Tese de mestrado. Engenharia Biomédica. 2006. Faculdade de Engenharia. Universidade do Porto, Universidade de Aveiro. Departamento de Engenharia Mecânica

Dissertação apresentada para obtenção do grau de Doutor em Engenharia Mecânica, na Faculdade de Engenharia da Universidade do Porto, sob a orientação dos Profs. Drs. António Torres Marques e George Jeronimidis

No description available.

Microscopic polyangiitis (MPA) is a rare condition characterized by nonspecific symptoms of fatigue, fever, or loss of appetite and additional symptoms related to areas of involvement. The rarity of the disease and its variable presentation can delay its diagnosis in the acute setting, potentially leading to delay in effective treatment and poor outcomes. This is a case report of a 68-year-old man, who presented to the hospital after being found poorly responsive at home. He was sedated and intubated in the field and brought to the ER. He was found to be hypotensive. History revealed abdominal aortic aneurysm, but no known history of GI bleed or use of blood-thinners. Physical exam revealed rhonchi throughout both lung fields, a soft, nondistended abdomen with normoactive bowel sounds, and cool extremities with weak palpable pulses. CBC was suggestive of anemia. BUN and creatinine were elevated indicating kidney injury. CT abdomen showed no acute pathology and an intact abdominal aortic aneurysm. CT chest revealed left lower lobe atelectasis and bilateral pulmonary edema. CT head was consistent with watershed infarction. Numerous blood transfusions were given. However, slow decline in hemoglobin continued to occur. Colonoscopy was considered for possible identification of the source of bleeding, however was not performed due to the family’s wish for conservative management. Bronchoscopy was performed, revealing extensive mucus plugging and bloody plugs. He was eventually weaned off sedation but remained largely unresponsive, occasionally opening his eyes to stimuli. On hospital day 7, necrotic lesions were noted on the patient’s ear and fingers. These findings prompted serologic studies for vasculitis that resulted in positive p-ANCA and myeloperoxidase antibodies, consistent with MPA. He was started on pulse dose IV steroids resulting in modest improvement in renal parameters and hemoglobin, but no discernible change in neurological status. The patient was transitioned to comfort care and palliative extubation was performed. Four days later, the patient expired. This case illustrates the potential for hypovolemic shock as a consequence of undiagnosed vasculitis. In this case, a definite diagnosis was not made, due to inability to perform a lung biopsy because of patient being on comfort measures. However, given that he was p-ANCA and myeloperoxidase positive, along with visualized blood on bronchoscopy, a presumptive diagnosis of MPA can be made. Although this patient may not have recovered even with early immunosuppressive treatment, vasculitis should be considered as a differential diagnosis in patients presenting in hypovolemic shock, especially if history, exam and noninvasive testing are unrevealing.

Gegenstand der vorliegenden Arbeit waren Untersuchungen zu zellulären Mechanismen ANCA-assoziierter systemischer Vaskulitiden. Es wurde dabei zunächst die ANCA-induzierte Aktivierung neutrophiler Granulozyten untersucht. Es konnte gezeigt werden, daß die Quervernetzung der ANCA-Antigene auf der Oberfläche neutrophiler Granulozyten die Generation von Sauerstoffradikalen auslöst. Der zweite Komplex beschäftigte sich mit der Regulation der Apoptose neutrophiler Granulozyten, die einen zentralen Mechanismus für die Auflösung von Entzündungen darstellt. Die Ergebnisse zeigen, daß die Apoptose neutrophiler Granulozyten durch IL-8 verzögert und durch TNF-alpha akzeleriert wird. Dabei führt die Interaktion mit extrazellulären Matrixsubstanzen wie Fibronektin zu einer weiteren Akzeleration der TNF-alpha-induzierten Apoptose. Die Bedeutung der Tyrosin Phosphorylierung für diesen Prozess wird gezeigt, und Ly-GDI, ein regulatorisches Protein für kleine GTPase-Proteine der Ras-Superfamilie, wurde als ein Substrat der Tyrosin-Kinasen charakterisiert. Letztlich konnten neue Kandidaten-Proteine für die Regulation der Lebensspanne neutrophiler Granulozyten identifiziert werden. / Mechanisms of ANCA-associated vasculitis were studied. First, the activation of human neutrophils by ANCA was investigated. The data demonstrate the important role of cross-linking ANCA-antigens by the auto-antibodies on the cell surface for the initiation of a respiratory burst. The second issue was the regulation of neutrophil apoptosis that is central to the resolution of inflammation. These experiments indicate that apoptosis was delayed by IL-8 and accelerated by TNF-alpha. Interaction with extracellular matrix proteins, such as fibronectin resulted in further acceleration of apoptosis. The important role of protein tyrosine phosphorylation was demonstrated, and Ly-GDI, a regulator of GTPase-proteins of the ras-superfamily was characterized. Finally, proteins that may control the life span of neutrophils were identified.

L’acide mycophénolique (MPA) est un immunosuppresseur très prometteur dans le traitement des maladies auto-immunes (MAI) telles que le lupus érythémateux disséminé (LED) et les vascularites à ANCA, et disponible sous deux formes pharmaceutiques : le mycophénolate mofétil (MMF) et le mycophénolate sodique (EC-MPS). Les études menées chez les patients transplantés recommandent le dosage plasmatique et le suivi pharmacocinétique (PK) du MPA, dans un objectif d’optimisation thérapeutique. A ce jour, ce suivi est encore inexistant dans les MAI, et les données de corrélation concentrations-efficacité thérapeutique, sur lesquelles se base l’optimisation, demeurent toujours rares dans ce domaine. Les travaux présentés dans cette thèse s’inscrivent dans l’étude des corrélations PK/pharmacodynamie (PD) du MPA dans les MAI. Ces travaux ont permis de proposer des schémas et des outils d’optimisation des traitements à base de MPA pour ces patients. Pour cela, les concentrations plasmatiques du MPA et de son métabolite 7-O-glucuronide (MPAG) ont été déterminées pour 53 patients présentant de manifestations extra-rénales de MAI à l’aide d’une méthode de chromatographie couplée à la spectrométrie de masse. Les paramètres PK ont été estimés pour MMF et EC-MPS dans les deux groupes de MAI. D’après ces travaux, l’optimisation du MMF chez les patients atteints de MAI peut reposer sur le suivi de la concentration à 12 h (C12) en MPA. Un seuil de 3 mg/L est proposé afin de maintenir la rémission dans le LED, mais reste à définir dans les vascularites. Pour EC-MPS, une stratégie de prélèvements limités basée sur la mesure de la concentration maximale et la C12 est nécessaire pour estimer l’aire sous la courbe des concentrations entre 0 et 12 h du MPA. / Mycophenolic acid (MPA), the active form of both mycophenolate mofetil (MMF) and enteric-coated mycophenolate sodium (EC-MPS), is an immunosuppressant increasingly used in the treatment of autoimmune diseases such as systemic lupus erythematosus (SLE) and ANCA-associated vasculitis. In transplant recipients, therapeutic drug monitoring (TDM) of MPA is widely used to prevent acute organ rejection. However, MPA TDM is currently not available in autoimmune diseases, as data on the pharmacokinetic (PK)/pharmacodynamic (PD) relationships are very sparse in this indication. Our aim was to study the possible PK/PD relationships of MPA in patients with non-renal manifestations of SLE or ANCA-associated vasculitis. An assay based on liquid chromatography coupled with mass spectrometry was applied to the PK study of MPA and its major glucuronide metabolite (MPAG) in 53 SLE and vasculitis patients receiving either MMF or EC-MPS. According to our results, in SLE patients with non-renal manifestations, TDM based on the measurement of MPA 12-h trough concentration (C12) would allow optimizing therapies with MMF. A 3-mg/L efficacy threshold could be proposed to prevent clinical flares under MMF maintenance therapy. For EC-MPS, a limited sampling strategy including MPA maximum concentration and C12 is necessary to estimate the area under the curve between 0 and 12-h of MPA.

Anti-Neutrophile Cytoplasmatische Antikörper verursachen nekrotisierende Vaskulitiden kleiner Blutgefäße. Die Serinprotease PR3 ist ein ANCA-Zielantigen, welches von zirkulierenden ANCA auf der Zellmembran erkannt wird. ANCA aktivieren neutrophile Granulozyten, die dann die nekrotisierende Vaskulitis verursachen. Das Membran-PR3 Expressionsmuster ist bimodal wobei mPR3-niedrig- und mPR3-hoch-exprimierende Zellen existieren. Wir testeten die Hypothese, dass ein Membranrezeptor eine hohe mPR3-Expression vermittelt. Wir verwendeten humane neutrophile Granulozyten, neutrophil-differenzierte Stammzellen und transfizierte HEK293 Zellen. Wir identifizierten das Glykoprotein CD177 als einen mPR3-präsentierenden Rezeptor. CD177 zeigte eine spezifische Bindung von reifem PR3-Protein, nicht aber von einem unprozessierten PR3. Wir separierten die mPR3-Zellpopulationen und führten Durchflusszytometrie, Giemsa-Färbung, Western Blot-Experimente und RT-PCR für die PR3 und CD177 mRNA-Expression durch. Wir fanden, dass die mPR3hoch neutrophilen Granulozyten PR3- und CD177-Protein enthielten, während in den mPR3niedrig neutrophilen Granulozyten nur PR3, aber kein CD177 detektierbar war. Die CD177-Regulation vollzog sich auf transkriptioneller Ebene, da die Zellen, die negativ für das CD177-Protein waren auch keine mRNA transkribierten. Um die Grundlage der fehlenden CD177-Transkription zu analysieren, identifizierten wir den Transkriptionsstart von CD177 für eine anschließende Mutations- und SNP-Analyse. Die CD177-Sequenzen der proteinkodierenden Regionen und der Intron-Exon-Übergänge der beiden Zellpopulationen waren identisch. Jedoch fanden wir, dass das CD177-Gen einer monoallelischen Expression unterliegt. Es wurde dabei maternale als auch paternale monoallelische Expression detektiert. In weiterführenden Untersuchungen soll der Regulationsmechanismus der monoallelischen CD177-Expression charakterisiert werden. / Anti-Neutrophil Cytoplasmic Antibodies cause necrotizing small-vessel vasculitis. The serine protease PR3 provides a main ANCA target antigen and is recognized by circulating ANCA on the neutrophil cell surface. ANCA activate neutrophils and activated neutrophils cause vasculitis. The membrane-PR3 expression pattern is bimodal in that low and high mPR3 expressing cells can be distinguished. We tested the hypothesis that a membrane receptor mediates mPR3high expression. We studied human neutrophils, neutrophilic differentiated CD34-positive hematopoietic stem cells and transfected HEK293 cells. We identified the glycoprotein CD177 as an mPR3 presenting receptor. CD177 demonstrated specific binding of mature, but not of unprocessed pro-PR3. We separated the two mPR3 populations and performed cytometry analysis, Giemsa staining, western blot analysis and RT-PCR for PR3 and CD177 expression. We detected PR3 and CD177 protein in mPR3high expressing neutrophils, whereas only PR3, but no CD177 was found in mPR3low expressing cells. Regulation took place on a transcriptional level because cells that were negative for CD177 protein were also negative for mRNA. To further study this finding, we identified the CD177 transcription start for a subsequent mutation and SNP analysis. CD177 sequences of the protein-coding regions and the intron-exon regions did not differ in both populations. However, we found a monoallelic CD177 expression and were able to detect maternal as well as paternal allele expression. Future experiments will elucidate the mechanisms that control monoallelic CD177 gene expression.

Dissertação apresentada para obtenção do grau de Mestre em Engenharia Biomédica, na Faculdade de Engenharia da Universidade do Porto, sob a orientação na FEUP do Professor Doutor António Torres Marques, e na Universidade de Aveiro do Professor Doutor José António Simões

Tese de mestr.. Engenharia Biomédica. Faculdade de Engenharia. Universidade do Porto. 2001

<p>Wegener´s granulomatosis (WG) is an unusual, serious, systemic vasculitis with specific clinical findings. The studies in this thesis aim at broadening our understanding of the aetiology and outcome of WG.</p><p>Patients with WG were identified in the In-patient Register 1975-2001. During this time the incidence increased three-fold, and neither ANCA-related increased awareness, nor diagnostic drift, seem to fully explain this trend, but it is still unclear if a true rise in incidence exists. </p><p>Anti- neutrophil cytoplasmic antibodies (ANCA) have been presented as highly specific for vasculitis. In a series of consecutive cANCA/PR3-ANCA positive patients, we investigated the positive predictive value for ANCA, and the outcome of patients with a positive cANCA/PR3-ANCA but not vasculitis. These patients have a low future risk of developing vasculitis, possibly indicating that ANCA, in this setting, reflects neutrophil activating properties not specific to vasculitis.</p><p>By linkage of the WG-cohort, and randomly selected population controls, to the Multi-generation register, we identified all first-degree relatives and spouses of patients and controls, totally encompassing some 2,000 patients and 70,000 relatives. Familial aggregation of WG was the exception, with absolute risks of < 1 per 1000.However, relative risks in first-grade relatives amounted to 1.56 (95% CI 0.35-6.90) such that a moderate familial aggregation cannot be excluded.</p><p>In the WG-cohort, cancer occurrence and risk was compared to that of the general population. Patients with WG have an overall doubled risk of cancer, with particularly increased risks of bladder-cancer, haematopoietic cancers including lymphomas and squamous skin-cancer. In a case-control study nested within the WG-cohort, treatment with cyclophosphamide was compared among bladder-cancer patients and matched cancer-free controls. Absolute risk of bladder cancer as high as 10% some years after diagnosis were found, and this risk can partly be attributed to cyclophosphamide-treatment, with a dose-response relationship.</p>