A New Method to Evaluate Lower Esophageal Distension Capacity in Eosinophilic Esophagitis by Using Functional Lumen Imaging Probe (EndoFLIP™).

Endoluminal functional lumen impedance planimetry (EndoFLIPTM) has become the gold standard to evaluate esophageal distensibility, although the study itself and its analysis present challenges. We propose here a new method to assess lower esophageal distension capacity that overcomes several limitations of prior approaches, including incomplete and corrupted EndoFLIPTM recordings. Esophageal distension capacity was evaluated with a 16-channel EndoFLIPTM in 10 controls and 14 patients with eosinophilic esophagitis (EoE). Controls were evaluated once. EoE patients were evaluated at baseline and after at least six weeks of treatment with orodispersible budesonide tablets, 1 mg bd. Balloon volumes were increased by 5 mL stepwise, either reaching a maximum volume of 60 mL or a maximum balloon pressure of 60 mmHg. Recordings were analyzed with a homemade R script. The mean esophageal diameter at 60 mL, D (60 mL), was calculated or extrapolated depending on whether the 60 mL volume was reached. By fitting a Michaelis-Menten curve across all measured diameters throughout all constant volume steps, the mean D (60 mL) was estimated. For control subjects, the mean ± SD value of D (60 mL) was 17.08 ± 1.69 mm, and for EoE patients at baseline, D (60 mL) was 14.51 ± 2.68 mm. After six weeks of treatment of EoE patients, D (60 mL) significantly increased to 16.22 ± 1.86 mm (paired Wilcoxon signed test: p = 0.0052), although the values for control subjects were not reached. The estimated mean esophageal diameter at 60 mL is a good proxy for esophageal distension capacity, which correlates with clinical outcomes in EoE. The method presented in this study overcomes difficulties encountered during the standard measurement protocol, allowing the analysis of recordings from incomplete and corrupted registries.

Tolerance to sterilised cow's milk in patients with eosinophilic oesophagitis triggered by milk.

BACKGROUND: Cow's milk protein is the main food trigger for eosinophilic oesophagitis (EoE) in children and adults and should be continuously avoided once identified as such. AIMS: To evaluate tolerance of sterilised cow's milk (boiled instead of UHT processing) with regard to maintenance of EoE remission, health-related quality of life (HRQoL), nutritional intake and allergic sensitisation in patients of all ages with milk-triggered EoE METHODS: We prospectively recruited patients in whom cow's milk was demonstrated to trigger EoE after an empirical food elimination diet-based study. They were given 200 ml of sterilised cow's milk twice daily for 8 weeks. Endoscopic assessment, peak eosinophil counts, oesophageal-related symptoms, HRQoL, blood eosinophils, eosinophil cationic protein (ECP), skin prick test and serum total and specific immunoglobulin E (IgE) to major milk proteins were monitored before and after sterilised milk intake. RESULTS: Eighteen patients (13 male) in EoE remission underwent a sterilised milk challenge. Twelve maintained EoE remission (<15 eos/hpf) while EoE recurred in the remainder. Endoscopic appearances deteriorated in non-tolerant patients. HRQoL scored well at baseline and was maintained among patients tolerant to sterilised milk, but deteriorated in reactive ones. No significant changes in blood eosinophil count, ECP, tryptase or total and milk-specific IgE serum levels were observed from baseline. However, cow's milk-specific IgE increased slightly in non-tolerant patients. Clinical and histological remission were maintained in patients who regularly consumed sterilised milk for 1 year. CONCLUSION: Sterilised milk did not trigger EoE in two-thirds of patients with documented milk-induced EoE, in either the short or long term.

Valvular aortic stenosis: a proteomic insight.

UNLABELLED: Calcified aortic valve disease is a slowly progressive disorder that ranges from mild valve thickening with no obstruction of blood flow, known as aortic sclerosis, to severe calcification with impaired leaflet motion or aortic stenosis. In the present work we describe a rapid, reproducible and effective method to carry out proteomic analysis of stenotic human valves by conventional 2-DE and 2D-DIGE, minimizing the interference due to high calcium concentrations. Furthermore, the protocol permits the aortic stenosis proteome to be analysed, advancing our knowledge in this area. SUMMARY: Until recently, aortic stenosis (AS) was considered a passive process secondary to calcium deposition in the aortic valves. However, it has recently been highlighted that the risk factors associated with the development of calcified AS in the elderly are similar to those of coronary artery disease. Furthermore, degenerative AS shares histological characteristics with atherosclerotic plaques, leading to the suggestion that calcified aortic valve disease is a chronic inflammatory process similar to atherosclerosis. Nevertheless, certain data does not fit with this theory making it necessary to further study this pathology. The aim of this study is to develop an effective protein extraction protocol for aortic stenosis valves such that proteomic analyses can be performed on these structures. In the present work we have defined a rapid, reproducible and effective method to extract proteins and that is compatible with 2-DE, 2D-DIGE and MS techniques. Defining the protein profile of this tissue is an important and challenging task that will help to understand the mechanisms of physiological/pathological processes in aortic stenosis valves.

Development of an optimal protocol for the proteomic analysis of stenotic and healthy aortic valves.

INTRODUCTION AND OBJECTIVES: For many years, degenerative aortic stenosis was thought to be a passive process secondary to calcium deposition in aortic valves. Although its etiology remains unknown, several authors have pointed out that degenerative aortic stenosis is associated with the same risk factors as coronary artery disease. Furthermore, histological similarities have been found between aortic valve stenosis and atherosclerotic plaque, giving rise to the hypothesis that degenerative aortic stenosis is an inflammatory process similar to atherosclerosis. Nevertheless, some data do not fit with this hypothesis and, consequently, greater understanding of the condition is needed. The main aim of this study was to develop a practical protocol for extracting protein for use in proteomic analysis from both stenotic and healthy aortic valves. METHODS: The study was carried out using a number of different proteomic methods: two-dimensional electrophoresis, mass spectrometry and additional techniques. RESULTS: We developed a simple and reproducible methodology in the laboratory for carrying out the proteomic analysis of human aortic valves and for identifying their component proteins. CONCLUSIONS: We developed a simple and reproducible method for extracting protein that can be used with mass spectrometry and that makes it possible to carry out large-scale proteomic analysis of stenotic aortic valves. Furthermore, the methodology will significantly increase our understanding of the valve proteome.

Obtención de un protocolo óptimo para el análisis proteómico de válvulas aórticas humanas sanas y estenóticas / Development of an optimal protocol for the proteomic analysis of stenotic and healthy aortic valves

Introducción y objetivos. Durante años, la estenosisaórtica (EA) degenerativa ha sido considerada como un proceso pasivo secundario al depósito de calcio en la válvula aórtica. Aunque no se conoce su etiología, diversos autores han señalado que comparte los mismos factores de riesgo que la enfermedad arterial coronaria. Además, se han encontrado similitudes histológicas en la válvula aórtica estenótica y la placa de ateroma, lo que ha llevado a la hipótesis de que la EA degenerativa es un proceso inflamatorio similar a la aterosclerosis. No obstante, existen algunos datos discordantes con esta teoría, lo que hace necesario profundizar en el conocimiento de esta patología. El principal objetivo de este trabajo es la puesta a punto de un protocolo de extracción proteínica eficaz paraválvulas estenóticas aórticas y válvulas aórticas sanas, compatible con la realización de estudios proteómicos. Métodos. Para el desarrollo del objetivo planteado, sehan utilizado diferentes abordajes proteómicos: electroforesis bidimensional, espectrometría de masas y técnicas complementarias. Resultados. Hemos puesto a punto una metodología, sencilla, reproducible y desarrollada en el laboratorio, que permite el análisis proteómico de la válvula aórtica humana, así como la identificación de las proteínas quelas componen. Conclusiones. Obtención de un método de extracción de proteínas sencillo, reproducible y compatible con la espectrometría de masas, que permite el análisis a gran escala del proteoma de válvulas con estenosis aórtica. Además, esta metodología aumentará de forma significativa nuestro conocimiento del proteoma valvular (AU)

Introduction and objectives. For many years, degenerative aortic stenosis was thought to be a passive process secondary to calcium deposition in aortic valves. Although its etiology remains unknown, several authors have pointed out that degenerative aortic stenosis is associated with the same risk factors as coronary artery disease. Furthermore, histological similarities have been found between aortic valve stenosis and atherosclerotic plaque, giving rise to the hypothesis that degenerative aortic stenosis is an inflammatory process similar to atherosclerosis. Nevertheless, some data do not fit with this hypothesis and, consequently, greater understanding of the condition is needed. The main aim of this study was to develop a practical protocol for extracting protein for use in proteomic analysis from both stenotic and healthy aortic valves. Methods. The study was carried out using a number of different proteomic methods: two-dimensional electrophoresis, mass spectrometry and additional techniques. Results. We developed a simple and reproducible methodology in the laboratory for carrying out the proteomic analysis of human aortic valves and for identifying their component proteins. Conclusions. We developed a simple and reproducible method for extracting protein that can be used with mass spectrometry and that makes it possible to carry outlarge-scale proteomic analysis of stenotic aortic valves. Furthermore, the methodology will significantly increase our understanding of the valve proteome (AU)

An optimum method designed for 2-D DIGE analysis of human arterial intima and media layers isolated by laser microdissection.

The formation and progression of atherosclerotic lesions involve complex mechanisms which are still not fully understood. A variety of cell types from the distinct arterial layers are implicated in the whole process from lipid accumulation within the vascular wall to plaque development and final rupture. In the present work, we employ the combination of laser microdissection and pressure catapulting and 2-D DIGE saturation labeling to investigate the human intima and media sub-proteomes isolated from atherosclerotic (coronary and aorta) or non-atherosclerotic vessels (preatherosclerotic coronary arteries). Laser microdissection and pressure catapulting allows the specific isolation of regions of interest. In turn, DIGE saturation labeling overcomes the limitation of extensive microdissection times to recover the protein amount required to perform comparative 2-DE, particularly when dealing with tissue regions rich in myofilament proteins, which result in low protein recovery. The compatibility and optimum performance of both techniques were investigated in detail, paying special attention to tissue staining and protein solubilization. Since scarce amount of protein obtained from microdissected tissue made it impossible to directly perform protein identification from 2-DE spots by MS, we performed in-solution digestion followed by LC-MS/MS analysis of total protein extracts from intima and media in order to get an overall picture of protein composition. Proteins so identified confirm the nature of the isolated regions. Finally, similar spot resolution on 2-D DIGE gels was obtained for the different human artery types (coronary, aorta) and studied layers (intima, media), setting the basis for future clinical comparative studies.

Permanent third-degree atrioventricular block as clinical presentation of an intracardiac bronchogenic cyst.

Bronchogenic cysts are the most common primary cysts in the mediastinum. However, intracardiac bronchogenic cysts are uncommon. The present case represents a unique situation, in which an intracardiac bronchogenic cyst at the region of the atrioventricular node presented as permanent complete atrioventricular block (AVB) and was associated with the presence of an ostium secundum atrial septal defect.