RESUMEN
Pompe disease is an autosomal recessive disorder in which deficiency of the lysosomal enzyme acid alpha-glucosidase results in the accumulation of glycogen mostly in muscle tissues. Several reports suggest a higher incidence of intracranial vascular abnormalities (IVAs) in this condition, as well as brain microbleeds and cerebral vasculopathy. The aim of our study was to evaluate through neuroimaging studies the incidence of these anomalies in our cohort of late-onset Pompe disease (LOPD) patients asymptomatic for cerebrovascular disease, looking for correlations with clinical and genetic data. We studied 18 LOPD patients with brain magnetic resonance angiography (MRA), or contrast-enhanced computed tomography (CECT). Diameters of individual arteries were measured and compared with average values as proposed in the literature. We found IVAs in 13 of the 18 patients, mostly dilatative arteriopathy affecting the vertebrobasilar system. The anterior circle was involved in seven of the 18 patients. The diameter of the basilar artery at 1 cm was found to correlate both with age (spearman rho, p = 0.037) and disease duration (p = 0.004), but no other statistically significant correlation was documented. The incidence of intracranial dilatative arteriopathy in LOPD was higher than in the general population, confirming the literature data. However, we did not find intracranial aneurysms microbleeds or significant cerebrovascular disease. Abnormalities in the anterior and the posterior circle of Willis correlated with age and disease duration, but not with the severity of muscle/respiratory involvement or with genetic data. Further studies in larger cohorts of patients are needed to confirm these findings.
Asunto(s)
Encéfalo/diagnóstico por imagen , Trastornos Cerebrovasculares/etiología , Glucosiltransferasas/genética , Enfermedad del Almacenamiento de Glucógeno Tipo II , Adulto , Edad de Inicio , Anciano , Arteria Basilar/diagnóstico por imagen , Arteria Basilar/patología , Trastornos Cerebrovasculares/diagnóstico por imagen , Trastornos Cerebrovasculares/epidemiología , Femenino , Enfermedad del Almacenamiento de Glucógeno Tipo II/complicaciones , Enfermedad del Almacenamiento de Glucógeno Tipo II/diagnóstico por imagen , Enfermedad del Almacenamiento de Glucógeno Tipo II/genética , Humanos , Masculino , Metaloproteinasa 3 de la Matriz/genética , Persona de Mediana Edad , Mutación/genética , Neuroimagen , Estadística como AsuntoRESUMEN
OBJECTIVE: A multicentre observational study was aimed to assess the prevalence of late-onset Pompe disease (LOPD) in a large high-risk population, using the dried blood spot (DBS) as a main screening tool. DESIGN/METHODS: 17 Italian neuromuscular centres were involved in the late-onset Pompe early diagnosis (LOPED) study. Inclusion criteria were: (1) age ≥5â years, (2) persistent hyperCKaemia and (3) muscle weakness at upper and/or lower limbs (limb-girdle muscle weakness, LGMW). Acid α-glucosidase (GAA) activity was measured separately on DBS by fluorometric as well as tandem mass spectrometry methods. A DBS retest was performed in patients resulted positive at first assay. For the final diagnosis, GAA deficiency was confirmed by a biochemical assay in skeletal muscle, whereas genotype was assessed by GAA molecular analysis. RESULTS: In a 14-month period, we studied 1051 cases: 30 positive samples (2.9%) were detected by first DBS screening, whereas, after retesting, 21 samples were still positive. Biochemical and molecular genetic studies finally confirmed LOPD diagnosis in 17 cases (1.6%). The median time from the onset of symptoms/signs to diagnosis was 5â years. Among those patients, 35% showed presymptomatic hyperCKaemia and 59% showed hyperCKaemia+LGMW, whereas 6% manifested with LGMW. CONCLUSIONS: LOPED study suggests that GAA activity should be accurately screened by DBS in all patients referring for isolated hyperCKaemia and/or LGMW. A timely diagnosis was performed in five patients with presymptomatic hyperCKaemia, but two had already manifested with relevant changes on muscle morphology and MRI. Consequently, enzyme replacement therapy was started in 14/17 patients, including the 2 patients still clinically presymptomatic but with a laboratory evidence of disease progression.
Asunto(s)
Enfermedad del Almacenamiento de Glucógeno Tipo II/diagnóstico , Adulto , Edad de Inicio , Creatina Quinasa/sangre , Diagnóstico Precoz , Femenino , Fluorometría , Enfermedad del Almacenamiento de Glucógeno Tipo II/genética , Enfermedad del Almacenamiento de Glucógeno Tipo II/terapia , Humanos , Masculino , Persona de Mediana Edad , Debilidad Muscular/etiología , Músculo Esquelético/patología , Patología Molecular/métodos , Reproducibilidad de los Resultados , Riesgo , Espectrometría de Masas en Tándem , alfa-Glucosidasas/genéticaRESUMEN
OBJECTIVE: We performed an electrophysiological study of swallowing (EPSS) in multiple sclerosis (MS) to describe oropharyngeal swallowing abnormalities and to analyze their correlations with dysphagia and with overall neurological impairment. METHODS: Neurological examinations were quantified using the Kurtzke Functional Systems and the Expanded Disability Status Scale (EDSS). Dysphagia was evaluated using the Dysphagia in Multiple Sclerosis (DYMUS) questionnaire, while fiberoptic endoscopic evaluation of swallowing (FEES) was used to establish the degree of aspiration and penetration, graded using the penetration-aspiration scale (PAS). The EPSS measured the duration of suprahyoid/submental muscle EMG activity (SHEMG-D), the duration of the laryngeal-pharyngeal mechanogram (LPM-D), and the duration of the pause in cricopharyngeal muscle EMG activity (CPEMG-PD); it also measured the interval between onset of the suprahyoid/submental muscle EMG activity (SHEMG) and onset of the laryngeal-pharyngeal mechanogram (I-SHEMG-LPM). RESULTS: 92% of patients showed at least one electrophysiological abnormality. I-SHEMG-LPM correlated positively with the DYMUS questionnaire. I-SHEMG-LPM, SHEMG-D, and DYMUS correlated positively with the PAS. Moderate to severe bladder sphincter dysfunction was associated with a significant reduction, or absence, of CPEMG-PD. CONCLUSION: EPSS improves our understanding of the pathophysiology of dysphagia in MS. SIGNIFICANCE: This investigation could be useful in MS patients with swallowing abnormalities.
Asunto(s)
Trastornos de Deglución/fisiopatología , Deglución , Esclerosis Múltiple/fisiopatología , Orofaringe/fisiopatología , Adulto , Anciano , Electromiografía , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Given the complexity of the respiratory chain structure, assembly and regulation, the diagnostic workout for the identification of defects of oxidative phosphorylation (OXPHOS) is a major challenge. Spectrophotometric assays, that measure the activity of individual respiratory complexes in tissue and cell homogenates or isolated mitochondria, are highly specific, but their utilization is limited by the availability of sufficient biological material and intrinsic sensitivity. A further limitation is tissue specificity, which usually determines attenuation, or disappearance, in cultured fibroblasts, of defects detected in muscle or liver. We used numerous fibroblast cell lines derived from patients with OXPHOS deficiencies to set up experimental protocols required for the direct readout of cellular respiration using the Seahorse XF96 apparatus, which measures oxygen consumption rate (OCR) and extra-cellular acidification rate (ECAR) in 96 well plates. Results demonstrate that first level screening based on microscale oxygraphy is more sensitive, cheaper and rapid than spectrophotometry for the biochemical evaluation of cells from patients with suspected mitochondrial disorders.
Asunto(s)
Técnicas de Laboratorio Clínico/métodos , Mitocondrias/enzimología , Mitocondrias/metabolismo , Enfermedades Mitocondriales/diagnóstico , Fosforilación Oxidativa , Técnicas de Laboratorio Clínico/economía , Humanos , Mitocondrias/genética , Sensibilidad y Especificidad , Factores de TiempoRESUMEN
The objective of this study was to describe a large Italian cohort of patients with late-onset glycogen storage disease type 2 (GSDII) at various stages of disease progression and to evaluate the clinical effectiveness of alglucosidase alpha enzyme replacement therapy (ERT). Previous studies showed in late-onset patients ERT efficacy against placebo and variable response in uncontrolled studies. Seventy-four juvenile or adult GSDII patients were treated with ERT in a multicenter open label, non-randomized study, from 12 months up to 54 months. Recombinant human alpha glucosidase (rh-GAA) was injected by intravenous route at 20 mg/kg every second week. Patients were divided into three groups according to ERT duration: Group A received treatment for 12-23 months (n = 16), Group B for 24-35 months (n = 14), and Group C for more than 36 months (n = 44). Clinical assessment included a 6-min walk test (6MWT), forced vital capacity (FVC), the Walton and Gardner-Medwin score, the number of hours of ventilation, body mass index, echocardiography and blood creatine kinase (CK). Included in our cohort were 33 males and 41 females (M:F = 0.8:1), with a mean age at first symptoms of 28.3 years (range 2-55 years) and a mean age of 43 years at study entry (range 7-72 years). Seven wheelchair bound patients, as well as 27 patients requiring ventilation support, were included. After treatment we could observe an increase in distance walked on the 6MWT in the large majority of patients (48/58; 83%), with an overall mean increase of 63 m (from 320 ± 161 to 383 ± 178 m). After treatment in the majority of patients FVC was improved or unchanged (45/69; 65%). In ventilated patients we observed an improvement in average number of hours off the ventilator (from 15.6 to 12.1 h). Six patients stopped mechanical ventilation and two others started it. The effect of therapy was not related to ERT duration. Nine of 64 patients (13%) that underwent to echocardiography showed a variable degree of cardiac hypertrophy (left ventriculum or septum), and a positive effect was observed after 36 months of ERT in one adult case. Discontinuation of treatment occurred in four patients: one drop-off case, one patient died for a sepsis after 34 months of treatment and two patients stopped ERT for worsening of general clinical condition. Mild adverse effects were observed in four cases (5%). This study represents the largest cohort of late-onset GSDII patients treated with ERT, and confirm a positive effect of treatment. These results, obtained in a large case series on therapy, indicate a favourable effect of ERT therapy, even in more advanced stage of the disease.
Asunto(s)
Terapia de Reemplazo Enzimático/métodos , Enfermedad del Almacenamiento de Glucógeno Tipo II/tratamiento farmacológico , Observación , alfa-Glucosidasas/uso terapéutico , Adolescente , Adulto , Anciano , Índice de Masa Corporal , Niño , Estudios de Cohortes , Ecocardiografía , Femenino , Enfermedad del Almacenamiento de Glucógeno Tipo II/fisiopatología , Frecuencia Cardíaca/efectos de los fármacos , Humanos , Italia , Masculino , Persona de Mediana Edad , Examen Físico , Respiración/efectos de los fármacos , Índice de Severidad de la Enfermedad , Estadísticas no Paramétricas , Factores de Tiempo , Resultado del Tratamiento , Capacidad Vital , Caminata/fisiología , Adulto JovenRESUMEN
BACKGROUND: In adult glycogen storage disease type II (GSDII), a single-gene mutation causes reduction of the lysosomal enzyme acid alpha-glucosidse. This produces a chronic proximal myopathy with respiratory involvement. Enzyme replacement treatment (ERT) has recently become available and is expected to improve muscle strength. This should result in increased lean body mass. In this study we evaluate body composition and nutritional status in GSDII, and assess whether these parameters changed during treatment. METHODS: Seventeen patients with late-onset GSDII, aged 52.6 +/- 16.8 years, received ERT for >18 months. Dietary habits and metabolic profiles of glucids, lipids, and proteins were assessed. Body composition was calculated using anthropometry and bioelectrical impedence analysis. RESULTS: On inclusion, we found increased fat mass (FM) in five patients in severe disease stage; all had normal body mass index (BMI). FM correlated inversely, and lean mass (LM) directly, with creatine kinase, prealbumin and albumin levels. After treatment, BMI and FM significantly increased, while LM only showed a trend toward increase. Prealbumin and albumin levels increased as early as after the first months of ERT. DISCUSSION: Body mass index value may underestimate FM in patients in severe stage of disease, due to altered body composition. In severely affected patients, laboratory parameters revealed a relative protein malnutrition, that was reversed by ERT, this reflecting restoration of normal muscle metabolic pathways. Increased BMI may indicate a reduction in energy consumption during exercise or respiration, along with clinical improvement.
Asunto(s)
Terapia de Reemplazo Enzimático/métodos , Enfermedad del Almacenamiento de Glucógeno Tipo II/tratamiento farmacológico , Enfermedad del Almacenamiento de Glucógeno Tipo II/fisiopatología , Estado Nutricional/efectos de los fármacos , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores/análisis , Biomarcadores/metabolismo , Composición Corporal/efectos de los fármacos , Composición Corporal/fisiología , Femenino , Enfermedad del Almacenamiento de Glucógeno Tipo II/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiopatología , Estado Nutricional/fisiología , Proteínas/análisis , Proteínas/metabolismoRESUMEN
Although it has been shown that muscle magnetic resonance imaging (MRI) improves the phenotypic characterization of patients with neuromuscular disorders and allows accurate quantification of muscle and adipose tissue distribution, to date quantitative MRI has not been used to assess the therapeutic response in clinical trials of neuromuscular diseases. We discuss quantitative MRI findings after a 6-month course of enzyme replacement therapy administered to nine patients with adult-onset glycogenosis II.
Asunto(s)
Enfermedad del Almacenamiento de Glucógeno Tipo II/tratamiento farmacológico , Enfermedad del Almacenamiento de Glucógeno Tipo II/patología , Imagen por Resonancia Magnética , Músculo Esquelético/patología , alfa-Glucosidasas/uso terapéutico , Edad de Inicio , Anciano , Femenino , Humanos , Imagen por Resonancia Magnética/métodos , Masculino , Persona de Mediana Edad , Proteínas Recombinantes/uso terapéuticoRESUMEN
OBJECTIVE: Idiopathic transverse myelitis (I-TM) is typically monophasic, while relapsing forms are usually referred to spinal cord-restricted neuromyelitis optica (NMO), atypical multiple sclerosis (MS), or myelitis during the course of infections and connectivitis. Our objective was to evaluate the frequency of recurrent I-TM; to clarify the nosology of these forms through comparison with NMO and post-infectious TM (P-TM). DESIGN: Prospective cohort study on patients presenting with I-TM was carried out inpatients of Infectious and Neurologic Disease Clinics, Italy. METHODS: Over an 8-year period, we recruited 13 patients with I-TM and 16 with P-TM. The patients were followed-up for at least 3 years with repeated brain and spinal cord magnetic resonance imaging (MRI) examinations, multimodal evoked potentials and serum screen for connectivitis. Relapses were defined on clinical and imaging criteria. RESULTS: Four patients with I-TM (31%) had a relapsing course . They were all males with age >50, and severe at-onset disability. The final outcome was poor in three out of four patients. Serum NMO-immunoglobulin G was undetectable in all patients. Longitudinally extensive myelitis was not predictive of relapses. I-TM and P-TM shared clinical, cerebrospinal fluid (CSF) and MRI features, as well as a similar rate (54 vs 38%) of peripheral nervous system involvement (polyradiculoneuritis), and an identical rate of relapses (31% for both forms). CONCLUSIONS: Our series support the existence of relapsing I-TM as a disease entity that does not appear related to NMO, nor to MS, cannot be further specified and shares many features with P-TM. The likelihood of relapses was unpredictable based on clinical, CSF and MRI findings.
Asunto(s)
Inmunoglobulinas/sangre , Mielitis Transversa/diagnóstico , Adulto , Anciano , Autoanticuerpos , Encéfalo/patología , Estudios de Cohortes , Femenino , Humanos , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Mielitis Transversa/sangre , Recurrencia , Estudios Retrospectivos , Pruebas Serológicas , Médula Espinal/patología , Adulto JovenRESUMEN
The diagnosis of glycogenosis type II is often complicated by the rarity of the condition and the heterogeneity of the clinical manifestations of the disease. It is a progressive, debilitating, and often fatal neuromuscular disorder that manifests as a continuum of clinical phenotypes, which vary with respect to organ involvement, age at onset, and severity. Early diagnosis requires both increased awareness among physicians regarding the clinical characteristics of the disease and fast and reliable acid alpha-glucosidase (GAA) enzyme activity assays to confirm the GAA deficiency. The clinical diagnosis of glycogenosis type II is confirmed by virtual absence (found in infants) and marked reduced activity (found in juveniles and adults) of GAA enzyme in blood samples, cultured fibroblasts, and muscle biopsies. This article specifically highlights the need for early recognition of the clinical manifestation of the disease in infants, juveniles, and adults. Descriptions of the main clinical features of the condition, as well as differential diagnosis are included. In addition, the tests required for a confirmed diagnosis are described, and use of muscle imaging to evaluate muscle pathology is reviewed.
Asunto(s)
Enfermedad del Almacenamiento de Glucógeno Tipo II/diagnóstico , Adolescente , Adulto , Edad de Inicio , Técnicas de Laboratorio Clínico/normas , Diagnóstico Diferencial , Diagnóstico Precoz , Enfermedad del Almacenamiento de Glucógeno Tipo II/enzimología , Enfermedad del Almacenamiento de Glucógeno Tipo II/fisiopatología , Humanos , Recién Nacido , Músculo Estriado/enzimología , Músculo Estriado/patología , Músculo Estriado/fisiopatología , Enfermedades Musculares/diagnóstico , Enfermedades Musculares/enzimología , Enfermedades Musculares/fisiopatología , Fenotipo , alfa-Glucosidasas/análisis , alfa-Glucosidasas/sangre , alfa-Glucosidasas/deficienciaRESUMEN
Glycogenosis type II is a multisystem disorder that requires management by a multidisciplinary team. The team should include several specialists, such as a metabolic disease specialist or biochemical geneticist, cardiologist, pulmonologist, neurologist, neuromuscular specialist, intensivist, orthopedist, respiratory therapist, physical therapist, occupational therapist, otolaryngologist speech therapist, audiologist, genetic counselor, and a metabolic dietician, who, as a team, will be capable of addressing the different manifestations of the condition. Aspects of functional assessment, rehabilitation, nutritional management, care coordination, nursing, genetic counseling, prenatal diagnosis, and screening are discussed in this article. In addition, treatment of glycogenosis type II is reviewed with attention to emerging therapeutic options.
Asunto(s)
Enfermedad del Almacenamiento de Glucógeno Tipo II/terapia , Encefalopatías Metabólicas Innatas/diagnóstico , Encefalopatías Metabólicas Innatas/fisiopatología , Encefalopatías Metabólicas Innatas/terapia , Cardiomiopatías/diagnóstico , Cardiomiopatías/fisiopatología , Cardiomiopatías/terapia , Terapia Genética/métodos , Terapia Genética/tendencias , Enfermedad del Almacenamiento de Glucógeno Tipo II/metabolismo , Enfermedad del Almacenamiento de Glucógeno Tipo II/fisiopatología , Humanos , Músculo Estriado/metabolismo , Músculo Estriado/fisiopatología , Enfermedades Musculares/metabolismo , Enfermedades Musculares/fisiopatología , Enfermedades Musculares/terapia , Grupo de Atención al Paciente/normas , Parálisis Respiratoria/diagnóstico , Parálisis Respiratoria/fisiopatología , Parálisis Respiratoria/terapia , alfa-Glucosidasas/deficiencia , alfa-Glucosidasas/genéticaAsunto(s)
Músculo Esquelético/efectos de los fármacos , Trastornos Miotónicos/tratamiento farmacológico , Propafenona/administración & dosificación , Bloqueadores de los Canales de Sodio/administración & dosificación , Canales de Sodio/efectos de los fármacos , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/genética , Adulto , Antiarrítmicos/administración & dosificación , Análisis Mutacional de ADN , Esquema de Medicación , Marcadores Genéticos/genética , Pruebas Genéticas , Humanos , Masculino , Hipertonía Muscular/tratamiento farmacológico , Hipertonía Muscular/genética , Hipertonía Muscular/fisiopatología , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiopatología , Mutación/genética , Trastornos Miotónicos/genética , Trastornos Miotónicos/fisiopatología , Canal de Sodio Activado por Voltaje NAV1.4 , Parálisis/tratamiento farmacológico , Parálisis/genética , Parálisis/fisiopatología , Canales de Sodio/genética , Canales de Sodio/metabolismo , Resultado del TratamientoRESUMEN
BACKGROUND: Acute disseminated encephalomyelitis (ADEM) refers to a monophasic acute multifocal inflammatory CNS disease. However, both relapsing and site-restricted variants, possibly associated with peripheral nervous system (PNS) involvement, are also observed, and a systematic classification is lacking. OBJECTIVE: To describe a cohort of postinfectious ADEM patients, to propose a classification based on clinical and instrumental features, and to identify subgroups of patients with different prognostic factors. METHODS: Inpatients of a Neurologic and Infectious Disease Clinic affected by postinfectious CNS syndrome consecutively admitted over 5 years were studied. RESULTS: Of 75 patients enrolled, 60 fulfilled criteria for ADEM after follow-up lasting from 24 months to 7 years. Based on lesion distribution, patients were classified as encephalitis (20%), myelitis (23.3%), encephalomyelitis (13.3%), encephalomyeloradiculoneuritis (26.7%), and myeloradiculoneuritis (16.7%). Thirty patients (50%) had a favorable outcome. Fifteen patients (25%) showed a relapsing course. Poor outcome was related with older age at onset, female gender, elevated CSF proteins, and spinal cord and PNS involvement. All but two patients received high-dose steroids as first-line treatment, with a positive response in 39 (67%). Ten of 19 nonresponders (53%) benefited from high-dose IV immunoglobulin; 9 of 10 had PNS involvement. The data were not controlled. CONCLUSIONS: A high prevalence of "atypical variants" was found in this series, with site-restricted damage or additional peripheral nervous system (PNS) involvement. Prognosis and response to steroids were generally good, except for some patient subgroups. In patients with PNS involvement and steroid failure, a favorable effect of IV immunoglobulin was observed.
Asunto(s)
Sistema Nervioso Central/fisiopatología , Encefalomielitis Aguda Diseminada/clasificación , Encefalomielitis Aguda Diseminada/diagnóstico , Nervios Periféricos/fisiopatología , Adulto , Factores de Edad , Anciano , Antiinflamatorios/uso terapéutico , Encéfalo/inmunología , Encéfalo/patología , Encéfalo/fisiopatología , Sistema Nervioso Central/inmunología , Sistema Nervioso Central/patología , Estudios de Cohortes , Encefalomielitis Aguda Diseminada/fisiopatología , Femenino , Estudios de Seguimiento , Humanos , Inmunoglobulinas Intravenosas/uso terapéutico , Masculino , Persona de Mediana Edad , Nervios Periféricos/inmunología , Nervios Periféricos/patología , Pronóstico , Estudios Prospectivos , Recurrencia , Factores Sexuales , Médula Espinal/inmunología , Médula Espinal/patología , Médula Espinal/fisiopatología , Raíces Nerviosas Espinales/inmunología , Raíces Nerviosas Espinales/patología , Raíces Nerviosas Espinales/fisiopatología , Esteroides/uso terapéutico , Resultado del TratamientoAsunto(s)
Amaurosis Fugax/diagnóstico , Demencia por Múltiples Infartos/diagnóstico , Trastornos Migrañosos/diagnóstico , Adulto , Amaurosis Fugax/etiología , Demencia por Múltiples Infartos/complicaciones , Humanos , Imagen por Resonancia Magnética , Masculino , Trastornos Migrañosos/genética , Linaje , SíndromeRESUMEN
Nitric oxide (NO) may participate in the mechanisms underlying vascular headaches, such as migraine and cluster headache (CH), by triggering neurogenic inflammation and activation of fibres conveying nociceptive inputs to the trigeminal ganglion. Similarly to migraine, the administration of the NO donor glyceryltrinitrate (GTN) to CH patients is a known model of inducing spontaneous-like attacks. We carried out a GTN test (0.9 mg, sublingually) in 18 patients with episodic CH in active phase and 12 controls. The plasma levels of NO metabolite nitrites (NO2-), after conversion of nitrates to NO2-, were measured spectrophotometrically at baseline, at the maximum intensity of the induced response (or 45 min after GTN in controls), and 120 min after GTN administration. The basal plasma levels of L-citrulline were also assayed in patients and controls using high-performance liquid chromatography. Basal NO2- levels, similar in GTN-responsive patients and controls (48.3 +/- 10.6 and 44.6 +/- 9.5 micromol/l, respectively) were found to be increased significantly at pain peak in patients (76.1 +/- 10.2 micromol/l) and after 45 min in controls (78.2 +/- 9.6 micromol/l) (P < 0.01 vs. respective baseline values), but not after 120 min, without differences between groups. L-citrulline levels in basal conditions showed no differences between groups (patients 64.8 +/- 11.7, controls 67.3 +/- 10.8 micromol/l). These data do not support the presence of a basal hyperactivity of the L-arginine-NO pathway in CH patients. Increased NO production may be of importance in the mechanisms leading to CH attacks, but other factors are likely to render CH patients hyperresponsive to NO, and ultimately to cause the occurrence of pain and associated features.
Asunto(s)
Citrulina/sangre , Cefalalgia Histamínica/sangre , Óxido Nítrico/fisiología , Nitritos/sangre , Nitroglicerina/farmacología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Adulto , Análisis de Varianza , Cefalalgia Histamínica/inducido químicamente , Femenino , Humanos , Masculino , Persona de Mediana Edad , Óxido Nítrico/sangre , Nitroglicerina/efectos adversosRESUMEN
We report here the complete coding sequence of a 203 cDNA, a member of the interferon-inducible Ifi 200 gene family. By combining reverse-transcriptase PCR and rapid amplification of cDNA ends (RACE) techniques we have obtained a 3.8-kb cDNA corresponding to a 203 mRNA. When used as a probe in northern analysis, its 3' segment hybridized to a 3.8-kb interferon-inducible mRNA, whereas the 5'-end additionally hybridized to a less abundant interferon-inducible 1.8-kb mRNA. Nucleotide sequence analysis revealed that the two mRNAs share the 5'-untranslated region and the same open reading frame, which encodes a hydrophilic protein composed of 408 amino acids. The difference between them is due to a 3'-untranslated region extended by alternative polyadenylation site selection. Furthermore, 203 mRNA was found to be inducible by interferon-alpha in various murine cell lines. Using polyclonal antibodies raised against a segment specific for the 203 protein, we established that p203 protein levels increase on treatment with interferon-alpha in murine fibroblasts and that p203 is located in the nucleus.
Asunto(s)
Proteínas Nucleares/genética , Fosfoproteínas/genética , Células 3T3 , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Núcleo Celular/metabolismo , Clonación Molecular , ADN Complementario/genética , Expresión Génica/efectos de los fármacos , Interferón Tipo I/farmacología , Ratones , Datos de Secuencia Molecular , Familia de Multigenes , ARN Mensajero/genética , Proteínas RecombinantesRESUMEN
The transcription of murine cytomegalovirus (MCMV) immediate-early (IE) genes is regulated by a large and complex enhancer containing several consensus binding sites for the ubiquitous transcription factor NF-kappa B. To verify whether MCMV, like the human CMV, can activate NF-kappa B-dependent transcription, we transfected murine embryo fibroblasts cells with a construct containing three copies of the NF-kappa B element in front of the homologous minimal MCMV IE1-3 promoter. Upon MCMV infection the reporter gene activity was transactivated to about three-fold above the basal level. The specificity of this transactivation was demonstrated by the lack of any significant effect on the activity of DNA constructs containing either a mutated NF-kappa B trimer or an ATF/CRE trimer. Gel shift assays with a NF-kappa B probe revealed that MCMV infection activated DNA binding proteins showing NF-kappa B characteristics. The DNA-binding activity remained elevated during the course of infection and was associated to an increase in the steady-state mRNA levels for the NF-kappa B subunit p105/p50. Since the promoter of the p105/p50 gene was transactivated by MCMV infection during the period in which the IE proteins are expressed, the role of the two major IE transcriptional regulatory proteins was examined. In cotransfection experiments, the IE1 protein transactivated the p105/p50 promoter, whereas the IE3 was ineffective in increasing the transcription of the reporter gene. Taken as a whole, these results demonstrate that MCMV, like its human counterpart, regulates the cellular NF-kappa B activity needed for the initial induction of the IE genes and the progression of the viral replicative cycle.
Asunto(s)
Citomegalovirus/fisiología , Genes Inmediatos-Precoces , Proteínas Inmediatas-Precoces/metabolismo , FN-kappa B/biosíntesis , Regiones Promotoras Genéticas , Activación Transcripcional , Proteínas Virales , Células 3T3 , Animales , Secuencia de Bases , Citomegalovirus/genética , Proteínas de Unión al ADN/biosíntesis , Genes Reporteros , Humanos , Proteínas Inmediatas-Precoces/biosíntesis , Ratones , Ratones Endogámicos BALB C , FN-kappa B/genética , Oligodesoxirribonucleótidos , Proteínas Recombinantes/biosíntesis , Especificidad por Sustrato , Transcripción Genética , Transfección , Rayos UltravioletaRESUMEN
Transcription of murine cytomegalovirus (MCMV) immediate-early (IE) genes is regulated by the interaction of cellular transcription factors with a strong viral enhancer controlling promoters flanking both sides of the regulatory sequence. We have previously demonstrated that interferon-alpha (IFN-alpha) inhibits MCMV replication by impairing the transcription of IE genes. To define the cis-acting elements and trans-acting factors involved in this inhibition, permissive murine fibroblasts were transferred with DNA constructs containing the chloramphenicol acetyl transferase reporter gene and portions of the IE enhanced. The region spanning -1185 to -259 relative to the IE1-3 promoter was sufficient to allow IFN-alpha-induced inhibition. Since this segment contains several NF-kappa B sites, cells were transfected with a construct containing three copies of NF-kappa B element in front of the homologous minimal IE1-3 promoter. Upon IFN-alpha treatment the reporter gene activity was strongly reduced, indicating that NF-kappa B binding site is sufficient to confer inhibition. The specificity of this inhibition was demonstrated by the lack of a significant effect on the activity of DNA constructs containing either a mutated NF-kappa B trimer or an ATF/CRE trimer. Gel shift assays with NF-kappa B probes revealed that MCMV infection activated NF-kappa B proteins, whereas IFN-alpha treatment significantly reduced their ability to bind NF-kappa B sites. In cotransfection experiments using various NF-kappa B subunit expression vectors and a reporter driven by three copies of an NF-kappa B element, activation of NF-kappa B-dependent transcription was observed with expression of p65 or combinations of p50-p65. Taken as a whole, these results suggest that IFN-alpha inhibits MCMV IE gene enhancer activity by mechanisms that decrease the availability of virus-induced NF-kappa B transcriptionally active in the nuclei of infected cells.