Decreased Expression of EC-SOD and Fibulin-5 in Alveolar Walls of Lungs From COPD Patients

Introduction: The aim of this study is to analyze the expression of the main oxidant scavenger superoxide dismutase (EC-SOD), its main binding protein Fibulin-5 and several oxidative and nitrosative-derived products in the lung of COPD patients and controls.Materials and methods: Lung tissue samples from 19 COPD patients and 20 control subjects were analyzed. The architecture of elastic fibres was assessed by light and electron microscope histochemical techniques, and levels of EC-SOD and fibulin-5 were analyzed by immunohistochemistry and RT-PCR. The impact of oxidative stress on the extracellular matrix was estimated by immunolocalization of 4-hydroxynonenal (4-HNE), malondialdehyde (MDA) and 3-nitrotyrosine (3-NYT) adducts.Results: Alveolar walls of COPD patients exhibited abnormal accumulations of collapsing elastic fibres, showing a pierced pattern in the amorphous component. The semiquantitative analysis revealed that COPD patients have a significantly reduced expression of both EC-SOD and fibulin-5 (0.59±0.64 and 0.62±0.61, respectively) in alveolar, bronchiolar and arteriolar walls compared to control subjects (1.39±0.63 and 1.55±0.52, respectively, p<0.05). No significant changes in mRNA levels of these proteins were observed between groups. Among the oxidation markers, malondialdehyde was the best in distinguishing COPD patients.Conclusions: COPD patients show a reduced expression of EC-SOD and fibulin-5 in the lung interstitium. Paralleling the reduction of EC-SOD levels, the decrease of fibulin-5 expression in COPD lungs supports the hypothesis of an impaired pulmonary antioxidant response in COPD patients. (AU)

Over-Mutated Mitochondrial, Lysosomal and TFEB-Regulated Genes in Parkinson's Disease.

The association between Parkinson's disease (PD) and mutations in genes involved in lysosomal and mitochondrial function has been previously reported. However, little is known about the involvement of other genes or cellular mechanisms. We aim to identify novel genetic associations to better understand the pathogenesis of PD. We performed WES in a cohort of 32 PD patients and 30 age-matched controls. We searched for rare variants in 1667 genes: PD-associated, related to lysosomal function and mitochondrial function and TFEB-regulated. When comparing the PD patient cohort with that of age matched controls, a statistically significant burden of rare variants in the previous group of genes were identified. In addition, the Z-score calculation, using the European population database (GnomAD), showed an over-representation of particular variants in 36 genes. Interestingly, 11 of these genes are implicated in mitochondrial function and 18 are TFEB-regulated genes. Our results suggest, for the first time, an involvement of TFEB-regulated genes in the genetic susceptibility to PD. This is remarkable as TFEB factor has been reported to be sequestered inside Lewy bodies, pointing to a role of TFEB in the pathogenesis of PD. Our data also reinforce the involvement of lysosomal and mitochondrial mechanisms in PD.

Decreased Expression of EC-SOD and Fibulin-5 in Alveolar Walls of Lungs From COPD Patients.

INTRODUCTION: The aim of this study is to analyze the expression of the main oxidant scavenger superoxide dismutase (EC-SOD), its main binding protein Fibulin-5 and several oxidative and nitrosative-derived products in the lung of COPD patients and controls. MATERIALS AND METHODS: Lung tissue samples from 19 COPD patients and 20 control subjects were analyzed. The architecture of elastic fibres was assessed by light and electron microscope histochemical techniques, and levels of EC-SOD and fibulin-5 were analyzed by immunohistochemistry and RT-PCR. The impact of oxidative stress on the extracellular matrix was estimated by immunolocalization of 4-hydroxynonenal (4-HNE), malondialdehyde (MDA) and 3-nitrotyrosine (3-NYT) adducts. RESULTS: Alveolar walls of COPD patients exhibited abnormal accumulations of collapsing elastic fibres, showing a pierced pattern in the amorphous component. The semiquantitative analysis revealed that COPD patients have a significantly reduced expression of both EC-SOD and fibulin-5 (0.59±0.64 and 0.62±0.61, respectively) in alveolar, bronchiolar and arteriolar walls compared to control subjects (1.39±0.63 and 1.55±0.52, respectively, p<0.05). No significant changes in mRNA levels of these proteins were observed between groups. Among the oxidation markers, malondialdehyde was the best in distinguishing COPD patients. CONCLUSIONS: COPD patients show a reduced expression of EC-SOD and fibulin-5 in the lung interstitium. Paralleling the reduction of EC-SOD levels, the decrease of fibulin-5 expression in COPD lungs supports the hypothesis of an impaired pulmonary antioxidant response in COPD patients.

Decreased Expression of EC-SOD and Fibulin-5 in Alveolar Walls of Lungs From COPD Patients.

INTRODUCTION: The aim of this study is to analyze the expression of the main oxidant scavenger superoxide dismutase (EC-SOD), its main binding protein Fibulin-5 and several oxidative and nitrosative-derived products in the lung of COPD patients and controls. MATERIALS AND METHODS: Lung tissue samples from 19 COPD patients and 20 control subjects were analyzed. The architecture of elastic fibres was assessed by light and electron microscope histochemical techniques, and levels of EC-SOD and fibulin-5 were analyzed by immunohistochemistry and RT-PCR. The impact of oxidative stress on the extracellular matrix was estimated by immunolocalization of 4-hydroxynonenal (4-HNE), malondialdehyde (MDA) and 3-nitrotyrosine (3-NYT) adducts. RESULTS: Alveolar walls of COPD patients exhibited abnormal accumulations of collapsing elastic fibres, showing a pierced pattern in the amorphous component. The semiquantitative analysis revealed that COPD patients have a significantly reduced expression of both EC-SOD and fibulin-5 (0.59±0.64 and 0.62±0.61, respectively) in alveolar, bronchiolar and arteriolar walls compared to control subjects (1.39±0.63 and 1.55±0.52, respectively, p<0.05). No significant changes in mRNA levels of these proteins were observed between groups. Among the oxidation markers, malondialdehyde was the best in distinguishing COPD patients. CONCLUSIONS: COPD patients show a reduced expression of EC-SOD and fibulin-5 in the lung interstitium. Paralleling the reduction of EC-SOD levels, the decrease of fibulin-5 expression in COPD lungs supports the hypothesis of an impaired pulmonary antioxidant response in COPD patients.

Correction: Deficient pulmonary IFN-ß expression in COPD patients.

[This corrects the article DOI: 10.1371/journal.pone.0217803.].

Deficient pulmonary IFN-ß expression in COPD patients.

COPD patients are prone to acute infectious exacerbations that impair their quality of life and hamper prognosis. The purpose of the present study was to investigate the in situ IFN-ß response in the lungs of stable COPD and non-COPD patients. Lung samples from 70 subjects (9 control never smokers, 19 control smokers without COPD, 21 patients with moderate COPD and 21 patients with very severe COPD) were studied for the expression of IFN-ß, its main transcription factor, IRF-7, and two products of its autocrine function, namely RIG-I and MDA-5, by immunohistochemical techniques and quantitative real-time PCR. IFN-ß, IRF-7, RIG-I and MDA-5 were widely detected in most lung cell types. In epithelial tissues and alveolar macrophages, IFN-ß and IRF-7 labeling scores were decreased up to 65% and 74%, respectively, for COPD patients, paralleling an analogous reduction (43% and 65%, respectively) in the amount of their lung mRNA. Moreover, this decreased production of IFN-ß in COPD patients correlated with a similar decrease in the expression of RIG-I and MDA-5, two essential members of the innate immune system. Our study indicates that most lung cells from stable COPD patients show a constitutive decreased expression of IFN-ß, IRF-7, RIG-I and MDA-5, suggesting that this deficiency is the main cause of their acute viral exacerbations.

¿Influye el consumo de comidas ricas en proteínas y grasas en la glucemia postprandial de pacientes con diabetes tipo 1 que realizan conteo de hidratos de carbono? / Does the consumption of meals rich in proteins and fats influence on the postprandial glucemia of patients with diabetes type 1 that follow carbohydrate counting?

Introducción: el tratamiento de la diabetes tipo 1 (DM1) requiere de la administración de insulina exógena; dentro de las variables a tener en cuenta para calcular la dosis se encuentra el contenido de hidratos de carbono (HC) de la comida a ingerir. Este macronutriente es considerado, desde hace varios años, el responsable del aumento de la glucemia postprandial (GPP). El conteo de hidratos de carbono (CHC) es el método más aceptado y utilizado actualmente en el tratamiento nutricional, aunque cada vez existe más evidencia de que hay otros macronutrientes, como las proteínas y las grasas, que pueden influir en la variación de la GPP. Objetivo: el objetivo de esta revisión bibliográfica es reunir los resultados de publicaciones científicas que analizaron la respuesta glucémica (RG) al consumo de comidas con alto contenido de proteínas y grasas y hacer un análisis de las diferentes intervenciones. Materiales y método: búsqueda bibliográfica en PUBMED, inicialmente 196 artículos. Luego de aplicar los criterios de inclusión y exclusión se seleccionaron 26 artículos realizados en personas con DM1 de los últimos 10 años (2007-2017) referidos al consumo de comidas altas en proteínas y grasas. Resultados: hay una significativa variación interpersonal en los requerimientos de insulina en respuesta a las grasas y proteínas dietarias, que puede fluctuar en un 65% ± 10%. En los estudios randomizados se logró determinar que en las comidas altas en grasas el pico de GPP fue demorado y la sensibilidad a la insulina fue menor. Uno de los estudios logró demostrar que el 100% de las comidas altas en grasa se asociaron con hiperglucemia tardía. En relación a las dos revisiones sistemáticas encontradas, se hace hincapié en la búsqueda de datos para mejorar el tratamiento intensificado de la DM1, siendo el control de la GPP el indicador principal, ponderando la importancia de considerar la ingesta proteica y grasa de manera adicional al CHC. Conclusión: se concluye que el efecto de una comida con un alto contenido en proteínas y grasas sobre la glucemia suele presentarse entre las 3 a 6 hs de consumidas, siempre teniendo en cuenta la respuesta individual y el modo de administrar la insulina. La tarea del equipo interdisciplinario es fundamental para conocer la respuesta individual en el paciente con DM1 ante el consumo de comidas altas en proteínas y grasas, pudiendo así orientar la toma de decisión(AU).

Introduction: the treatment of type 1 diabetes (DM1) requires the administration of exogenous insulin, being the carbohydrate (HC) content of the meal to be ingested one of the variables to be considered to calculate the insulin dose. For several years, this macronutrient has been considered responsible for the increase in postprandial glycemia (PPG). Carbohydrate Counting (CHC) is the most accepted and currently used method in the nutritional treatment, although there is enough evidence that other macronutrients, such as protein and fat, can influence on the variation of PPG. Objective: to gather the results of scientific publications which analysed the glycemic response (GR) to the consumption of high-protein and high-fat meals and to analyse de different interventions. After applying the inclusion and exclusion criteria, 24 articles were selected including those with individuals with DM1 from the past 10 years (with the exception of one) referring to the consumption of high-protein and high-fat meals. Results: there is a significant interpersonal variation in insulin requirements in response to dietary fat and protein, which can fluctuate by 65% +/- 10%. Randomized studies showed that in the high-fat meals, the peak of PPG was delayed and insulin sensitivity was lower. One of the studies showed that 100% of high-fat meals were associated with late hyperglycemia. Both systematic reviews emphasize the need to search for data to improve the intensive treatment of DM1, with the control of PPG being the main indicator, considering protein and fat intake, in addition to CHC. Conclusion: the effect on blood glucose of high-protein and high-fat meals usually occurs between 3 to 6 hours after being consumed, always considering the individual response and the insulin administration method. The task of the interdisciplinary team is essential to know the individual response in the DM1 patient to the consumption of high-protein and high-fat meals, thus being able to guide the decision-making process(AU).

Mutations in TIMM50 cause severe mitochondrial dysfunction by targeting key aspects of mitochondrial physiology.

3-Methylglutaconic aciduria (3-MGA-uria) syndromes comprise a heterogeneous group of diseases associated with mitochondrial membrane defects. Whole-exome sequencing identified compound heterozygous mutations in TIMM50 (c.[341 G>A];[805 G>A]) in a boy with West syndrome, optic atrophy, neutropenia, cardiomyopathy, Leigh syndrome, and persistent 3-MGA-uria. A comprehensive analysis of the mitochondrial function was performed in fibroblasts of the patient to elucidate the molecular basis of the disease. TIMM50 protein was severely reduced in the patient fibroblasts, regardless of the normal mRNA levels, suggesting that the mutated residues might be important for TIMM50 protein stability. Severe morphological defects and ultrastructural abnormalities with aberrant mitochondrial cristae organization in muscle and fibroblasts were found. The levels of fully assembled OXPHOS complexes and supercomplexes were strongly reduced in fibroblasts from this patient. High-resolution respirometry demonstrated a significant reduction of the maximum respiratory capacity. A TIMM50-deficient HEK293T cell line that we generated using CRISPR/Cas9 mimicked the respiratory defect observed in the patient fibroblasts; notably, this defect was rescued by transfection with a plasmid encoding the TIMM50 wild-type protein. In summary, we demonstrated that TIMM50 deficiency causes a severe mitochondrial dysfunction by targeting key aspects of mitochondrial physiology, such as the maintenance of proper mitochondrial morphology, OXPHOS assembly, and mitochondrial respiratory capacity.

Consumo de bebidas endulzadas artificialmente con edulcorantes no nutritivos y su relación con el desarrollo de síndrome metabólico y diabetes tipo 2 / Consumption of artificially sweetened beverages (BEA)with non-nutritive sweeteners and its relationship to the development of metabolic syndrome and type 2 diabetes

Introducción: En Argentina un 37,1% de la población padece sobrepeso, un 20,8% obesidad y un 9,8% diabetes. La prevalencia de síndrome metabólico oscila entre el 20 y 25%. Según el estudio HIDRATAR, el consumo promedio de agua, bebidas e infusiones era de 2.050 ml/día, de los cuales un 29% correspondió a bebidas endulzadas artificialmente (BEA). No existe consenso de los organismos internacionales para su consumo. Metodología: Se llevó a cabo una búsqueda bibliográfica en PubMed, Scielo y Cochrane, de artículos publicados a partir del año 2005, con el objetivo de conocer la relación entre el consumo de BEA y el riesgo de desarrollo de síndrome metabólico y diabetes mellitus tipo 2. Resultados: Se encontraron 12 trabajos que señalan los efectos adversos de las BEA. Los mismos representan una mayor población en estudio, seguida durante más tiempo. Es un tema controvertido con mucha disparidad en la obtención de la información, teniendo en cuenta además que, en el caso de estudios con humanos, son muchos los componentes del plan alimentario que hay que considerar. Conclusiones: Ha quedado demostrado que los edulcorantes no nutritivos no son sustancias metabólicamente inertes y hay evidencia que sugiere que las BEA no son completamente inocuas, siendo primordial y necesaria la educación para limitar su consumo y promover la ingesta de agua.

Ecto-nucleotidases activities in the contents of ovarian endometriomas: potential biomarkers of endometriosis.

Endometriosis, defined as the growth of endometrial tissue outside the uterus, is a common gynecologic condition affecting millions of women worldwide. It is an inflammatory, estrogen-dependent complex disorder, with broad symptomatic variability, pelvic pain, and infertility being the main characteristics. Ovarian endometriomas are frequently developed in women with endometriosis. Late diagnosis is one of the main problems of endometriosis; thus, it is important to identify biomarkers for early diagnosis. The aim of the present work is to evaluate the ecto-nucleotidases activities in the contents of endometriomas. These enzymes, through the regulation of extracellular ATP and adenosine levels, are key enzymes in inflammatory processes, and their expression has been previously characterized in human endometrium. To achieve our objective, the echo-guided aspirated fluids of endometriomas were analyzed by evaluating the ecto-nucleotidases activities and compared with simple cysts. Our results show that enzyme activities are quantifiable in the ovarian cysts aspirates and that endometriomas show significantly higher ecto-nucleotidases activities than simple cysts (5.5-fold increase for ATPase and 20-fold for ADPase), thus being possible candidates for new endometriosis biomarkers. Moreover, we demonstrate the presence of ecto-nucleotidases bearing exosomes in these fluids. These results add up to the knowledge of the physiopathologic mechanisms underlying endometriosis and, open up a promising new field of study.

Tablas de la Relación Fósforo/Proteína de los Alimentos y su Aplicación en la Enfermedad Renal Crónica / Table showing dietary phosphorus to protein ratio

El control del fósforo dietario es una de las intervenciones más importantes en el cuidado nutricional de los pacientes con Enfermedad Renal Crónica, lo que implica dificultad en la adherencia al tratamiento. La proteína y el fósforo están estrechamente relacionados en los alimentos. La guía K/DOQI recomienda la estimación de la relación fósforo/proteína de los alimentos. Existe evidencia de tablas publicadas con la relación fósforo/proteína. Hasta el momento no se dispone de una tabla de alimentos habituales en la población argentina con la estimación de la relación. Objetivo: estimar la relación fósforo/proteína de alimentos de uso habitual en la población y establecer su aplicación en el tratamiento nutricional de la Enfermedad Renal Crónica. Material y método: las tablas se elaboraron a partir de la Base de Datos de Composición de Alimentos ARGENFOODS, Vademecum Nutrinfo, SARA-Ministerio de Salud de la Nación Argentina e información de etiquetas nutricionales. Resultados: se encuentran comprendidos en las tablas. Conclusiones: La selección de alimentos del plan alimentario del paciente con Enfermedad Renal Crónica, debería considerar la relación fósforo/proteína de los mismos. Las tablas proporcionan un análisis de los alimentos permitiendo adecuar la variedad de la alimentación de los pacientes. La relación fósforo/proteína es una herramienta valiosa para el tratamiento dietético y la educación alimentaria.

Tablas de la Relación Fósforo/Proteína de los Alimentos y su Aplicación en la Enfermedad Renal Crónica / Table showing dietary phosphorus to protein ratio

El control del fósforo dietario es una de las intervenciones más importantes en el cuidado nutricional de los pacientes con Enfermedad Renal Crónica, lo que implica dificultad en la adherencia al tratamiento. La proteína y el fósforo están estrechamente relacionados en los alimentos. La guía K/DOQI recomienda la estimación de la relación fósforo/proteína de los alimentos. Existe evidencia de tablas publicadas con la relación fósforo/proteína. Hasta el momento no se dispone de una tabla de alimentos habituales en la población argentina con la estimación de la relación. Objetivo: estimar la relación fósforo/proteína de alimentos de uso habitual en la población y establecer su aplicación en el tratamiento nutricional de la Enfermedad Renal Crónica. Material y método: las tablas se elaboraron a partir de la Base de Datos de Composición de Alimentos ARGENFOODS, Vademecum Nutrinfo, SARA-Ministerio de Salud de la Nación Argentina e información de etiquetas nutricionales. Resultados: se encuentran comprendidos en las tablas. Conclusiones: La selección de alimentos del plan alimentario del paciente con Enfermedad Renal Crónica, debería considerar la relación fósforo/proteína de los mismos. Las tablas proporcionan un análisis de los alimentos permitiendo adecuar la variedad de la alimentación de los pacientes. La relación fósforo/proteína es una herramienta valiosa para el tratamiento dietético y la educación alimentaria.(AU)

High expression of ecto-nucleotidases CD39 and CD73 in human endometrial tumors.

One of the strategies used by tumors to evade immunosurveillance is the accumulation of extracellular adenosine, which has immunosupressive and tumor promoting effects. The study of the mechanisms leading to adenosine formation at the tumor interstitium are therefore of great interest in oncology. The dominant pathway generating extracellular adenosine in tumors is the dephosphorylation of ATP by ecto-nucleotidases. Two of these enzymes acting sequentially, CD39 and CD73, efficiently hydrolyze extracellular ATP to adenosine. They have been found to play a crucial role in a variety of tumors, but there were no data concerning endometrial cancer, the most frequent of the invasive tumors of the female genital tract. The aim of the present work is to study the expression of CD39 and CD73 in human endometrial cancer. We have analyzed protein and gene expression, as well as enzyme activity, in type I endometrioid adenocarcinomas and type II serous adenocarcinomas and their nonpathological endometrial counterparts. High levels of both enzymes were found in tumor samples, with significantly increased expression of CD39 in type II serous tumors, which also coincided with the higher tumor grade. Our results reinforce the involvement of the adenosinergic system in cancer, emphasizing the relevance of ecto-nucleotidases as emerging therapeutic targets in oncology.

Fuentes de fósforo, aditivos alimentarios y Enfermedad Renal Crónica / Sources of phosphorus, food additives

La hiperfosfatemia en la Enfermedad Renal Crónica está asociada a enfermedad ósea y a incremento del riesgo de morbilidad y mortalidad cardiovascular. Su tratamiento consiste en la disminución de la absorción intestinal del fósforo a través de una dieta controlada, el uso de quelantes y la eliminación del fósforo a través de la diálisis. Existen tres fuentes básicas de fósforo dietético: el fósforo orgánico se encuentra naturalmente en los alimentos fuentes de proteínas. El fósforo proveniente de los alimentos de origen vegetal se encuentra en forma de ácido fítico cuya biodisponibilidad es de menos del 50%. El fósforo inorgánico es el componente principal de aditivos alimentarios y altamente biodisponible. Las sales de fosfato presentan numerosas aplicaciones en la industria alimentaria. Se ha elaborado un glosario de aditivos admitidos por el Código Alimentario Argentino para su adición en los alimentos industrializados. Según la legislación, no es obligatoria la inclusión del fósforo en las etiquetas nutricionales, por lo que es decisión de la industria su información. Resulta útil el conocimiento del número INS. El fósforo añadido durante el procesamiento alimentario es una importante fuente del mineral por su magnitud y su elevada biodisponibilidad. Es precisa la incorporación del fósforo en la Información nutricional de los rótulos alimentarios con el objetivo de un mejor control de la fosfatemia, sin afectar la ingesta proteica de los pacientes. Se recomienda implementar estrategias educativas para ayudar a los pacientes a identificar los aditivos con fósforo en los alimentos. El consejo nutricional debería hacer énfasis en el consumo de alimentos naturales como base de la alimentación diaria. La inclusión del fosforo total en la información nutricional ayudaría a mejorar la estimación del aporte de fósforo de la dieta

Fuentes de fósforo, aditivos alimentarios y Enfermedad Renal Crónica / Sources of phosphorus, food additives

La hiperfosfatemia en la Enfermedad Renal Crónica está asociada a enfermedad ósea y a incremento del riesgo de morbilidad y mortalidad cardiovascular. Su tratamiento consiste en la disminución de la absorción intestinal del fósforo a través de una dieta controlada, el uso de quelantes y la eliminación del fósforo a través de la diálisis. Existen tres fuentes básicas de fósforo dietético: el fósforo orgánico se encuentra naturalmente en los alimentos fuentes de proteínas. El fósforo proveniente de los alimentos de origen vegetal se encuentra en forma de ácido fítico cuya biodisponibilidad es de menos del 50%. El fósforo inorgánico es el componente principal de aditivos alimentarios y altamente biodisponible. Las sales de fosfato presentan numerosas aplicaciones en la industria alimentaria. Se ha elaborado un glosario de aditivos admitidos por el Código Alimentario Argentino para su adición en los alimentos industrializados. Según la legislación, no es obligatoria la inclusión del fósforo en las etiquetas nutricionales, por lo que es decisión de la industria su información. Resulta útil el conocimiento del número INS. El fósforo añadido durante el procesamiento alimentario es una importante fuente del mineral por su magnitud y su elevada biodisponibilidad. Es precisa la incorporación del fósforo en la Información nutricional de los rótulos alimentarios con el objetivo de un mejor control de la fosfatemia, sin afectar la ingesta proteica de los pacientes. Se recomienda implementar estrategias educativas para ayudar a los pacientes a identificar los aditivos con fósforo en los alimentos. El consejo nutricional debería hacer énfasis en el consumo de alimentos naturales como base de la alimentación diaria. La inclusión del fosforo total en la información nutricional ayudaría a mejorar la estimación del aporte de fósforo de la dieta (AU)

Sera from amyotrophic lateral sclerosis patients induce the non-canonical activation of NMDA receptors "in vitro".

Amyotrophic lateral sclerosis (ALS) is a neuromuscular disease characterized by the selective loss of both upper and lower motoneurons (MNs). The familial form of the illness is associated with mutations in the gene encoding Cu/Zn superoxide dismutase 1 (SOD-1) enzyme, but it accounts for fewer than 10% of cases; the rest, more than 90%, correspond to the sporadic form of ALS. Although many proposals have been suggested over the years, the mechanisms underlying the characteristic selective killing of MN in ALS remain unknown. In this study we tested the effect of sera from sporadic ALS patients on NMDA receptors (NMDAR). We hypothesize that an endogenous seric factor is implicated in neuronal death in ALS, mediated by the modulation of NMDAR. Sera from ALS patients and from healthy subjects were pretreated to inactivate complement pathways and dialyzed to remove glutamate and glycine. IgGs from ALS patients and healthy subjects were obtained by affinity chromatography and dialyzed against phosphate-buffered saline. Human NMDAR were expressed in Xenopus laevis oocytes, and ionic currents were recorded using the two-electrode voltage clamp technique. Sera from sporadic ALS patients induced transient oscillatory currents in oocytes expressing NMDAR with a significantly higher total electrical charge than that induced by sera from healthy subjects. Sera from patients with other neuromuscular diseases did not exert this effect. The currents were inhibited by MK-801, a noncompetitive blocker of NMDAR. The PLC inhibitor, U-73122, and the IP(3) receptor antagonist, 2-APB, also inhibited the sera-induced currents. The oscillatory signal recorded was due to internal calcium mobilization. Isolated IgGs from ALS patients significantly affected the activity of oocytes injected with NMDAR, causing a 2-fold increase over the response recorded for IgGs from healthy subjects. Our data support the notion that ALS sera contain soluble factors that mobilize intracellular calcium, not opening directly the ionic conductance, but through the non-canonical activation of NMDAR.

Amyloid ß peptide oligomers directly activate NMDA receptors.

Amyloid beta (Aß) oligomers accumulate in the brain tissue of Alzheimer disease patients and are related to disease pathogenesis. The precise mechanisms by which Aß oligomers cause neurotoxicity remain unknown. We recently reported that Aß oligomers cause intracellular Ca(2+) overload and neuronal death that can be prevented by NMDA receptor antagonists. This study investigated whether Aß oligomers directly activated NMDA receptors (NMDARs) using NR1/NR2A and NR1/NR2B receptors that were heterologously expressed in Xenopus laevis oocytes. Indeed, Aß oligomers induced inward non-desensitizing currents that were blocked in the presence of the NMDA receptor antagonists memantine, APV, and MK-801. Intriguingly, the amplitude of the responses to Aß oligomers was greater for NR1/NR2A heteromers than for NR1/NR2B heteromers expressed in oocytes. Consistent with these findings, we observed that the increase in the cytosolic concentration of Ca(2+) induced by Aß oligomers in cortical neurons is prevented by AP5, a broad spectrum NMDA receptor antagonist, but slightly attenuated by ifenprodil which blocks receptors with the NR2B subunit. Together, these results indicate that Aß oligomers directly activate NMDA receptors, particularly those with the NR2A subunit, and further suggest that drugs that attenuate the activity of such receptors may prevent Aß damage to neurons in Alzheimers disease.

Increased intramuscular nerve branching and inhibition of programmed cell death of chick embryo motoneurons by immunoglobulins from patients with motoneuron disease.

Massive programmed cell death (PCD) of developing chick embryo motoneurons (MNs) occurs in a well defined temporal and spatial sequence between embryonic day (E) 6 and E10. We have found that, when administered in ovo, either circulating immunoglobulins G (IgGs) or cerebrospinal fluid from patients with MN disease can rescue a significant number of chick embryo MNs from normally occurring PCD. An increase of branching of intramuscular nerves was also observed that may account for the rescuing effects of pathologic IgGs. Proteomic analysis and further analysis by ELISA indicated that these effects may be mediated by the interaction of circulating human immunoglobulins with proteins of the semaphorin family.

Association of Kv1.5 and Kv1.3 contributes to the major voltage-dependent K+ channel in macrophages.

Voltage-dependent K(+) (Kv) currents in macrophages are mainly mediated by Kv1.3, but biophysical properties indicate that the channel composition could be different from that of T-lymphocytes. K(+) currents in mouse bone marrow-derived and Raw-264.7 macrophages are sensitive to Kv1.3 blockers, but unlike T-cells, macrophages express Kv1.5. Because Shaker subunits (Kv1) may form heterotetrameric complexes, we investigated whether Kv1.5 has a function in Kv currents in macrophages. Kv1.3 and Kv1.5 co-localize at the membrane, and half-activation voltages and pharmacology indicate that K(+) currents may be accounted for by various Kv complexes in macrophages. Co-expression of Kv1.3 and Kv1.5 in human embryonic kidney 293 cells showed that the presence of Kv1.5 leads to a positive shift in K(+) current half-activation voltages and that, like Kv1.3, Kv1.3/Kv1.5 heteromers are sensitive to r-margatoxin. In addition, both proteins co-immunoprecipitate and co-localize. Fluorescence resonance energy transfer studies further demonstrated that Kv1.5 and Kv1.3 form heterotetramers. Electrophysiological and pharmacological studies of different ratios of Kv1.3 and Kv1.5 co-expressed in Xenopus oocytes suggest that various hybrids might be responsible for K(+) currents in macrophages. Tumor necrosis factor-alpha-induced activation of macrophages increased Kv1.3 with no changes in Kv.1.5, which is consistent with a hyperpolarized shift in half-activation voltage and a lower IC(50) for margatoxin. Taken together, our results demonstrate that Kv1.5 co-associates with Kv1.3, generating functional heterotetramers in macrophages. Changes in the oligomeric composition of functional Kv channels would give rise to different biophysical and pharmacological properties, which could determine specific cellular responses.