Tesis por compendio / En la presente Tesis Doctoral se utilizaron cebada y remolacha como fuentes alternativas de carbohidratos en dietas para trucha arcoíris con el objetivo de evaluar su efecto sobre parámetros productivos, histología hepática e intestinal, estrés y calidad de la carne. Los marcadores estudiados fueron los mismo tanto para los experimentos de cebada como de remolacha. En las pruebas de la cebada se utilizaron concentraciones crecientes de este ingrediente (0-32%) en la dieta, se inició con un peso medio de 127.72 ± 5.65 g y se finalizó cuando alcanzaron el peso comercial al cabo de 84 días. En las pruebas de remolacha se utilizaron dos niveles de remolacha (14% y 28%) y dos de betaína (0.9% y 1.63%), se inició con un peso medio de 69 ± 2.2 g y se finalizó cuando los peces alcanzaron el peso comercial al cabo de 105 días. La inclusión de cebada en dietas de trucha arcoíris no mostró diferencias significativas en los parámetros de crecimiento ni biométricos. La inclusión de 14% remolacha y 0.9% betaína no afectó al crecimiento, parámetros nutritivos, biométricos y retenciones de nutrientes en comparación con el control, concentraciones mayores de remolacha y betaína tuvieron un efecto negativo. Cuando se estudiaron los parámetros histológicos y morfométricos en hígado e intestino, los resultados mostraron que los peces alimentados con concentraciones crecientes de cebada mostraron hepatocitos más pequeños, mientras que los peces alimentados con remolacha y betaína presentaban hepatocitos más grandes, en ambos casos el hígado mostró un menor nivel de vacuolización. La concentración de cebada de un 8% produjo un efecto negativo a nivel morfológico del intestino, sin embargo, la inclusión de remolacha no produjo ningún efecto. Los peces fueron sometidos a estrés por ausencia de oxígeno y aumento de densidad durante 10 minutos. Cuando el estrés fue analizado a nivel bioquímico los resultados mostraron que a niveles basales, la inclusión de cebada y remolacha no causó ningún cambio en los niveles de cortisol, glucosa y MDA, aunque los valores basales de lactato fueron significativamente más bajos en los peces alimentados con remolacha y betaína. Treinta minutos después del estrés la concentración de cortisol, glucosa y lactato aumentó significativamente en los peces de todos los grupos y la concentración de MDA disminuyó. La inclusión de cebada mostró valores más bajos de cortisol, glucosa y lactato que la dieta control. Cuando los peces alcanzaron el peso comercial se analizó la calidad de los filetes y las propiedades antioxidantes. En ambas pruebas los peces alimentados con los ingredientes objeto de estudio mostraron valores de actividad de agua más bajos y una mejora en la textura y el color de los filetes comparado con los peces alimentados con la dieta control. La inclusión de remolacha y betaína no tuvo ningún efecto sobre los parámetros oxidativos del filete, mientras que la inclusión de cebada inhibió su oxidación lipídica. Concentraciones de cebada por encima del 8% mejoraron la actividad antioxidante de los filetes y aumentaron el contenido en alpha-tocoferol, sin embargo, los peces alimentados con remolacha y betaína mostraron un mayor contenido en flavonoides y fenólico pero no se observó ningún efecto sobre las propiedades antioxidantes del filete. Por último, cuando se llevó a cabo el análisis sensorial se observó que los peces alimentados con dietas con más de un 8% de cebada mostraron unas agallas más rojas y mejor textura, además de un color rojo del filete más intenso, parámetros que se correlacionan con una mejora de la frescura del pescado. Sin embargo, la inclusión de cebada o remolacha en dietas de trucha arco iris no tuvo ningún efecto sobre la aceptabilidad de los filetes. / The current Doctoral Thesis used barley and red beet as alternative carbohydrate sources in rainbow trout diets. The aim was to evaluate their effect on productive, histological and morphometric parameters, their effect on biochemical indexes after an acute stress and their effect on the quality of rainbow trout (Oncorhynchus mykiss). The different markers studied were the same in the barley and red beet experiments. In the barley experiment, increasing concentrations of barley (0-32%) were used in the diet, starting with an average weight of 127.72 ± 5.65 g and ending when they reached commercial weight after 84 days. In the red beet experiment, two red beet levels (14% and 28%) and two betaine levels (0.9% and 1.63%) were used, starting with an average weight of 69 ± 2.2 g and finishing when they reached commercial weight at the end of 105 days. The inclusion of barley in rainbow trout diets did not show significant differences in growth and biometric parameters. While the inclusion of 14% of red beet and 0.9% betaine did not affect growth, nutritive or biometric parameters and nutrient retentions compared to control, higher red beet and betaine concentrations showed a negative effect. When the histological and morphometric parameters were studied in liver and intestine, the results showed that fish fed at high barley concentrations showed smaller hepatocytes than control, while hepatocytes were larger in fish fed with red beet and betaine than control, however, in both cases, the liver had a lower level of vacuolization. Barley inclusion at 8% produced a negative effect at intestine morphological level; however, no effects were observed with the inclusion of red beet. Fish were submitted to stress, a lack of oxygen and increased of fish density, during 10 minutes. The results showed that at basal levels, the inclusion of barley and red beet did not cause any change in cortisol, glucose and MDA levels, although basal lactate values were significantly lower in the fish fed with red beet and betaine. Thirty minutes after stress the concentration of cortisol, glucose and lactate increased significantly in the fish of all groups and the concentration of MDA decreased. The inclusion of barley showed lower cortisol, glucose and lactate values than control. When fish reached commercial weight, the quality of the fillets and antioxidant properties were analysed. In both trials, fish fed with the experimental ingredients showed lower water activity values and textural and colour properties were enhanced. Red beet and betaine inclusion did not show any effect on the oxidative parameters of the fillets, while the inclusion of barley showed an inhibitory effect on fillets lipid oxidation. Concentrations of barley above 8% improved the antioxidant activity of the fillets and increased the content of alpha-tocopherol, however, fish fed with red beet and betaine showed a higher content of flavonoids and phenolics but no effect on the antioxidant properties of the fillet. Finally, when the sensory analysis was carried out, it was observed that fish fed diets with more than 8% barley showed redder gills and better texture than control, in addition to a more intense red colour of the fillet, these characteristics are correlated with an improvement of fish freshness. However, the inclusion of barley or red beet in rainbow trout diets had no effect on the acceptability of fillets. / En la present tesi doctoral es van utilitzar ordi i remolatxa com a fonts alternatives de carbohidrats en dietes per a truita amb l'objectiu d'avaluar el seu efecte sobre paràmetres productius, histologia hepàtica i intestinal, estrès i qualitat de la carn. Els marcadors estudiants fòren els mateixos en els experiments d`ordi I remolatxa. En les proves de l'ordi es van utilitzar concentracions creixents d'aquest ingredient (0-32%), es va iniciar amb un pes mitjà de 127.72 ± 5.65 g i es va finalitzar quan van arribar al pes comercial al cap de 84 dies. En les proves de remolatxa es van utilitzar dos nivells de remolatxa (14% i 28%) i dos de betaïna (0.9% i 1.63%), es va iniciar amb un pes mitjà de 69 ± 2.2 g i es va finalitzar quan els peixos van aconseguir el pes comercial al cap de 105 dies. La inclusió d'ordi en dietes de truita no va mostrar diferències significatives en els paràmetres de creixement i biomètrics. La inclusió de 14% remolatxa i 0,9% betaïna no va afectar el creixement, paràmetres nutritius, biomètrics i retencions de nutrients en comparació amb el control, concentracions majors de remolatxa i betaïna van tenir un efecte negatiu. Quan es van estudiar els paràmetres histològics i morfomètrics en fetge i intestí, els resultats van mostrar que els peixos alimentats amb concentracions creixents d'ordi tenien hepatòcits més petits, mentres que els peixos alimentats amb remolatxa i betaïna presentaven hepatòcits més grans, i en ambdós casos el fetge va mostrar un menor nivell de vacuolització. La concentracion d'ordi d'un 8% va produir un efecte negatiu a nivell morfològic de l'intestí, mentres que la inclusió de remolatxa no va produir cap efecte a nivell d'intestí. Els peixos van ser sotmesos a estrès per absència d'oxigen i augment de densitat durant 10 minuts. Quan l'estrès va ser analitzat a nivell bioquímic els resultats van mostrar que a nivells basals, la inclusió d'ordi i remolatxa no va causar cap canvi en els nivells de cortisol, glucosa i MDA, encara que els valors basals de lactat van ser significativament més baixos en els peixos alimentats amb remolatxa i betaïna. 30 minuts després de l'estrès la concentració de cortisol, glucosa i lactat va augmentar significativament en els peixos de tots els grups i la concentració de MDA va disminuir. La inclusió d`ordi va mostrar valors més baixos de cortisol, glucosa i lactat que la dieta control. Quan els peixos van aconseguir el pes comercial es va analitzar la qualitat dels filets i propietats antioxidants. En ambdues proves els peixos alimentats amb els ingredients objecte d'estudi van mostrar valors d'activitat d'aigua més baixos i una millora en la textura i el color dels filets comparat amb els peixos alimentats amb la dieta control. La inclusió de remolatxa i betaïna no va tenir cap efecte sobre els paràmetres oxidatius del filet, mentre que la inclusió d'ordi va inhibir l'oxidació lipídica dels filets. Concentracions d'ordi per sobre del 8% van millorar l'activitat antioxidant dels filets i van augmentar el contingut en alpha-tocoferol, però, els peixos alimentats amb remolatxa i betaïna van mostrar un major contingut en flavonoides i fenòlic però no es va observar cap efecte sobre les propietats antioxidants del filet. Finalment, quan es va dur a terme l'anàlisi sensorial es va observar que els peixos alimentats amb dietes amb més d'un 8% d'ordi van mostrar unes ganyes més vermelles i millor textura, a més d'un color vermell del filet més intens, paràmetres que es correlacionen amb una millora de la frescor del peix. No obstant això, la inclusió d'ordi o remolatxa en dietes de truita no va tenir cap efecte sobre l'acceptabilitat dels filets. / Pinedo Gil, J. (2018). Study of novel carbohydrate sources on rainbow trout (Oncorhynchus mykiss) diets [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/107364 / Compendio

Les ß-glucanes (BG) sont les polysaccharides les plus abondants de la paroi de Saccharomyces cerevisiae. Depuis des millénaires, ils sont utilisés pour leurs propriétés immunostimulantes et leurs potentiels thérapeutiques. L'objectif de ce travail était de caractériser la réponse immunitaire induite par les BG et de comprendre leurs modes d'action sur les macrophages murins en contexte infectieux. Nous avons montré que (i) les extraits de paroi enrichis en BG n'induisent qu'une faible production de cytokines par les macrophages contrairement aux extraits bruts, (ii) la réponse inflammatoire médiée par les extraits bruts résulte de la signalisation des TLRs et non de Dectin-1 et (iii) les BG stimulent la synthèse tardive de GM-CSF via Dectin-1. En conditions infectieuses, les BG enrichis confèrent une forte signature inflammatoire aux macrophages prétraités conduisant à l'amplification de la production cytokinique, à la synthèse de ROS et l'optimisation de la clairance bactérienne. En conclusion, cette étude souligne les enjeux de l'utilisation des BG enrichis comme adjuvants dans l'amélioration de la résistance des individus aux infections. / ß-glucans (BG) are the most abundant polysaccharides of the Saccharomyces cerevisiae cell wall. For decades, they have been extensively used because of their immuno-modulatory properties and their potential therapeutic effects. The aim of this study was to characterize the immune response induced by BG and to understand their mechanisms of action on murine macrophages occurring upon bacterial infections. We demonstrated that (i) BG-enriched extracts trigger low amounts of cytokine production in contrast with crude products, (ii) the immune response mediated by crude extracts results from TLRs and not from Dectin-1 signaling and (iii) BG-enriched compounds stimulate the late and strong induction of GM-CSF in a Dectin-1-dependent manner. Upon bacteria exposure, BG-enriched extracts confer a strong inflammatory to pretreated macrophages leading to synergistic increase of cytokine release, ROS production and better clearance of pathogens. Altogether, our findings emphasize the relevancy of using BG-enriched extracts for the design of novel adjuvant formulations contributing to individuals' resistance to infections.

Les glucane-saccharases sont des transglucosidases de la famille 70 des glycoside-hydrolases. A partir de saccharose, ces enzymes catalysent la synthèse d’alpha -glucanes, polymères de haute masse molaire formés d’unités glucosyle. Elles sont aussi capables de synthétiser des oligosaccharides ou glucoconjugués par réaction de transglucosylation sur des accepteurs exogènes de natures variées. De par la diversité de leurs spécificités, tant au niveau des liaisons osidiques synthétisées [alpha (1→2) ; alpha (1→3) ; alpha (1→4) ou alpha (1→6)] que de l’organisation de ces liaisons au sein des produits formés, ces biocatalyseurs peuvent être mis à profit pour produire des hydrates de carbones d'intérêt pour les secteurs de l'alimentation, de la santé et de l'environnement. L'objectif de ces travaux de thèse était de générer par ingénierie enzymatique de nouvelles glucane-saccharases capables de synthétiser des alpha -glucanes et des gluco-oligosaccharides de structures et propriétés innovantes, afin d’élargir le panel d'applications de ces molécules. Sur le plan fondamental, l'enjeu était aussi d'améliorer la compréhension des relations entre structure et spécificité des glucane-saccharases. Pour atteindre nos objectifs, nous avons utilisé une stratégie d'ingénierie combinatoire de la dextrane-saccharase DSR-S de Leuconostoc mesenteroides NRRL B-512F, qui catalyse la synthèse d’un dextrane formé à 95 % de liaisons alpha (1→6) et 5 % alpha (1→3). Le travail a tout d'abord consisté à développer une méthode de criblage multi-étapes et à haut-débit, pour isoler et trier les variants de spécificités de liaisons variées. La stratégie comprend une première étape de sélection in vivo sur milieu solide suivie d’un criblage par RMN 1D 1H automatisé au format microplaque. Il s'agit de la première méthode de criblage à haut-débit de la spécificité de liaison des transglucosidases et glycosyltransferases. Cette stratégie a ensuite été appliquée au criblage de deux banques de variants de la DSR-S, totalisant plus de 36 000 clones obtenus par mutagénèse de saturation et recombinaison simultanée de 8 résidus du domaine catalytique. Au total, 82 mutants capables de synthétiser entre 2 et 8 fois plus de liaisons alpha (1→3) par rapport à l’enzyme parentale ont été isolés. La caractérisation des propriétés catalytiques de sept mutants représentatifs de la diversité de spécificité générée a permis d’identifier un nouveau motif peptidique 460DYVHT464 impliqué dans la spécificité de DSR-S. Enfin, la caractérisation de la structure et des propriétés rhéologiques et mécaniques des dextranes synthétisés par ces 7 mutants a mis en évidence les différences de taille et de conformation de ces macromolécules en solution et révélé l’aptitude du polymère synthétisé par le variant H463R/T464V/S512T à former des films bio-sourcés innovants, dont les propriétés mécaniques sont remarquables en comparaison de celles d'autres biopolymères extraits de plantes ou produits par fermentation / Glucansucrases are transglucosidases from glycoside hydrolase family 70. From sucrose, these enzymes catalyse the synthesis of alpha -glucans, high molecular weight polymers formed of glucosyl units. Glucansucrases are also able to synthesize oligosaccharides or glucoconjugates by transglucosylation reaction onto various exogenous acceptors. Due to the large specificity, in terms of osidic linkages synthesized [alpha (1→2); alpha (1→3); alpha (1→4) or alpha (1→6)] and their organization within the formed products, these biocatalysts can be used to produce carbohydrates of interest in food, health and environment fields. The aim of this research work was to generate, by enzyme engineering, new glucansucrases able to synthesize alpha -glucans and gluco-oligosaccharides with novel structures and properties, to enlarge applications of these molecules. At fundamental level, the work was to improve the understanding of the relationships between structure and specificity of glucansucrases. To reach our objectives, we used a combinatorial engineering strategy on the dextransucrase DSR-S of Leuconostoc mesenteroides NRRL B-512F, which catalyses the synthesis of a dextran formed by 95 % of alpha (1→6) and 5 % alpha (1→3) linkages. The work consisted first in the development of a multi-step high throughput screening methodology to sort out and isolate variants with altered linkage specificities. The strategy includes a first stage of in vivo selection on solid medium followed by an automated 1D 1H NMR-based screening using microplates. To date, this is the first high-throughput screening method for linkage specificity determination of transglucosidases and glycosyltransferases. This strategy was applied to the screening of two DSR-S variants libraries, totalizing more than 36,000 clones obtained by saturation mutagenesis and simultaneous recombination of 8 residues from the catalytic domain. Eighty two mutants able of synthesize 2 to 8 times more alpha (1→3) linkages compared to the parental enzyme were isolated. The characterization of the catalytic properties of 7 representative mutants enabled the identification of a new peptide motif 460DYVHT464, involved in DSR-S specificity. Finally, the characterization of structural, rheological and mechanical properties of dextrans synthetized by these 7 mutants highlighted the differences in size and conformation of these macromolecules in solution and revealed the capacity of the polymer synthesized by H463R/T464V/S512T variant to form biofilms, whose mechanical properties are remarkable in comparison to those of other biopolymers extracted from plants or produced by fermentation.

The Masters thesis deals with dependence of quality parameters of malt on the temperature of mashing at sweet wort preparation. The theoretical part focuses on the technology of malt production including description of ingredients, production parts, some kinds of malt and the quality parameters. There is also described the technology of sweet wort preparation and the methods of determination of the malt quality parameters in the sweet wort there. The experimental part focuses on determination of malt extract, viscosity, haze, colour, time of starch cleavage, pH, degree of fermentation and beta-glucans content. Based on temperature dependence of parameters, the individual samples were compared and the effects of temperature were discussed. The biggest extract of light and caramel malts was explored at 70 °C, of coloured malt at 50 °C. The lowest viscosity of light and coloured malts was explored at 65 °C, of caramel malt at 50 °C. The lowest haze of wort was at 65 °C. Ideal colour of all malts was at all mashing temperatures. Ideal pH of all malts was explored at 70 °C. Ideal time of starch cleavage was explored at 70-85 °C. The biggest achievable degree of fermentation and the hugest increase of beta-glucans content were explored at 65 °C.

Bacterial infections are a common and deadly threat to vulnerable patients. Alternative strategies to fight infection are needed. β-Glucan, an immunomodulator derived from the fungal cell wall, provokes resistance to infection by inducing trained immunity, a phenomenon that persists for weeks to months. Given the durability of trained immunity, it is unclear which leukocyte populations sustain this effect. Macrophages have a life span that surpasses the duration of trained immunity. Thus, we sought to define the contribution of differentiated macrophages to trained immunity. Our results show that β-glucan protects mice from infection by augmenting recruitment of innate leukocytes to the site of infection and facilitating local clearance of bacteria, an effect that persists for more than 7 d. Adoptive transfer of macrophages, trained using β-glucan, into naive mice conferred a comparable level of protection. Trained mouse bone marrow-derived macrophages assumed an antimicrobial phenotype characterized by enhanced phagocytosis and reactive oxygen species production in parallel with sustained enhancements in glycolytic and oxidative metabolism, increased mitochondrial mass, and membrane potential. β-Glucan induced broad transcriptomic changes in macrophages consistent with early activation of the inflammatory response, followed by sustained alterations in transcripts associated with metabolism, cellular differentiation, and antimicrobial function. Trained macrophages constitutively secreted CCL chemokines and robustly produced proinflammatory cytokines and chemokines in response to LPS challenge. Induction of the trained phenotype was independent of the classic β-glucan receptors Dectin-1 and TLR-2. These findings provide evidence that β-glucan induces enhanced protection from infection by driving trained immunity in macrophages.

A aspergilose invasiva (AI) é a infecção por fungos filamentosos mais comum em pacientes imunodeprimidos, especialmente em transplantes de células tronco hematopoiético e neoplasias hematológicas. Objetivo: Geral: Estabelecer a comparação entre a dosagem de Galactomanana (GM), 1,3betaD-glucana (BDG) e dados tomográficos no diagnóstico da AI bem como seu papel no desfecho clínico. Específicos: 1. Verificar a sensibilidade e especificidade dos ensaios de Galactomanana e de 1,3betaD-glucana no soro e lavado broncoalveolar. 2. Comparar os resultados da galatomanana e 1,3betaD-glucana com os dados de imagem em pacientes com suspeita de AI. 3. Verificar a relação entre a evolução dos níveis de GM e desfecho clínico (óbito e sobrevida). Casuística, Materiais e Métodos: Realizou-se um estudo tipo coorte prospectiva, incluindo 398 sujeitos das diversas enfermarias de pacientes imunodeprimidos do HCFMUSP, sendo incluídos dois grupos de pacientes: 202(51%) AI e 198(49%) controles. Resultados: Dos casos, 18 (8,8%) tinham aspergilose provada, 28 (13,7%) provável e 158 possível (77,5%), de acordo a classificação de 2002 EORTC/MSG (European Organization for Research and Treatment of Cancer / Mycoses Study Group). Os sujeitos submetidos ao TCTH eram 42,7%, com neoplasias hematológicas 37%, TOS 9% e outras doenças 11,3%. Os fatores de risco associados ao desenvolvimento da AI foram neutropenia, monocitopenia, uso de corticóide, presença de doença pelo citomegalovírus e rejeição ou doença do enxerto contra o hospedeiro. O fator de risco associado à evolução para o óbito foi a presença de AI. Foram observados bons desempenhos para a GM tanto no soro como no LBA com LR menores que os registrados na literatura. O melhor desempenho da GM no soro para aspergilose+provável ocorreu com LR de 0,35 com sensibilidade-S, especificidade-E, valor preditivo positivo- VPP), valor preditivo negativo-VPN) e área sob a curva-ASC de 54,4%, 73,4%, 50,8%, 76,2% e 0,64, sendo os valores superiores para aspergilose provada tanto na S, como E, VPN. No LBA os valores de S-E-VPP-VPN-ASC para GM para LR de 0,65 para aspergilose provável + provada foram 58,3%, 92,6%, 87,5%,71,4% e 0,75, sendo na aspergilose provada os valores de S, e VPN superiores. Nesta casuística, o melhor desempenho para BDG no soro apontou para uma LR de 100 pg/mL na aspergilose provável+provada, com 54,5%, 73,4%, 50,8% e 76,2%, 0,64 respectivamente para S-E-VPP-VPN-ASC. Para BDG no LBA, a LR na aspergilose provável + provada foi de 140 pg/mL, com os mesmos valores de 46,7%, 76,7%, 70%, 55,6% e 0.62, respectivamente. Conclusão: A GM no LBA e no soro foram úteis no diagnóstico da aspergilose mediante emprego de LR menores, sendo mais sensível na LBA, principalmente em estágios iniciais da forma angioinvasiva. A persistência de GM sérica foi relacionada ao óbito em relação à negativação da mesma. A proporção de concordância entre a TC e os biomarcadores no soro e no LBA variou de 0,5 a 0,6, com pequena concordância na estatística kappa. Excelente concordância foi observada entre dois radiologistas independentes, que analisaram de maneira cega as TC de sujeitos com aspergilose provada. Nesta casuística com inclusão de doenças sistêmicas e endêmicas, a BDG teve baixo desempenho diagnóstico / Invasive aspergillosis (IA) has become the leading infectious cause of death in immunocompromised hosts, particularly in subjects under SCTH and hematologic neoplasias. Objectives: General: To compare the performance of GM and BG tests in serum and bronchoalveolar lavage fluid (BAL) and computer tomography (CT) scans in the diagnosis of IA in immunocompromised hosts as well as their role in the patient outcome. Specific: 1. To analyse the sensitivity and specificity of Galactomannan and 1,3 betaD-glucan assays in the serum and bronchoalveolar lavage. 2. To compare the results of Galactomannan and 1,3betaD-glucan assays with CT scans in patients with invasive aspergilosis. 3. To analyse the relationship between the evolution of galactomannan levels and clinical outcome (death or survival). Patients, Materials and Methods: From December 2008 to March 2013, a prospective cohort of 398 patients from several wards of immunocompromised patients of Hospital das Clínicas, Faculdade de Medicina, University of São Paulo was included classified in two groups of patients: 202 (51%) with invasive aspergillosis (IA) and 198 (49%) control patients. Results: Considering 202 cases, 18(8.8%) were subjects with proven, 28(13.7%) with probable aspergillosis and 156(77.5%), with possible aspergillosis, according to 2002 EORTC/MSG (European Organization for Research and Treatment of Cancer/Mycoses Study Group) criteria. The most common underlying disease were: HSCT (42.7%), hematologic malignancy (37%), SOT (9%), or other diseases (11.3%). The main risk factors associated with IA were neutropenia, monocytopenia, patients under corticosterois, presence of CMV disease, and rejection or graft versus host disease. The risk factor associated with death was the presence of invasive aspergillosis. Good performances for serum and BAL GM were registered with lower cutoffs in the present workin relationship to those found in the literature. The best cutoff for proven + probable aspergillosis for serum GM was observed at 0.35 vallue with Sensitivity-S, Specificity-Sp, Positive Predictive value-PPV), Negative Predictive Value-NPV) and AUC of 54.4%, 73.4%, 50.8%, 76.2% and 0.64; the values for proven aspergillosis alone were higher for S, Sp and NPV. On BAL tests for GM (cutoff value of 0.65) in proven+probable aspergillosis we observed 58.3%, 92.6%, 87.5%,71.4%, 0.75, respectively as S-Sp-PPV-NPVAUC; the sensitivity and VPN were higher in proven aspergillosis alone. In this work, the best performance in proven+probable aspergillosis for serum BDG showed 100 pg/ML as cutoff value, with 54.5%, 73.4%, 50.8%,76.2%, 0.64 for S-Sp-PPVNPV- AUC, respectively. For BAL- BDG, the cut off for proven+probable aspergillosis was 140 pg/mL, and we observed 46.7%, 76.7%, 70.0%, 55.,6%, 0.62, respectively for for S-Sp-PPV-NPV-AUC. Conclusion: The serum and BAL GM are useful tests for diagnosis in early stages of angioinvasive form at lower cutoffs; BAL GM is more sensitive. Agreement proportion between CT scan and each biomarker in the serum or BAL ranged from 0.5-0.6, with low ? index. Perfect ? statistic was observed for analysis of CT scan of subjects in proven aspergillosis by two independent radiologists, blinded for diagnosis. Persistence of serum GM was associated to death in relationship with its negativation. BDG test showed low performance in this work, where systemic and endemic diseases were included

A aspergilose invasiva (AI) é a infecção por fungos filamentosos mais comum em pacientes imunodeprimidos, especialmente em transplantes de células tronco hematopoiético e neoplasias hematológicas. Objetivo: Geral: Estabelecer a comparação entre a dosagem de Galactomanana (GM), 1,3betaD-glucana (BDG) e dados tomográficos no diagnóstico da AI bem como seu papel no desfecho clínico. Específicos: 1. Verificar a sensibilidade e especificidade dos ensaios de Galactomanana e de 1,3betaD-glucana no soro e lavado broncoalveolar. 2. Comparar os resultados da galatomanana e 1,3betaD-glucana com os dados de imagem em pacientes com suspeita de AI. 3. Verificar a relação entre a evolução dos níveis de GM e desfecho clínico (óbito e sobrevida). Casuística, Materiais e Métodos: Realizou-se um estudo tipo coorte prospectiva, incluindo 398 sujeitos das diversas enfermarias de pacientes imunodeprimidos do HCFMUSP, sendo incluídos dois grupos de pacientes: 202(51%) AI e 198(49%) controles. Resultados: Dos casos, 18 (8,8%) tinham aspergilose provada, 28 (13,7%) provável e 158 possível (77,5%), de acordo a classificação de 2002 EORTC/MSG (European Organization for Research and Treatment of Cancer / Mycoses Study Group). Os sujeitos submetidos ao TCTH eram 42,7%, com neoplasias hematológicas 37%, TOS 9% e outras doenças 11,3%. Os fatores de risco associados ao desenvolvimento da AI foram neutropenia, monocitopenia, uso de corticóide, presença de doença pelo citomegalovírus e rejeição ou doença do enxerto contra o hospedeiro. O fator de risco associado à evolução para o óbito foi a presença de AI. Foram observados bons desempenhos para a GM tanto no soro como no LBA com LR menores que os registrados na literatura. O melhor desempenho da GM no soro para aspergilose+provável ocorreu com LR de 0,35 com sensibilidade-S, especificidade-E, valor preditivo positivo- VPP), valor preditivo negativo-VPN) e área sob a curva-ASC de 54,4%, 73,4%, 50,8%, 76,2% e 0,64, sendo os valores superiores para aspergilose provada tanto na S, como E, VPN. No LBA os valores de S-E-VPP-VPN-ASC para GM para LR de 0,65 para aspergilose provável + provada foram 58,3%, 92,6%, 87,5%,71,4% e 0,75, sendo na aspergilose provada os valores de S, e VPN superiores. Nesta casuística, o melhor desempenho para BDG no soro apontou para uma LR de 100 pg/mL na aspergilose provável+provada, com 54,5%, 73,4%, 50,8% e 76,2%, 0,64 respectivamente para S-E-VPP-VPN-ASC. Para BDG no LBA, a LR na aspergilose provável + provada foi de 140 pg/mL, com os mesmos valores de 46,7%, 76,7%, 70%, 55,6% e 0.62, respectivamente. Conclusão: A GM no LBA e no soro foram úteis no diagnóstico da aspergilose mediante emprego de LR menores, sendo mais sensível na LBA, principalmente em estágios iniciais da forma angioinvasiva. A persistência de GM sérica foi relacionada ao óbito em relação à negativação da mesma. A proporção de concordância entre a TC e os biomarcadores no soro e no LBA variou de 0,5 a 0,6, com pequena concordância na estatística kappa. Excelente concordância foi observada entre dois radiologistas independentes, que analisaram de maneira cega as TC de sujeitos com aspergilose provada. Nesta casuística com inclusão de doenças sistêmicas e endêmicas, a BDG teve baixo desempenho diagnóstico / Invasive aspergillosis (IA) has become the leading infectious cause of death in immunocompromised hosts, particularly in subjects under SCTH and hematologic neoplasias. Objectives: General: To compare the performance of GM and BG tests in serum and bronchoalveolar lavage fluid (BAL) and computer tomography (CT) scans in the diagnosis of IA in immunocompromised hosts as well as their role in the patient outcome. Specific: 1. To analyse the sensitivity and specificity of Galactomannan and 1,3 betaD-glucan assays in the serum and bronchoalveolar lavage. 2. To compare the results of Galactomannan and 1,3betaD-glucan assays with CT scans in patients with invasive aspergilosis. 3. To analyse the relationship between the evolution of galactomannan levels and clinical outcome (death or survival). Patients, Materials and Methods: From December 2008 to March 2013, a prospective cohort of 398 patients from several wards of immunocompromised patients of Hospital das Clínicas, Faculdade de Medicina, University of São Paulo was included classified in two groups of patients: 202 (51%) with invasive aspergillosis (IA) and 198 (49%) control patients. Results: Considering 202 cases, 18(8.8%) were subjects with proven, 28(13.7%) with probable aspergillosis and 156(77.5%), with possible aspergillosis, according to 2002 EORTC/MSG (European Organization for Research and Treatment of Cancer/Mycoses Study Group) criteria. The most common underlying disease were: HSCT (42.7%), hematologic malignancy (37%), SOT (9%), or other diseases (11.3%). The main risk factors associated with IA were neutropenia, monocytopenia, patients under corticosterois, presence of CMV disease, and rejection or graft versus host disease. The risk factor associated with death was the presence of invasive aspergillosis. Good performances for serum and BAL GM were registered with lower cutoffs in the present workin relationship to those found in the literature. The best cutoff for proven + probable aspergillosis for serum GM was observed at 0.35 vallue with Sensitivity-S, Specificity-Sp, Positive Predictive value-PPV), Negative Predictive Value-NPV) and AUC of 54.4%, 73.4%, 50.8%, 76.2% and 0.64; the values for proven aspergillosis alone were higher for S, Sp and NPV. On BAL tests for GM (cutoff value of 0.65) in proven+probable aspergillosis we observed 58.3%, 92.6%, 87.5%,71.4%, 0.75, respectively as S-Sp-PPV-NPVAUC; the sensitivity and VPN were higher in proven aspergillosis alone. In this work, the best performance in proven+probable aspergillosis for serum BDG showed 100 pg/ML as cutoff value, with 54.5%, 73.4%, 50.8%,76.2%, 0.64 for S-Sp-PPVNPV- AUC, respectively. For BAL- BDG, the cut off for proven+probable aspergillosis was 140 pg/mL, and we observed 46.7%, 76.7%, 70.0%, 55.,6%, 0.62, respectively for for S-Sp-PPV-NPV-AUC. Conclusion: The serum and BAL GM are useful tests for diagnosis in early stages of angioinvasive form at lower cutoffs; BAL GM is more sensitive. Agreement proportion between CT scan and each biomarker in the serum or BAL ranged from 0.5-0.6, with low ? index. Perfect ? statistic was observed for analysis of CT scan of subjects in proven aspergillosis by two independent radiologists, blinded for diagnosis. Persistence of serum GM was associated to death in relationship with its negativation. BDG test showed low performance in this work, where systemic and endemic diseases were included

Indiana University-Purdue University Indianapolis (IUPUI) / Recent advancements in the management of systemic glucose regulation in obesity/T2DM include drug therapies designed to utilize components of the incretin system specifically related to glucagon-like peptide 1 (GLP-1). More recently, GLP-1 has been investigated for potential cardioprotective effects. Several investigations have revealed that acute/sub-acute intravenous administration of GLP-1 significantly reduces myocardial infarct size following ischemia/reperfusion injury and improves cardiac contractile function in the settings of coronary artery disease, myocardial ischemia/reperfusion injury, and heart failure. Despite an abundance of data indicating that intravenous infusion of GLP-1 is cardioprotective, information has been lacking on the cardiac effects of iv GLP-1 in the MetS or T2DM population. Some important questions this study aimed to address are 1) what are the direct, dose-dependent cardiac effects of GLP-1 in-vivo 2) are the cardiac effects influenced by cardiac demand (MVO2) and/or ischemia, 3) does GLP-1 effect myocardial blood flow, glucose uptake or total oxidative metabolism in human subjects, and 4) are the cardiac effects of GLP-1 treatment impaired in the settings of obesity/MetS and T2DM. Initial studies conducted in canines demonstrated that GLP-1 had no direct effect on coronary blood flow in-vivo or vasomotor tone in-vitro, but preferentially increased myocardial glucose uptake in ischemic myocardium independent of effects on cardiac contractile function or coronary blood flow. Parallel translational studies conducted in the humans and Ossabaw swine demonstrate that iv GLP-1 significantly increases myocardial glucose uptake at rest and in response to increases in cardiac demand (MVO2) in lean subjects, but not in the settings of obesity/MetS and T2DM. Further investigation in isolated cardiac tissue from lean and obese/MetS swine indicate that this impairment in GLP-1 responsiveness is related to attenuated activation of p38-MAPK, independent of alterations in GLP-1 receptor expression or PKA-dependent signaling. Our results indicate that the affects of GLP-1 to reduce cardiac damage and increase left ventricular performance may be impaired by obesity/MetS and T2DM.