Characterization of CD4 and CD8 T cell responses in MuSK myasthenia gravis.

Muscle specific tyrosine kinase myasthenia gravis (MuSK MG) is a form of autoimmune MG that predominantly affects women and has unique clinical features, including prominent bulbar weakness, muscle atrophy, and excellent response to therapeutic plasma exchange. Patients with MuSK MG have predominantly IgG4 autoantibodies directed against MuSK on the postsynaptic muscle membrane. Lymphocyte functionality has not been reported in this condition. The goal of this study was to characterize T cell responses in patients with MuSK MG. Intracellular production of IFN-gamma, TNF-alpha, IL-2, IL-17, and IL-21 by CD4+ and CD8+ T cells was measured by polychromatic flow cytometry in peripheral blood samples from 11 Musk MG patients and 10 healthy controls. Only one MuSK MG patient was not receiving immunosuppressive therapy. Regulatory T cells (Treg) were also included in our analysis to determine if changes in T cell function were due to altered Treg frequencies. CD8+ T cells from MuSK MG patients had higher frequencies of polyfunctional responses than controls, and CD4+ T cells had higher IL-2, TNF-alpha, and IL-17. MuSK MG patients had a higher percentage of CD4+ T cells producing combinations of IFN-gamma/IL-2/TNF-gamma, TNF-alpha/IL-2, and IFN-gamma/TNF-alpha. Interestingly, Treg numbers and CD39 expression were not different from control values. MuSK MG patients had increased frequencies of Th1 and Th17 cytokines and were primed for polyfunctional proinflammatory responses that cannot be explained by a defect in CD39 expression or Treg number.

Drug Delivery through the Blood-Brain Barrier through Use of Vascular Cell Adhesion Molecule 1- Targeted Nanoparticles

The blood brain barrier (BBB) is a semi-permeable membrane separating the brain from the bloodstream, preventing many drugs that treat neurological diseases, such as Alzheimer’s and Parkinson’s, from reaching the brain. Our project aimed to create a novel drug delivery system targeting the brain during neural inflammation. We developed a cationic solid lipid nanoparticle (CSLN) complex composed of cationic nanoparticles, biotin, streptavidin, and anti-vascular cell adhesion molecule-1 (anti- VCAM-1) antibodies. The anti-VCAM-1 antibody is used to target VCAM-1, a cell adhesion protein found on the BBB endothelium. VCAM-1 expression is elevated in the presence of inflammatory molecules, such as tumor necrosis factor-alpha (TNF- α). Through the use of a simple BBB model, results showed that our novel drug delivery system experienced some level of success in targeting the brain inflammation due to increasing TNF-α concentrations. This is promising for drug delivery research and provides support for VCAM-1 targeting using more robust and complex BBB models.

Human Vascular Microphysiological System for in vitro Drug Screening.

In vitro human tissue engineered human blood vessels (TEBV) that exhibit vasoactivity can be used to test human toxicity of pharmaceutical drug candidates prior to pre-clinical animal studies. TEBVs with 400-800 μM diameters were made by embedding human neonatal dermal fibroblasts or human bone marrow-derived mesenchymal stem cells in dense collagen gel. TEBVs were mechanically strong enough to allow endothelialization and perfusion at physiological shear stresses within 3 hours after fabrication. After 1 week of perfusion, TEBVs exhibited endothelial release of nitric oxide, phenylephrine-induced vasoconstriction, and acetylcholine-induced vasodilation, all of which were maintained up to 5 weeks in culture. Vasodilation was blocked with the addition of the nitric oxide synthase inhibitor L-N(G)-Nitroarginine methyl ester (L-NAME). TEBVs elicited reversible activation to acute inflammatory stimulation by TNF-α which had a transient effect upon acetylcholine-induced relaxation, and exhibited dose-dependent vasodilation in response to caffeine and theophylline. Treatment of TEBVs with 1 μM lovastatin for three days prior to addition of Tumor necrosis factor - α (TNF-α) blocked the injury response and maintained vasodilation. These results indicate the potential to develop a rapidly-producible, endothelialized TEBV for microphysiological systems capable of producing physiological responses to both pharmaceutical and immunological stimuli.

A novel inflammatory biomarker, GlycA, associates with disease activity in rheumatoid arthritis and cardio-metabolic risk in BMI-matched controls

BACKGROUND: RA and CVD both have inflammation as part of the underlying biology. Our objective was to explore the relationships of GlycA, a measure of glycosylated acute phase proteins, with inflammation and cardiometabolic risk in RA, and explore whether these relationships were similar to those for persons without RA. METHODS: Plasma GlycA was determined for 50 individuals with mild-moderate RA disease activity and 39 controls matched for age, gender, and body mass index (BMI). Regression analyses were performed to assess relationships between GlycA and important markers of traditional inflammation and cardio-metabolic health: inflammatory cytokines, disease activity, measures of adiposity and insulin resistance. RESULTS: On average, RA activity was low (DAS-28 = 3.0 ± 1.4). Traditional inflammatory markers, ESR, hsCRP, IL-1β, IL-6, IL-18 and TNF-α were greater in RA versus controls (P < 0.05 for all). GlycA concentrations were significantly elevated in RA versus controls (P = 0.036). In RA, greater GlycA associated with disease activity (DAS-28; RDAS-28 = 0.5) and inflammation (RESR = 0.7, RhsCRP = 0.7, RIL-6 = 0.3: P < 0.05 for all); in BMI-matched controls, these inflammatory associations were absent or weaker (hsCRP), but GlycA was related to IL-18 (RhsCRP = 0.3, RIL-18 = 0.4: P < 0.05). In RA, greater GlycA associated with more total abdominal adiposity and less muscle density (Rabdominal-adiposity = 0.3, Rmuscle-density = -0.3, P < 0.05 for both). In BMI-matched controls, GlycA associated with more cardio-metabolic markers: BMI, waist circumference, adiposity measures and insulin resistance (R = 0.3-0.6, P < 0.05 for all). CONCLUSIONS: GlycA provides an integrated measure of inflammation with contributions from traditional inflammatory markers and cardio-metabolic sources, dominated by inflammatory markers in persons with RA and cardio-metabolic factors in those without.

Competing Actions of Type 1 Angiotensin II Receptors Expressed on T Lymphocytes and Kidney Epithelium during Cisplatin-Induced AKI.

Inappropriate activation of the renin-angiotensin system (RAS) contributes to many CKDs. However, the role of the RAS in modulating AKI requires elucidation, particularly because stimulating type 1 angiotensin II (AT1) receptors in the kidney or circulating inflammatory cells can have opposing effects on the generation of inflammatory mediators that underpin the pathogenesis of AKI. For example, TNF-α is a fundamental driver of cisplatin nephrotoxicity, and generation of TNF-α is suppressed or enhanced by AT1 receptor signaling in T lymphocytes or the distal nephron, respectively. In this study, cell tracking experiments with CD4-Cre mT/mG reporter mice revealed robust infiltration of T lymphocytes into the kidney after cisplatin injection. Notably, knockout of AT1 receptors on T lymphocytes exacerbated the severity of cisplatin-induced AKI and enhanced the cisplatin-induced increase in TNF-α levels locally within the kidney and in the systemic circulation. In contrast, knockout of AT1 receptors on kidney epithelial cells ameliorated the severity of AKI and suppressed local and systemic TNF-α production induced by cisplatin. Finally, disrupting TNF-α production specifically within the renal tubular epithelium attenuated the AKI and the increase in circulating TNF-α levels induced by cisplatin. These results illustrate discrepant tissue-specific effects of RAS stimulation on cisplatin nephrotoxicity and raise the concern that inflammatory mediators produced by renal parenchymal cells may influence the function of remote organs by altering systemic cytokine levels. Our findings suggest selective inhibition of AT1 receptors within the nephron as a promising intervention for protecting patients from cisplatin-induced nephrotoxicity.

Identification of pertussis-specific effector memory T cells in preschool children

Whooping cough remains a problem despite vaccination, and worldwide resurgence of pertussis is evident. Since cellular immunity plays a role in long-term protection against pertussis, we studied pertussis-specific T-cell responses. Around the time of the preschool acellular pertussis (aP) booster dose at 4 years of age, T-cell memory responses were compared in children who were primed during infancy with either a whole-cell pertussis (wP) or an aP vaccine. Peripheral blood mononuclear cells (PBMCs) were isolated and stimulated with pertussis vaccine antigens for 5 days. T cells were characterized by flow-based analysis of carboxyfluorescein succinimidyl ester (CFSE) dilution and CD4, CD3, CD45RA, CCR7, gamma interferon (IFN-γ), and tumor necrosis factor alpha (TNF-α) expression. Before the aP preschool booster vaccination, both the proliferated pertussis toxin (PT)-specific CD4⁺ and CD8⁺ T-cell fractions (CFSE^dim) were higher in aP-than in wP-primed children. Post-booster vaccination, more pertussis-specific CD4⁺ effector memory cells (CD45RA^- CCR7^-) were induced in aP-primed children than in those primed with wP. The booster vaccination did not appear to significantly affect the T-cell memory subsets and functionality in aP-primed or wP-primed children. Although the percentages of Th1 cytokine-producing cells were alike in aP- and wP-primed children pre-booster vaccination, aP-primed children produced more Th1 cytokines due to higher numbers of proliferated pertussis-specific effector memory cells. At present, infant vaccinations with four aP vaccines in the first year of life result in pertussis-specific CD4⁺ and CD8⁺ effector memory T-cell responses that persist in children until 4 years of age and are higher than those in wP-primed children. The booster at 4 years of age is therefore questionable; this may be postponed to 6 years of age.

Immunological Signatures Identifying Different Stages of Latent Mycobacterium tuberculosis Infection and Discriminating Latent from Active Tuberculosis in Humans

Objectives: One third of the world population is considered latently infected with Mycobacterium tuberculosis(LTBI) and sterilizing this reservoir of bacteria that may reactivate is required for tuberculosis (TB) elimination. Thegroup of individuals with LTBI is heterogeneous with some of them being more at risk to develop TB disease thanothers. Improved diagnosis of subjects with LTBI is needed, allowing to differentiate subjects with LTBI from thosewith active TB, and to select among LTBI subjects those who are more at risk to develop active TB. We havecharacterized at the cellular level both the quantitative and qualitative T cell responses to different mycobacterialantigens in selected populations of infected subjects in order to identify new biomarkers that could help to identify M.tuberculosis-infected subjects and to stratify them in risk groups for reactivation of the infection.Methods: Lymphoblast frequencies and cytokine production (IFN-γ, TNF-α, IL-2) among CD4+ and CD8+ T cellswere analyzed by flow cytometry after in vitro stimulation with the latency antigen heparin-binding haemagglutinin(HBHA) or early-secreted antigen Target-6 (ESAT-6) of peripheral blood mononuclear cells from clinically wellcharacterized M. tuberculosis-infected humans (28 LTBI, 22 TB disease,12 controls). The LTBI group definedaccording to the Center for Disease Control guidelines was subdivided into QuantiFERON-TB Gold in-Tube (QFT)positive and negative subgroups.Results: Similar to TB patients, QFT+ LTBI subjects had higher proportions of HBHA-induced TNF-αsingle+ CD4+lymphocytes than QFT- LTBI subjects (p<0.05). Compared to LTBI subjects, TB patients had higher frequencies ofESAT-6-induced CD8+ lymphoblasts (p<0.001), higher proportions of ESAT-6-induced IFN-γ+TNF-α+ CD4+ Tlymphocytes (p<0.05), and lower proportions of HBHA-induced IFN-γ+TNF-α+IL-2+ (p<0.05) CD4+ T lymphocytes.Conclusions: These data provide new biomarkers to discriminate active TB from LTBI, and more interestingly,help to identify LTBI subjects with increased likelihood to develop TB disease.

Promedio bayesiano de modelos para la predicción del consumo máximo de oxígeno en deportistas con datos de no-ejercicio

Históricamente, los modelos de no-ejercicio para predecir el consumo máximo de oxígeno (VO2max) han sido construidos mediante regresión lineal frecuentista, usando técnicas estándar de selección de modelos. Sin embargo, existe incertidumbre acerca de la estructura estadística en el proceso de selección del modelo. En este estudio se propuso construir un modelo de no-ejercicio para predecir el VO2max en deportistas orientados al rendimiento, considerando la incertidumbre de modelo a través del Promedio Bayesiano de Modelos (BMA). Un objetivo adicional fue comparar la performance predictiva del BMA con las de los modelos derivados de varias técnicas frecuentistas usuales de selección de variables. Con tal fin, se implementó un submuestreo aleatorio estratificado repetido. Los datos incluyeron observaciones de la variable respuesta (en L·min-1), así como registros de Género, Deporte, Edad, Peso, Talla e Índice de masa corporal (BMI) (Edad = 22.1 ± 4.9 años, media ± SD; n = 272). Se propuso una clasificación de deportes con el objetivo de incluirla dentro del proceso de construcción del modelo: Combate, Juego, Resistencia 1 y Resistencia 2. El enfoque BMA se implementó en base a dos métodos: Occam's window y Composición de Modelo mediante el método de Monte Carlo con Cadenas de Markov (MC²). Se observaron discrepancias en la selección de variables entre los procedimientos frecuentistas. Ambos métodos de BMA produjeron resultados muy similares. Los modelos que incluyeron Género y las variables dummies para Resistencia 1 y Resistencia 2 acumularon virtualmente toda la probabilidad de modelo a posteriori. El Peso fue el predictor continuo con la más alta probabilidad de inclusión a posteriori (menor a 0.8). Las combinaciones de variables que involucraron predictores con un alto nivel de multicolinealidad fueron desacreditadas. Los modelos con sustancial contribución para el BMA presentaron un ajuste apreciable (R² ajustado menor a 0.8). Entre los modelos seleccionados por estrategias frecuentistas, el obtenido mediante el método de regresión por pasos (Stepwise regression method) con alfa igual a 0.05 fue el más respaldado por los datos, en términos de probabilidad de modelo a posteriori. En concordancia con la literatura, el BMA tuvo mejor performance predictiva de los datos fuera de la muestra que los modelos seleccionados por técnicas frecuentistas, medida por la cobertura del intervalo de predicción de 90 por ciento. La clasificación de deportes reveló resultados consistentes.

Caracterización ecofisiológica de líneas elite de trigo : identificación de atributos vinculados con el rendimiento potencial

Las actuales tasas de progreso en rendimiento de trigo son inferiores a las requeridas para satisfacer la demanda de alimentos proyectada. Hasta la fecha las mejoras del rendimiento potencial han sido basadas en la selección por rendimiento per se, sin embargo hay evidencias de que criterios de selección indirecta basados en atributos fisiológicos asociados al rendimiento podrían acelerar el progreso de mejoramiento. Para ello, es necesario identificar estos atributos y comprender su base genética a fin de facilitar su incorporación en nuevos genotipos. En este sentido, una evaluación fenotípica correcta y detallada resulta ser el factor limitante a la hora de incrementar nuestro conocimiento sobre los atributos fisiológicos que determinan el rendimiento potencial. El objetivo general del trabajo fue realizar la fenotipificación de un set de líneas elite de trigo primaveral (denominadas CIMCOG) proveniente de CIMMYT, de modo de caracterizar la variabilidad genotípica e identificar atributos fisiológicos asociados con el rendimiento y sus componentes numéricos y fisiológicos. El set de líneas CIMCOG se evaluó a campo, sin limitaciones hídrico-nutricionales, ni bióticas, bajo un diseño alfa lattice con 2 repeticiones. El set de líneas presentó baja variabilidad en el tiempo a antesis debido a que se encontraron en el rango de 54-58 días con una diferencia (aproximadamente 4 días), demostrando que es un set adecuado para identificar atributos favorables para aumentar el rendimiento. La biomasa fue el principal componente fisiológico que explicó los cambios en el rendimiento y estuvo asociada a cambios en la eficiencia de uso de la radiación (EUR), mientras que la variación en el índice de cosecha fue sustancialmente menor que la observada en biomasa. El número de granos por unidad de área fue el principal componente que explicó las variaciones en el rendimiento, siendo el número de espigas por unidad de área su principal determinante. Teniendo en cuenta este set de genotipos, futuros progresos en el rendimiento de trigo podrían estar asociados a mejoras en la EUR y a un mayor establecimiento de espigas, lo cual requiere una mayor comprensión de aspectos

Fisiología del fruto de kiwi (Actinidia deliciosa) durante su desarrollo y ablandamiento : expresión de genes asociados y su modulación por etileno y 1-metilciclopropeno

A fin de investigar el efecto fisiológico del etileno y del 1-metilciclopropeno en el progreso del ablandamiento del kiwi, se trató los frutos con esos reguladores inmediatamente después de la cosecha, o con 1-metilciclopropeno en diferentes estadios de la maduración después de 40, 80 o 120 d de almacenamiento refrigerado (0ºC). El tratamiento con etileno inmediatamente después de la cosecha estimuló el ablandamiento de la pulpa, incrementó y adelantó el pico de producción de etileno y la expresión de los genes KWACS1 y KWACO1 involucrados en la biosíntesis del etileno. En cambio, el tratamiento con 1-metilciclopropeno en el mismo estadio retrasó marcadamente el ablandamiento e inhibió la producción de etileno. Los incrementos en la abundancia de transcriptos de KWACS1 y KWACO1 fueron bloqueados por el tratamiento con 1-metilciclopropeno, indicando que estos genes son regulados positivamente por el etileno. Los kiwis almacenados en frío (0 ºC) por 40, 80 o 120 d y luego tratados con 1-metilciclopropeno antes de su retorno a 20 ºC para su maduración ulterior mostraron una tasa reducida de ablandamiento de la pulpa y un estadio de madurez de consumo extendido. Estos resultados indican claramente que la aplicación de 1-metilciclopropeno puede jugar un papel significativo en el inicio y en el progreso del ablandamiento del kiwi. El 1-metilciclopropeno inhibió o restringió severamente la producción autocatalítica de etileno en cualquier estado de maduración. La transcripción de los genes KWACS1 y KWACO1 resulto inhibida por el tratamiento con 1-metilciclopropeno después de 40 y 80 d de almacenamiento en frío, sugiriendo que existe una regulación por retroalimentación positiva para la producción de etileno, incluso después del almacenamiento refrigerado. Para investigar los niveles de expresión de genes relacionados con la pared celular durante la ontogenia del kiwi y en respuesta a la aplicación de etileno y de 1-metilciclopropeno, se obtuvo una secuencia completa de cDNA a la cual se denominó AdGAL1, determinándose por análisis bioinformático que es un homólogo de ß-D-galactosidasa de kiwi. El producto deducido de la traducción de AdGAL1 consta de 728 aminoacidos de longitud mientras que laproteina madura posee una masa molecular predicha de 81,12 kDa y un pI teorico de 7,5. Se efectuaron reacciones de RT-PCR semicuantitativas para evaluar la expresión de AdGAL1 y de una serie de secuencias de ADN. Los transcriptos que hibridizan con AdGAL1 resultaron apenas detectables durante el crecimiento del fruto pero se observaron tanto en mesocarpo externo como en columela al comienzo del ablandamiento del fruto (Fase IV, Estadio 1), y durante el ablandamiento tardío (Estadio 3) sugiriendo su injerencia en las grandes pérdidas de galactosa de la pared celular durante el ablandamiento del kiwi. La abundancia de transcriptos que hibridizan con AdARF1 y AdARF/XYL (codificantes de alfa-L-arabinofuranosidasa y de alfa-L-arabinofuranosidasa/ß-D-xilosidasa putativas) permaneció relativamente constante a traves de todo el crecimiento y la maduración(...)

Control de avena negra (Avena fatua L.) con aplicación de trifluralina en cultivos de cebada cervecera (Hordeum vulgare L.)

p.149-156

Teorema de Bayes y lenguaje matemático Maple. Estimación de la función de densidad posterior

El objetivo de este trabajo es explicar el uso del teorema de Bayes en la estimación de la función de densidad posterior (fdp) de parámetros de interés, usando el software matemático Maple. Se presenta el caso de la distribución de Pareto como una aproximación a la distribución de los ingresos de una población. Se estima la fdp del parámetro alfa de la distribución de Pareto para el caso de datos agrupados.

Polyunsaturated fatty acids in the pathogenesis and treatment of multiple sclerosis

Epidemiological, biochemical, animal model and clinical trial data described in this overview strongly suggest that polyunsaturated fatty acids, particularly n-6 fatty acids, have a role in the pathogenesis and treatment of multiple sclerosis (MS). Data presented provides further evidence for a disturbance in n-6 fatty acid metabolism in MS. Disturbance of n-6 fatty acid metabolism and dysregulation of cytokines are shown to be linked and a "proof of concept clinical trial" further supports such a hypothesis. In a randomised double-blind, placebo controlled trial of a high dose and low dose selected GLA (18:3n-6)-rich oil and placebo control, the high dose had a marked clinical effect in relapsing-remitting MS, significantly decreasing the relapse rate and the progression of disease. Laboratory findings paralleled clinical changes in the placebo group in that production of mononuclear cell pro-inflammatory cytokines (TNF-alpha, IL-1 beta) was increased and anti-inflammatory TGF-beta markedly decreased with loss of membrane n-6 fatty acids linoleic (18:2n-6) and arachidonic acids (20:4n-6). In contrast there were no such changes in the high dose group. The improvement in disability (Expanded Disability Status Scale) in the high dose suggests there maybe a beneficial effect on neuronal lipids and neural function in MS. Thus disturbed n-6 fatty acid metabolism in MS gives rise to loss of membrane long chain n-6 fatty acids and loss of the anti-inflammatory regulatory cytokine TGF-beta, particularly during the relapse phase, as well as loss of these important neural fatty acids for CNS structure and function and consequent long term neurological deficit in MS.

TRANSIENT LIPOPOLYSACCHARIDE-INDUCED CYTOKINE RESPONSES IN THE MATERNAL AND FETAL GUINEA PIG

The aim of this study was to further investigate the role of pro-inflammatory cytokines in the pathogenesis of fetal cererbral white matter injury associated with chorioamnionitis by charaterizing the time course of the cytokine response in the pregnant guinea pig following a maternal inflammatory insult. Chorioamnionitis increases the risk for fetal brain injury. In the guinea pig, a threshold maternal inflammatory response must be reached for significant fetal brain injury to occur. However, a previous study demonstrated that, by seven days after an acute maternal inflammatory insult, cytokine levels in both maternal and fetal compartments are not different from controls. The purpose of this study, therefore, was to test the hypothesis that a significant cytokine response occurs within the first seven days following an acute maternal inflammatory response. Pregnant guinea pigs (n=34) were injected intraperitoneally with 100µg/kg lipopolysaccharide (LPS) at 70% gestation and euthanized at 24 hours, 48 hours or 5 days following endotoxin exposure. Control animals were euthanized at 70% gestation without exposure. Concentrations of interleukin-6, interleukin 1-β and tumour necrosis factor-α (IL-6, IL-1β, TNF-α) were quantified in the maternal serum and amniotic fluid by enzyme-linked immunosorbent assay. IL-6 and IL-1β concentrations were elevated in the maternal serum at 24 hours and returned to control levels by five days. In the amniotic fluid, IL-6 peaked at 48 hours and IL-1β at 24 hours. TNF-α levels were not significantly increased. A single maternal LPS injection produces transient increases in cytokine concentrations in the maternal serum and amniotic fluid. This further implicates the cytokines as potential mediators of fetal white matter damage. Although this response might not be sufficient to produce the brain injury itself, it may initiate harmful pro-inflammatory cytokine cascades, which could even continue to harm the fetus following delivery. A human diagnostic protocol was developed to assess the use of serial serum biomarkers, including IL-6 and TNF-α, in the prediction of histological chorioamnionitis. Preliminary analysis of the pilot study suggests that certain biomarkers might be worthy of further investigation in a larger-scale study.

Enhanced inflammatory responses in alpha-tocopherol transfer protein null mice

The liver preferentially secretes alpha-tocopherol into plasma under the control of the hepatic alpha-tocopherol transfer protein (alpha-TTP). alpha-TTP-null mice (Ttpa(-/-) mice) are vitamin E deficient, therefore were used for investigations of in vivo responses to sub-normal tissue alpha-tocopherol concentrations during inflammation. Increased basal oxidative stress in Ttpa(-/-) mice was documented by increased plasma lipid peroxidation, and superoxide production by bone marrow-derived neutrophils stimulated in vitro with phorbol 12-myristate 13-acetate. Lipopolysaccharide (LPS) injected intraperitoneally induced increases in lung and liver HO-1 and iNOS, as well as plasma NO(x) in Ttpa(+/+) mice. LPS induced more modest increases in these markers in Ttpa(-/-) mice, while more marked increases in plasma IL-10 and lung lavage TNF alpha were observed. Taken together, these results demonstrate that alpha-tocopherol is important for proper modulation of inflammatory responses and that sub-optimal alpha-tocopherol concentrations may derange inflammatory-immune responses.