Recapitulation of tumor heterogeneity and molecular signatures in a 3D brain cancer model with decreased sensitivity to histone deacetylase inhibition

INTRODUCTION Physiologically relevant pre-clinical ex vivo models recapitulating CNS tumor micro-environmental complexity will aid development of biologically-targeted agents. We present comprehensive characterization of tumor aggregates generated using the 3D Rotary Cell Culture System (RCCS). METHODS CNS cancer cell lines were grown in conventional 2D cultures and the RCCS and comparison with a cohort of 53 pediatric high grade gliomas conducted by genome wide gene expression and microRNA arrays, coupled with immunohistochemistry, ex vivo magnetic resonance spectroscopy and drug sensitivity evaluation using the histone deacetylase inhibitor, Vorinostat. RESULTS Macroscopic RCCS aggregates recapitulated the heterogeneous morphology of brain tumors with a distinct proliferating rim, necrotic core and oxygen tension gradient. Gene expression and microRNA analyses revealed significant differences with 3D expression intermediate to 2D cultures and primary brain tumors. Metabolic profiling revealed differential profiles, with an increase in tumor specific metabolites in 3D. To evaluate the potential of the RCCS as a drug testing tool, we determined the efficacy of Vorinostat against aggregates of U87 and KNS42 glioblastoma cells. Both lines demonstrated markedly reduced sensitivity when assaying in 3D culture conditions compared to classical 2D drug screen approaches. CONCLUSIONS Our comprehensive characterization demonstrates that 3D RCCS culture of high grade brain tumor cells has profound effects on the genetic, epigenetic and metabolic profiles of cultured cells, with these cells residing as an intermediate phenotype between that of 2D cultures and primary tumors. There is a discrepancy between 2D culture and tumor molecular profiles, and RCCS partially re-capitulates tissue specific features, allowing drug testing in a more relevant ex vivo system

Functional diversity and co-operativity between subclonal populations of paediatric glioblastoma and diffuse intrinsic pontine glioma cells

The failure to develop effective therapies for pediatric glioblastoma (pGBM) and diffuse intrinsic pontine glioma (DIPG) is in part due to their intrinsic heterogeneity. We aimed to quantitatively assess the extent to which this was present in these tumors through subclonal genomic analyses and to determine whether distinct tumor subpopulations may interact to promote tumorigenesis by generating subclonal patient-derived models in vitro and in vivo. Analysis of 142 sequenced tumors revealed multiple tumor subclones, spatially and temporally coexisting in a stable manner as observed by multiple sampling strategies. We isolated genotypically and phenotypically distinct subpopulations that we propose cooperate to enhance tumorigenicity and resistance to therapy. Inactivating mutations in the H4K20 histone methyltransferase KMT5B (SUV420H1), present in <1% of cells, abrogate DNA repair and confer increased invasion and migration on neighboring cells, in vitro and in vivo, through chemokine signaling and modulation of integrins. These data indicate that even rare tumor subpopulations may exert profound effects on tumorigenesis as a whole and may represent a new avenue for therapeutic development. Unraveling the mechanisms of subclonal diversity and communication in pGBM and DIPG will be an important step toward overcoming barriers to effective treatments

Mammalian sex determination—insights from humans and mice

Disorders of sex development (DSD) are congenital conditions in which the development of chromosomal, gonadal, or anatomical sex is atypical. Many of the genes required for gonad development have been identified by analysis of DSD patients. However, the use of knockout and transgenic mouse strains have contributed enormously to the study of gonad gene function and interactions within the development network. Although the genetic basis of mammalian sex determination and differentiation has advanced considerably in recent years, a majority of 46,XY gonadal dysgenesis patients still cannot be provided with an accurate diagnosis. Some of these unexplained DSD cases may be due to mutations in novel DSD genes or genomic rearrangements affecting regulatory regions that lead to atypical gene expression. Here, we review our current knowledge of mammalian sex determination drawing on insights from human DSD patients and mouse models

Indoleamine 2,3-Dioxygenase (IDO) Enzyme Links Innate Immunity and Altered T-Cell Differentiation in Non-ST Segment Elevation Acute Coronary Syndrome

Atherosclerosis is a chronic inflammatory disease characterized by a complex interplay between innate and adaptive immunity. Dendritic cells (DCs) play a key role in T-cell activation and regulation by promoting a tolerogenic environment through the expression of the immunosuppressive enzyme indoleamine 2,3-dioxygenase (IDO), an intracellular enzyme involved in tryptophan catabolism. IDO expression and activity was analyzed in monocytes derived DCs (MDDCs) from non-ST segment elevation myocardial infarction (NSTEMI) patients, stable angina (SA) patients and healthy controls (HC) by real-time quantitative polymerase chain reaction (RT-qPCR) before and after in vitro maturation with lipopolysaccharide (LPS). The amount of tryptophan catabolite; kynurenine; was evaluated in the culture supernatants of mature-MDDCs by ELISA assay. Autologous mixed lymphocyte reaction (MLR) between mature-MDDCs and naive T-cells was carried out to study the differentiation towards T-helper 1 (Th1) and induced regulatory T-cells (iTreg). Analysis of IDO mRNA transcripts in mature-MDDCs revealed a significant reduction in cells isolated from NSTEMI (625.0 +/- 128.2; mean +/- SEM) as compared with those from SA (958.5 +/- 218.3; p = 0.041) and from HC (1183.6 +/- 231.6; p = 0.034). Furthermore; the concentration of kynurenine was lower in NSTEMI patients (2.78 +/- 0.2) and SA (2.98 +/- 0.25) as compared with HC (5.1 +/- 0.69 ng/mL; p = 0.002 and p = 0.016; respectively). When IDO-competent mature-MDDCs were co-cultured with allogeneic naive T-cells, the ratio between the percentage of generated Th1 and iTreg was higher in NSTEMI (4.4 +/- 2.9) than in SA (1.8 +/- 0.6; p = 0.056) and HC (0.9 +/- 0.3; p = 0.008). In NSTEMI, the tolerogenic mechanism of the immune response related to IDO production by activated MDDCs is altered, supporting their role in T-cell dysregulation

Las competencias profesionales del nutricionista deportivo The professional competences of the sports dietitian

OBJETIVO: El artículo presenta los resultados obtenidos en la investigación que dio origen a la tesis doctoral defendida por la autora en la Universitat de Lleida (España), cuyo objetivo fue identificar las Competencias Profesionales de los nutricionistas que trabajan en el ámbito de la Nutrición Deportiva. MÉTODOS: Fueron investigados 14 expertos provenientes de Australia (n=1), Brasil (n=7), España (n=3) y Estados Unidos (n=3). La herramienta metodológica utilizada fue la técnica Delphi, compuesta de tres rondas de cuestionarios. En la primera ronda los expertos proporcionaron, a través de sus discursos, la identificación de un listado de Competencias Profesionales, información que en la segunda y tercera ronda pudieron ser evaluadas y posteriormente analizadas a través de cálculos estadísticos descriptivos (media, moda, mediana y desviación Standard). RESULTADOS: De esta manera, se llegó al consenso entre los expertos sobre 147 competencias profesionales identificadas. Las competencias fueron clasificadas en cuatro macro categorías de Competencias Profesionales: Competencias Técnicas (38), Metodológicas (62), Participativas (24) y Personales (23). CONCLUSIÓN: Los resultados demostraron que el estudio sistematizado de las Competencias Profesionales del Nutricionista Deportivo contribuye para el establecimiento de los contenidos que deben componer la disciplina de Nutrición Deportiva a ser incorporada en los itinerarios curriculares de las carreras de Nutrición Humana y Dietética.<br>OBJECTIVE: This article presents the results of the doctoral research developed by the author at the University of Lleida (Spain) in June 2006. The main goal was to investigate the professional competences of dietitians who work in the field of Sports Nutrition. METHODS: Fourteen experts in Sports Nutrition from 4 countries were investigated, Australia (n=1), Brazil (n=7), Spain (n=3) and the United States (n=3). The methodological tool applied was the Delphi Technique, consisting of three rounds of questionnaires. In the first round, the experts identified a number of Professional Competences, and on the following second and third rounds these competences were assessed and analyzed by descriptive statistics (mean, mode, median and standard deviation). RESULTS: A consensus was reached by the experts on 147 identified competences. The competences were classified into four macro categories of professional competences: Technical (38), Methodological (62), Participative (24) and Personal (23) competences. CONCLUSION: The results showed that the systemized study of the professional competences of the Sports Dietitian contributes to the establishment of the contents to be taught in Sports Nutrition classes that are now being incorporated in the curriculum of undergraduate nutrition courses

Immunoglobulin A antibodies to oxidized collagen type II as a potential biomarker for the stratification of spondyloarthritis from rheumatoid arthritis

Objectives The discovery of diseased tissue-specific neoantigens offers the opportunity to develop important disease tissue-specific biomarkers that can help in the prediction, diagnosis, and stratification of diseases. This opportunity is specifically significant for autoimmune diseases where diagnostic biomarkers are not available. Inflammatory autoimmune diseases are commonly associated with local generation of large amounts of reactive oxidants. We have previously identified oxidative post-translationally modified (oxPTM) tissue-specific neoantigens in rheumatoid arthritis (RA) and type 1 diabetes that elicit an immune response. In the current study, we studied the presence and clinical significance of antibodies to oxPTM collagen type II (CII) in patients with spondyloarthritis (SpA). Method Levels of antibodies specific to native CII and oxPTM-CII were assessed by enzyme-linked immunosorbent assay. Results Immunoglobulin G (IgG) binding to oxPTM-CII was observed in 52%, 83%, and 28% of serum samples from patients with axial spondyloarthritis (axSpA), RA, and psoriatic arthritis (PsA), respectively. Importantly, while strong IgA anti-oxPTM-CII responses were detected in axSpA and PsA patients, with 47% and 84% respective binders, no IgA anti-oxPTM-CII was detected in RA patients. IgA anti-oxPTM-CII reactivity in axSpA patients treated with biologics was higher and more frequent, with 85% binders compared to 9% binders in patients treated with synthetic disease-modifying anti-rheumatic drugs. Conclusion Our data imply that SpA and PsA are associated with the presence of antibodies to oxPTM-CII, suggesting that there may be a humoral component that may distinguish patients with SpA from RA. Our approach could be adapted to other diseases, particularly to inflammatory autoimmune diseases.</p