analyzed TGF-b family members on RNA and protein level in vivo and in vitro. This high expression suggests a functional involvement of GDF-15 in ovarian carcinogenesis. The immune system is able to eliminate altered cells but sometimes tumor cells escape from this immune surveillance. Thus, this work focuses on the contribution of GDF-15 to the immune escape of OvCA. NK cells act as early cytotoxic effectors against tumors and represent important immune mediators with impact on innate and adaptive immunity. In vitro, exogenous GDF-15 inhibits NK cell lysis against OvCA cells. Additionally, GDF-15 deficient OvCA cells are killed more efficiently in NK lysis assays than GDF-15 over-expressing OvCA targets. Inhibition of NK cell lysis can occur by different synergistic mechanisms: receptor modulation, direct inhibition of cytotoxic functions and apoptosis regulation. Like TGF-b GDF-15 down-regulates NKG2D receptor expression and furthermore, induces the expression of the inhibitory receptor CD305 and the NKG2A and NKG2C associated receptor CD94. Additionally, GDF-15 interferes directly with the cytolytic activity of NK cells by reducing granzyme B expression. Moreover, GDF-15 sensitizes immune cells for apoptosis by up-regulation of CD95/Fas. Signal transduction analyses reveal a delay in GDF-15 dependent SMAD signaling in immune cells compared to that of TGF-b suggesting a rather indirect effect of GDF-15. But GDF-15 can additionally activate p38/MAPK in immune cells. GDF-15 and TGF-b dependent gene regulation in NK cells is different although regulated genes cluster in the same functional groups. Only three genes (CD55, caspase-8 and Apolipoprotein 6) are regulated in a similar way. In summary, GDF-15 and TGF-b show similar immune modulating characteristics. TGF-b seems to be more potent mostly achieving a more powerful effect but here, GDF-15 showed a broader mechanistic spectrum. The functional role of GDF-15 as an immune mediator in tumor context reveals an interesting new target for experimental therapy not only for OvCA but for all GDF-15 producing tumors

Background GDF-15 is a divergent member of the TGF-superfamily, which was first described as macrophage inhibitory cytokine-1 (MIC-1), revealing an immune modulatory function. GDF-15 is a soluble protein which is, under physiological conditions, highly expressed in the placenta and found in elevated levels in blood sera of pregnant women. Apart from the placenta, GDF-15 is expressed in healthy tissue, albeit to a lower extent and overexpressed in many solid tumors. A variety of different functions are attributed to GDF-15 in healthy as well as diseased humans. On the one hand, GDF-15 is required for successful pregnancy and low GDF-15 serum levels during pregnancy correlate with fetal abortion. On the other hand, overexpression of GDF-15, which can be observed in several malignancies is correlated with a poor prognosis. Furthermore, tumor derived GDF-15 leads to cancer associated anorexia-cachexia syndrome in mice. The aim of my PhD thesis was to further investigate the role of GDF-15 as an immune modulatory factor in cancer, in particular, by inhibiting the target molecule in vitro and in vivo. Therefore, the main focus was placed on the generation and characterization of monoclonal GDF-15 specific blocking antibodies, which were tested in vitro and in vivo, which represents a substantial part of my work. Results Here, GDF-15 was shown to be highly expressed in human gynecological cancer and brain tumors. We could then demonstrate that GDF-15 modulates effector immune cells in vitro. GDF-15 mediated a slight downregulation of the activating NKG2D receptor on NK and CD8+ T cells, which is crucial for proper anti-tumoral immune responses. Furthermore, we could demonstrate that GDF-15 reduces the adhesion of CD4+ and CD8+ T cells on endothelial cells in vitro. A negatively affected trans-endothelial migration of leukocytes into inflamed tissue could explain the low T cell infiltration in GDF-15 expressing tumors, which were observed in vivo, where mice bearing (shRNA mediated) GDF-15 deficient glioma cells revealed enhanced immune cell infiltrates in the tumor microenvironment, compared with the GDF-15 expressing control group. Those animals further exhibited a decreased tumor growth and prolonged survival. GDF-15 is a soluble protein, secreted by more than 50 % of solid tumors and associated with grade of malignancy. Therefore a neutralizing monoclonal antibody to GDF-15 was assumed to be an auspicious therapeutically anti-cancer tool. Such an antibody was thus generated in GDF-15 knock out mice against human GFD-15. Amongst many clones, the GDF-15 antibody clone B1-23 was found to be applicable in Western Blot as well as in ELISA techniques, detecting a three-dimensional epitope of the mature GDF-15 dimer with high affinity and specificity. To enable the humanization for a later administration in humans, the variable regions of antibody B1-23 were identified by a special PCR method using degenerate primers and cloned into a sequencing vector. The sequence obtained thereby enabled the generation of chimeric and humanized B1-23 variants. After further comprehensive characterization, the original mouse antibody B1-23 as well as the chimeric antibody (ChimB1-23) and the humanized B1-23 antibody (H1L5) were applied in a melanoma xenograft study in vivo. None of the antibodies could significantly inhibit tumor growth. .However of utmost importance, body weight loss mediated by tumor derived GDF-15 could be significantly prevented upon administration of all three GDF-15 specific antibodies, which confirmed the antagonizing functionality of the immunoglobulin. Conclusion GDF-15 is a promising cancer target, involved in tumor progression and cancer related cachexia. A monoclonal GDF-15 antibody was generated, which served on one hand as a tool for molecular biological applications (Western Blot, ELISA, etc.) and on the other hand was applied as an antagonizing antibody in vitro and in vivo. Even though tumor growth inhibition by GDF-15 depletion in T cell deficient athymic mice failed using B1-23, the same antibody and derivates thereof (chimeric and humanized) impressively prevented tumor associated cachexia in UACC-257 melanoma bearing nude mice. The missing anti-tumor effect in our own melanoma model in nude mice can only partially be explained by the missing secondary immunity, in particular cytotoxic T cells, in the athymic animals, since in a similar melanoma model, performed by an external company, a tumor reduction in immunocompromised animals was observed, when B1-23 was administered. These findings support the idea that T cells are substantial for an effective tumor immunity and are in line with the results of the syngeneic, T cell comprising, mouse glioma model, where silencing of tumor expressed GDF-15 led to an enhanced intratumoral T cell infiltration and a prolonged survival. Taken together our data allow for the conclusion that tumor associated cachexia can be combatted with the GDF-15 antibody B1-23. Further, B1-23 might elicit direct anti-tumor effects in immune competent models, which contain T cells, rather than in an athymic, T cell deficient nude mouse model
Hintergrund GDF-15 ist ein divergentes Mitglied der TGF-Superfamilie, welches zuerst als „macrophage inhibitory cytokine-1“ (MIC-1) mit immunmodulatorischen Eigenschaften beschrieben wurde. GDF-15 ist ein lösliches Protein, das unter physiologischen Bedingungen hauptsächlich in der Plazenta exprimiert wird und welches im Serum von Schwangeren in erhöhten Konzentrationen nachgewiesen werden kann. Mit Ausnahme der Plazenta wird GDF-15 in verschiedenen gesunden Geweben gefunden, hier jedoch in deutlich niedrigeren Konzentrationen, und ist in vielen soliden Tumoren überexprimiert. GDF-15 werden sowohl bei gesunden, als auch bei kranken Menschen, unterschiedlichste Funktionen zugeschrieben. Zum einen ist GDF-15 für eine erfolgreiche Schwangerschaft notwendig. Niedrige GDF-15 Spiegel im Serum während der Schwangerschaft korrelieren mit dem Verlust des Fötus. Zum anderen korreliert die Überexpression von GDF-15, welche bei unterschiedlichen Malignitäten beobachtet werden kann, mit einer schlechten Prognose. Darüber hinaus verursacht das von Tumorzellen sezernierte GDF-15 das sogenannte „Anorexie-Kachexie Syndrom“ in Mäusen. Das Ziel meiner Arbeit war es, die immunmodulatorische Funktion von GDF-15 im Tumorkontext zu untersuchen, insbesondere durch eine Hemmung des Zielmoleküls in vitro und in vivo. Aus diesem Grund wurde der Schwerpunkt auf die Generierung und Charakterisierung monoklonaler, GDF-15 spezifischer, blockierender Antikörper gelegt. Diese wurden sowohl in vitro als auch in vivo getestet, was einen großen Teil dieser Arbeit darstellt. Ergebnisse Es konnte gezeigt werden, dass GDF-15 in humanen gynäkologischen Tumoren wie auch in Hirntumoren überexprimiert ist. Weiterhin ließ sich zeigen, dass GDF-15 Effektorzellen des Immunsystems in vitro moduliert. Dabei verursacht GDF-15 eine moderate Herunterregulation des aktivierenden Killing Rezeptors NKG2D auf NK und CD8+ T Zellen, welcher eine hohe Bedeutung für eine effektive anti-tumorale Immunantwort hat. Darüber hinaus konnten wir zeigen, dass GDF-15 die Adhäsion von CD4+ und CD8+ T Zellen auf Endothelzellen in vitro herabsetzt. Eine daraus resultierende Reduktion der trans-endothelialen Migration von Leukozyten in entzündetes Gewebe erklärt möglicherweise die niedrige T Zell Infiltration in GDF-15 exprimierenden Tumoren, welche in vivo beobachtet werden konnten. Mäuse, denen (auf shRNA basierende) GDF-15-defiziente Gliomzellen appliziert wurden, zeigten im Vergleich zur Kontrollgruppe, welche GDF-15-exprimierenden Gliomzellen erhalten hatte, ein verlängertes Überleben, vermindertes Tumorwachstum und eine erhöhte Immunzellinfiltration in das Tumormikromillieu. GDF-15 ist ein lösliches Protein, das von mehr als 50 % aller soliden Tumore sezerniert wird und mit dem Grad der Malignität korreliert. Daher wurde postuliert, dass ein neutralisierender monoklonaler Antikörper gegen GDF-15 eine effektive neue Antikrebstherapie ermöglichen sollte. Solch ein Antikörper wurde entsprechend in GDF-15-defizienten Mäusen generiert. Unter verschiedenen Klonen wurde der Antikörper Klon B1-23 identifiziert, welcher sowohl im Western Blot als auch im ELISA anwendbar ist. Dieser Klon detektiert ein drei-dimensionales Epitop des maturen GDF-15 Dimers mit hoher Affinität und Spezifität. Um den Antikörper für eine spätere Anwendung im Menschen humanisieren zu können, wurden die variablen Regionen des Klons B1-23 durch eine spezielle PCR Methode unter Verwendung degenerierter Primer und nachfolgender Klonierung in einen Sequenzierungsvektor identifiziert. Die hierdurch gewonnenen Sequenzen ermöglichten die Generierung von chimären und humanisierten Varianten von B1-23. Nach anschließender intensiver Charakterisierung konnte sowohl der ursprüngliche Maus-Antikörper B1-23 als auch der chimäre B1-23 Antikörper (ChimB1-23) und der humanisierte B1-23 Antikörper (H1L5) in einer Melanom Xenograft Studie in vivo getestet werden. Zwar ließ sich mit keinem der Antikörper eine signifikante Hemmung des Tumorwachstums beobachten. Als herausragendes Ergebnis zeigte sich allerdings, dass der durch GDF-15 induzierte Gewichtsverlust signifikant durch die Verabreichung der GDF-15 spezifischen Antikörper verhindert werden konnte, was die antagonisierende Funktionalität des entwickelten Immunglobulins bestätigte. Schlussfolgerung GDF-15 ist ein vielversprechendes Zielmolekül bei Krebserkrankungen, welches bei der Tumorprogression und Tumor-assoziierter Kachexie beteiligt ist. Es konnte ein monoklonaler Anti-GDF-15 Antikörper generiert werden, welcher zum einen molekularbiologisch zum Einsatz kam (z.B. Western Blot, ELISA, etc.) und zum anderen als antagonisierender Antikörper sowohl in vitro als auch in vivo Anwendung fand. Auch wenn B1-23 scheinbar keine Tumorwachstumshemmung durch die Depletion von GDF-15 in T Zell defizienten athymischen Mäusen zeigte, konnte derselbe Antikörper wie auch die abgeleiteten Varianten (chimärisiert und humanisiert) eindrücklich die Tumor assoziierte Kachexie im UACC-257 Melanom Modell verhindern. Der ausgebliebene antitumorale Effekt in unserem Melanom Modell in Nacktmäusen lässt sich nur zum Teil durch eine fehlende sekundäre Immunkomponente, insbesondere das Fehlen zytotoxischer T Zellen, erklären, da es in einem ähnlichen Xenograft Melanom Modell, welches in Auftragsforschung (CRO) durchgeführt wurde, zu einer Reduktion des Tumorwachstums durch die Applikation von B1-23 kam. Diese Ergebnisse lassen vermuten, dass T Zellen unerlässlich für eine effektive antitumorale Antwort sind, eine Annahme, die durch die Ergebnisse des syngenen Gliom Maus-Modells unterstützt wird, in welchem es durch das Ausschalten von Tumor produziertem GDF-15 zu einer erhöhten intratumoralen T Zell Infiltration und einem längeren Überleben kam. Zusammengenommen erlauben uns diese Daten den Schluss, dass eine tumorbedingte Kachexie durch den GDF-15-Antikörper B1-23 bekämpft werden kann. Allerdings sind direkte B1-23 vermittelte antitumorale Effekte eher in immunkompetenten Modellen mit T Zellen als in einem athymischen, T Zell defizienten Nacktmaus-Modell zu erwarten

GDF-15 wird seit wenigen Jahren als prognostischer und prädiktiver Marker in der Tumortherapie diskutiert. Diese Pilotstudie sollte erstmals GDF-15 bei Patienten mit HER2/neu positivem Mammakarzinom im frühen Stadium im klinischen Verlauf untersuchen. Dazu wurden 22 Patienten rekrutiert und die GDF-15-Spiegel mittels ELISA vor und während einer Antikörpertherapie bestimmt. Um GDF-15 als prädiktiven Marker zu testen, wurde nach neoadjuvanter Therapie und anschließender Operation der Regressionsgrad nach Sinn bewertet. In der untersuchten Kohorte wurde ein medianer GDF 15-Spiegel von 0,33 ng/ml ermittelt. Im Therapieverlauf kam es zu keiner signifikanten Veränderung des Spiegels. Höhere GDF-15-Spiegel konnten allerdings bei größeren Tumoren und bei einem höheren BMI analysiert werden. Ebenfalls konnten wir zeigen, dass der GDF-15-Spiegel signifikant mit dem Alter steigt. Nicht signifikant, aber von Bedeutung ist der Zusammenhang zwischen GDF-15 und dem Regressionsgrad nach Sinn. Die untersuchten Patienten wiesen tendenziell höhere GDF-15-Werte bei niedrigem Regressionsgrad auf. Ein schlechteres Ansprechen auf eine Antikörpertherapie bei höheren GDF 15-Spiegeln ist somit anzunehmen
GDF-15 as a prognostic and predictive marker in tumortherapy is actively discussed. In this pilot study we analysed the GDF-15 levels before an while antibody-therapy in patients with early her2-positiv breast cancer and correlated those with clinical variables. Overall we found relatively low GDF-15 levels. The correlation of GDF-15 with the sinn regression was not significant but showed a tendency. This indicates a worse response rate to a antibody-therapy for patients with higher GDF-15 serum levels

Na prática clínica, a questão central relativamente à terapêutica com anticoagulantes orais tem sido como maximizar os seus benefícios, ou seja, prevenir eventos trombóticos, sem incorrer no aumento dos seus riscos, nomeadamente eventos hemorrágicos. Diversas escalas de risco hemorrágico têm sido desenvolvidas no sentido de permitir tomar decisões clínicas bem fundamentadas. O ABC-Bleeding é um score recente com resultados bastante promissores e que se destaca por considerar no cálculo do risco o Growth differentiation factor 15, uma citocina que aparenta tornar esta escala mais eficaz. Este estudo analisa as escalas de risco hemorrágico ABC-Bleeding, HAS-BLED e ORBIT e uma escala de risco trombótico, o CHA2DS2-VASc numa população com fibrilhação auricular medicada com anticoagulantes, seguida nas consultas de coagulação do serviço de Imunohemoterapia do Centro Hospitalar Universitário da Cova da Beira. A população considerada é de 150 doentes, 30 doentes de cada um dos 5 anticoagulantes: apixabano, edoxabano, dabigatrano, rivaroxabano e varfarina. Foram analisadas as suas características clínicas e laboratoriais e destaca-se a existência de vários fatores de risco cardiovasculares e comorbilidades na amostra, o que se reflete nas pontuações nas várias escalas e nos níveis séricos de GDF-15. A probabilidade de evento hemorrágico previsto pelo ABC-Bleeding é em média de 2,63%, e a pontuação no HAS-BLED de 1,96, no ORBIT de 1,73 e no CHA2DS2-VASc de 4,16. O valor médio de GDF-15 é de 2111,68 ng/l e através do teste de Qui-Quadrado de Pearson verifica-se uma associação moderada deste biomarcador com a idade, insuficiência cardíaca, diabetes mellitus, função renal (clearance de creatinina e taxa de filtração glomerular estimada) e hemoglobina bem como uma associação relativamente forte com os valores de TnT-hs. Decorridos três meses desde a avaliação inicial foi verificada a ocorrência ou não de hemorragia major, para a qual apenas dois doentes cumpriram os critérios, pelo que não é possível concluir sobre a eficácia das escalas. Nas análises sanguíneas realizadas à data da identificação do evento, em ambos os doentes identificou-se função renal significativamente alterada o que poderá ter aumentado as concentrações sanguíneas do anticoagulante e portanto justificar a ocorrência destes eventos. Este estudo revalida o papel importante do GDF-15 enquanto biomarcador.
In clinical practice, the main question regarding oral anticoagulant therapy has been how to maximize its benefits, to prevent thrombotic events, without increasing risks, including bleeding events. Several hemorrhagic risk scores have been developed in order to make informed clinical decisions. The ABC-Bleeding is a recent score with highly promising results and stands out by including the Growth differentiation factor 15, a cytokine that appears to make this scale more effective. This study analyzes the ABC-Bleeding, HAS-BLED and ORBIT hemorrhagic risk scores and a thrombotic risk score, CHA2DS2-VASc in a population with atrial fibrillation medicated with anticoagulants, followed in the coagulation consultations of the Immunohemotherapy service of the Centro Hospitalar Universitário da Cova da Beira. The study population includes 150 patients, distributed by 5 groups according to the anticoagulant (apixaban, edoxaban, dabigatran, rivaroxaban and warfarin), each one with 30 patients. Their clinical and laboratory characteristics were analyzed and it stands out that there are several cardiovascular risk factors and comorbidities in the study sample, which is reflected in scores within the various scales and serum levels of GDF-15. The mean probability of hemorrhagic event predicted by ABC-Bleeding is 2.63%, 1.96 in the HAS-BLED, 1.73 in the ORBIT and 4.16 in the CHA2DS2-VASc scores. The mean value of GDF-15 is 2111.68 ng / l and a moderate association between this cytokine and age, heart failure, diabetes mellitus, renal function (creatinine clearance and estimated glomerular filtration rate) and hemoglobina, as well as a relatively strong association with TnT-hs values, are shown by Pearson's Chi-Square test. Three months after the initial evaluation, the occurrence or not of major haemorrhage was evaluated, for which only two patients fulfilled the criteria, so it was not possible to state about the efficacy of the scales. Blood tests performed at the time of the event identification in both patients demonstrated significantly altered renal function, which may have increased the anticoagulant blood concentrations and therefore justify the occurrence of these events. This study revalidates the important role of GDF-15 as a biomarker.

Cancers represent second the most common cause of death in the Czech Republic. The most common are breast and colorectal cancers. Identification of prognostic factors improving decision-making approaches for treatment optimization belongs to the key aims of clinical research in oncology. Carriers of mutation in cancer-susceptibility genes represent a small but clinically important group of high-risk patients. The implementation of NGS have accelerated predisposing genes analyses. The large extent of data about the presence of variants in predisposing genes is in striking contrast to only a very limited information available about clinico-pathological characteristics of mutation carriers. Determination of the risk of tumor development in carriers of rare mutations or variants of unclear significance in genes with incomplete penetrance represent substantial drawbacks of current NGS analyses. To address these issues, we have attempted i) to introduce a unified approach to NGS analysis in breast cancer patients, ii) to characterize importance of prognostic factors in BRCA1/BRCA2 mutation carriers, and iii) to identify the cancer risks in carriers of germline mutations in the CHEK2 gene. Colorectal cancer represents seemingly histologically homogeneous disease. However, at the molecular level it can be...