Estudio de los mecanismos implicados en la génesis y evolución del asma (proyecto MEGA): creación y seguimiento a largo plazo de una cohorte de pacientes asmáticos

The general aim of this study is to create a cohort of asthma patients with varying grades of severity in order to gain greater insight into the mechanisms underlying the genesis and course of this disease.The specific objectives focus on various studies, including imaging, lung function, inflammation, and bronchial hyperresponsiveness, to determine the relevant events that characterize the asthma population, the long-term parameters that can determine changes in the severity of patients, and the treatments that influence disease progression. The study will also seek to identify the causes of exacerbations and how this affects the course of the disease.Patients will be contacted via the outpatient clinics of the 8 participating institutions under the auspices of the Spanish Respiratory Diseases Networking System (CIBER). In the inclusion visit, a standardized clinical history will be obtained, a clinical examination, including blood pressure, body mass index, complete respiratory function tests, and FENO will be performed, and the Asthma Control Test (ACT), Morisky-Green test, Asthma Quality of Life Questionnaire (Mini AQLQ), the Sino-Nasal Outcome Test 22 (SNOT-22), and the Hospital Anxiety and Depression scale (HADS) will be administered. A specific electronic database has been designed for data collection. Exhaled breath condensate, urine and blood samples will also be collected. Non-specific bronchial hyperresponsiveness testing with methacholine will be performed and an induced sputum sample will be collected at the beginning of the study and every 24 months. A skin prick test for airborne allergens and a chest CT will be performed at the beginning of the study and repeated every 5 years.     

Intranuclear filamentous inclusion in human oedematous cerebellar Golgi cells

The presence of intranuclear filamentous inclusions in cerebellar Golgi cells is reported in three patients with cerebellar tumours. Samples of cerebellar cortex were processed for conventional transmission electron microscopy. Cerebellar biopsies were performed according to the basic principles of Helsinki declaration. Intranuclear inclusions observed in oedematous Golgi cells, appeared as straight rodlets up to 3 microm in length and up to 0.4 microm in width, characterized by a periodic or crystalloid structure formed by dense bands, 9.2 nm thick separated by clear spaces, 5.4 nm in width. These structures are considered abnormal protein aggregates apparently induced by excitotoxicity, oxidative stress and impaired energy metabolism.     

Morphological changes of dendrites in the human edematous cerebral cortex. A transmission electron microscopic study

The structural pathology of dendritic processes has been examined in 38 patients with clinical diagnosis of brain trauma, brain tumours and congenital malformations. Cortical biopsies of frontal, parietal, temporal and occipital cortex were conventionally processed for transmission electron microscopy. Isolated ultrathin sections and montages of electron micrographs were used to trace the intracortical dendritic course. Swollen and beaded dendrites were observed in all cases examined, which exhibited fragmentation of limiting plasma membrane and cytoskeletal structures. The swollen dendrites showed vacuolization, dense residual bodies, enlarged rough and smooth endoplasmic reticulum, edematous clear and dark mitochondria, a decreased synaptic density of shaft synapses, edematous and dystrophic changes of spine apparatus and a partial loss of dendritic spines. A wide variety of dendritic spine shapes were observed: mushroom-type, stubby, gem-like filiform spine, and megaspine, considered as spine dysgenesis in the congenital malformations and spine pathology and spine plasticity in brain traumatic injuries and brain tumours. The multifactorial processes associated with brain edema and brain ischemia, such as calcium overload, activation of calcium-dependent proteolytic enzymes, protein aggregation, glutamate-induced neurotoxicity, release of lysosomal enzymes, deficit of ATP, stress oxidative and lipid peroxidation have been considered in relation with the pathological dendritic changes. Dendrotoxicity due to brain edema and brain ischemia seems to be the fundamental pathogenetic mechanism.     

Nerve cell death types in the edematous human cerebral cortex

Cortical biopsies of 18 patients with clinical diagnosis of congenital hydrocephalus, brain trauma, and vascular anomaly were examined with the transmission electron microscope to study the distinct and overlapped morphological cell types of nerve cell death in the human edematous cerebral cortex. The nerve cells showed lobulated and shrunken nucleus, irregular enlargement and fragmentation of perinuclear cistern, with areas of apparently intact nuclear pore complexes alternating with regions of nuclear pore complex disassembly. The nucleolus appears unaltered in moderate edema and with distorted nucleolar subcompartments in severe edema. Most nonpyramidal nerve cells, astrocytes and oligodendrocytes underwent an oncotic-apoptotic-necrotic continuum featured by swollen nucleoplasm, cytoplasm, and cell organelles, chromatin condensation and marginalization, and formation of apoptotic bodies. In a lesser proportion other nonpyramidal nerve cells, astrocytes and oligodendrocytes only showed apoptosis or oncosis. Autophagic cell death characterized by presence of autophagic vacuoles of lysosomal origin was rarely seen. The above findings suggest that different mechanisms of nerve cell death occur in the human edematous cerebral cortex related with brain trauma, congenital hydrocephalus, vascular anomaly, and their anoxic-ischemic conditions. An oncotic-apoptotic continuum process leading to necrosis predominates in human cerebral cortex nerve cell populations. The nerve cell death is discussed in relation with the severity of brain edema, anoxic-ischemic conditions of brain parenchyma, oxidative stress, glutamate excitotoxicity, calcium overload, and caspase dependent and independent mechanisms.     

The inflammatory reaction in human traumatic oedematous cerebral cortex

The inflammatory reaction surrounding hemorrhagic and perihematomal brain parenchyma has been studied by means of light and transmission electron microscopy in 12 patients with severe traumatic head injuries complicated with subdural or extradural hematoma or hygroma. Perivascular cells, ameboid phagocytic microglial cells, and infiltrated macrophage/monocyte system were observed surrounding perivascular and intraparenchymal hemorrhagic foci. They showed phagocytic activity of degenerated nerve cell processes, and organized proteinaceous edema fluid present in the enlarged extracellular space. Endocytosis by means of clathrin coated vesicles also was observed. Facultative and professional phagocytes exhibited a full repertoire of lysosomes, phagosomes containing nerve cell debris, lipid droplets, and lipofucsin granules. Phagocytic pericytes remaining within the capillary basement membrane were also observed around perivascular hemorrhages. The inflammatory reaction was examined in young and old patients with an evolution time of brain injury ranging from 1 day to 2 years. The inflammatory process developed according to the intensity of traumatic insult, patient age, associated hematoma or hygroma, severity of vasogenic and cytotoxic oedema, and anoxic-ischemic conditions of brain parenchyma.     

Nerve cell nuclear and nucleolar abnormalities in the human oedematous cerebral cortex. An electron microscopic study using cortical biopsies

Cerebral cortical biopsies of 17 patients with clinical diagnosis of congenital hydrocephalus, complicated brain trauma, cerebellar syndrome and vascular anomaly were examined with the transmission electron microscope to study the nuclear and nucleolar abnormalities induced by moderate and severe brain oedema, and the associated anoxic-ischemic conditions of brain tissue. In infant patients with congenital hydrocephalus and Arnold-Chiari malformation two different structural patterns of immature chromatin organization were found: the clear type characterized by a clear granular and fibrillar structure of euchromatin, scarce heterochromatin masses and few perichromatin granules, and a dense granular and fibrillar euchromatin with abundant and scattered heterochromatin masses, and increased number of perichromatin granules. The lobulated nuclei exhibited an irregularly dilated and fragmented perinuclear cistern, and areas of apparently intact nuclear pore complexes alternating with regions of nuclear pore complex disassembly. In moderate traumatic brain injuries some nucleoli exhibit apparent intact nucleolar substructures, and in severe brain oedema some nucleoli appeared shrunken and irregularly outlined with one or two fibrillar centers, and others were disintegrated. The nuclear and nucleolar morphological alterations are discussed in relation with oxidative stress, peroxidative damage, hemoglobin-induced cytotoxicity, calcium overload, glutamate excitotoxicity, and caspase activation.