Tesis sobre el tema "Eosinophilic granulomatosis with polyangiitis"

Granulomatosis with polyangiitis (GPA, formerly known as Wegener’s granulomatosis) is a rare and sometimes fatal systemic autoimmune disease. Anti-neutrophil cytoplasmic antibodies (ANCAs) specific for proteinase 3 (PR3) are associated with GPA. However, the pathogenesis of GPA is not yet clear. Our aim was to investigate the local autoimmune response, circulating immune modulatory cells and cells expressing the immune suppressor molecules programmed death 1 (PD-1) and its ligands in GPA. In mucosa from GPA patients, activated B cells were observed located alongside PR3 expressing cells and B cell survival factors BAFF and APRIL, which was produced by the granulomas and giant cells. B cells were proliferating and persistent in biopsies. However no evidence of B cell clones from the mucosal biopsies circulating in peripheral blood was observed in GPA. An increased frequency of circulating TFH cells and a reduced frequency of Treg cells was observed in peripheral blood from GPA patients on conventional therapies compared to healthy controls. No such difference was found in GPA patients treated with rituximab. The frequency of circulating TFH and Treg cells was found to be inversely correlated in human peripheral blood. No difference in the relative quantity of mRNA encoding PD-1 in lymphocytes and monocytes was found in GPA patients compared with healthy controls. Lower percentage of CD14+ monocytes expressing PD-1 was observed in GPA patients. Lower relative quantity of mRNA encoding PD-1 ligands PD-L1 and PD-L2 in T cells and monocytes was observed in GPA patients. In conclusion, data in this thesis identifies activated B cells alongside auto-antigens and B cell survival factors in the mucosa in GPA. A negative correlation between TFH and Treg cells is observed that implies the balance between T cell subsets and its B cell dependence are associated with disease activity in GPA. The deficiency of PD-L1 and PD-L2 mRNA in lymphocytes and monocytes may contribute to the pathogenesis of GPA.

The full abstract for this thesis is available in the body of the thesis, and will be available when the embargo expires.
Medicine, Faculty of
Experimental Medicine, Division of
Medicine, Department of
Graduate

Granulomatosis with Polyangiitis (GPA) is a granulomatous inflammatory disease. It is generally described as a systemic disorder which can present with a localized presentation like the orbit. Orbital GPA can be the initial manifestation of GPA where over time the disease may progress and become severe, involving vital organs. This study aimed to look for biomarkers in orbital GPA biopsies that could indicate diagnosis and be a predictor for the progression of the disease. To identify GPA patients, retrospective examination of patients’ medical records, who had undergone orbital biopsies for orbital inflammatory disease (OID), over a 21 year period, was performed. Long term outcomes of these patients were studied. Further subjective and objective histology analyses were done on haemotoxylin and eosin (H&E) tissue preparations. Comparison of cellular activity in biopsies of GPA and other OID were performed. Further T cells, B cells and macrophage phenotypes and their cytokines, were investigated with immunohistochemistry (IHC). IHC cell count comparisons were performed between GPA, sarcoidosis and idiopathic inflammatory orbital diseases (IIOD) biopsies. Results showed that in patients who presented with orbital GPA with no systemic manifestations, the disease remained localised and did not progress to systemic form, over time. H&E tissue biopsies examination showed that GPA tissues had a higher cellular activity compared to OIDs. Vasculitis and necrosis were found to be independently associated with the diagnosis of orbital GPA but these features were unreliable for diagnosis as a number of the biopsies did not exhibit these features. In immunohistochemistry staining, T cells, B cells and macrophage subtypes counts were comparable between GPA, sarcoidosis and IIOD. Nonetheless cytokines IL-17, IL-23 and BAFF-receptor (BAFF-R), were found significantly more in GPA compared to sarcoidosis and IIOD. This suggests that these cytokines possibly have a role in the pathogenesis of GPA and may have diagnostic value.

INTRODUÇÃO: Eventos vasculares e imunológicos são centrais na patogênese da granulomatose com poliangeíte (GPA). Moléculas de adesão celular tem papel fundamental no recrutamento de células inflamatórias do sangue para os tecidos. Diferentes leitos vasculares apresentam particularidades na expressão de moléculas de adesão celular explicando talvez parte da especificidade da GPA por determinados órgãos. A elevação no nível sérico de moléculas de adesão celular e aumento da expressão destas em amostras de biópsia renal já foram demonstrados em pacientes com vasculite ANCA associada. No entanto, o fenômeno ainda não foi estudado in situ no pulmão. OBJETIVO: O objetivo deste estudo foi analisar o padrão de expressão endotelial pulmonar de três moléculas de adesão celular na GPA, in situ: molécula de adesão intercelular 1 (ICAM-1), molécula e adesão vascular 1 (VCAM-1) e E-selectina. MÉTODOS: Examinou-se a expressão endotelial de ICAM-1, VCAM-1, E-selectina usando marcação imuno-histoquímica em secções de pulmão fixadas e parafinadas de lesões de GPA (n = 8 casos, 90 secções transversais de vasos analisados). Foram também analisados controles positivos: amostras de pulmão de doença intersticial associada à esclerodermia (SScl) (n = 8 casos, 96 secções transversais de vasos analisados) e controles negativos (n = 9 casos, 90 secções transversais de vasos analisados). A quantificação imuno-histoquímica foi realizada no aumento de 400x usando a técnica de point-counting. RESULTADOS: ICAM-1: A expressão endotelial mediana de ICAM-1 esteve aumentada de forma semelhante na GPA e na SScl (81% and 73%, respectivamente; p = 0.97). A comparação com o grupo controle (26.3%) revelou diferença estatisticamente significativa entre controle e GPA (p <0.001) quanto entre controle e SScl (p = 0.017). VCAM-1: A expressão mediana de VCAM-1 esteve significativamente aumentada na GPA se comparada a SScl (79.5% vs 41.4%; p = 0.012), no entanto, a expressão endotelial de VCAM-1 nos controles também esteve moderadamente aumentada (49.8%) e não houve diferença estatística entre SScl e controles ( p = 0.549) ou entre GPA e controles (p = 0.242). E-selectina: A expressão endotelial mediana de E-selectina esteve aumentada de forma semelhante na GPA e SScl (100% e 88.2%, respectivamente; p = 0.272). A comparação com o grupo controle (13.8%) revelou diferença estatisticamente significativa entre controle e GPA (p < 0.001) e controle e SScl (p = 0.045). CONCLUSÃO: Esses achados evidenciam o fenômeno de ativação endotelial pulmonar in situ em lesões de GPA. O perfil de expressão de moléculas de adesão parece ter particularidades em diferentes doenças a exemplo da maior expressão de VCAM-1 na GPA em relação à SScl. Essas observações contribuem para o conhecimento fisiopatogênico na GPA.
INTRODUCTION: Vascular and immunologic processes are central to the pathogenesis of granulomatosis with polyangiitis (GPA). Endothelial cellular adhesion molecules have a central role in recruiting leukocytes to sites of inflammation. Moreover, different vascular beds are phenotypically and functionally distinct with regard to expression of cellular adhesion molecules. They have been shown to be elevated in sera and in renal biopsies of patients with active ANCA-associated vasculitis. Despite of that, the expression of cellular adhesion molecules has not been studied in situ in the lungs. OBJECTIVE: Within this context, the aim of this study was to analyze the in situ pulmonary endothelial immunohistochemical pattern of expression of three cellular adhesion molecules in GPA: intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1) and E-selectin . METHODS: For such, we examined formalin-fixed, paraffin-embedded sections of lung lesions of GPA (n = 8 cases, 90 transverse sections of pulmonary vessels), negative controls which were obtained from autopsies ( n = 9 cases, 90 transverse sections of pulmonary vessels analyzed) and positive controls which were cases of interstitial lung disease associated with systemic sclerosis (SScl) (n = 8 cases, 96 transverse sections of pulmonary vessels). The quantification of the immunohistochemical staining was performed at x400 magnification using the technique of point-counting, previously described. RESULTS: ICAM-1: Median endothelial expression of ICAM-1 was similarly enhanced in GPA and SScl (81% and 73%, respectively; p = 0.97). When compared to controls (26.3%), both GPA (p <0.001) and SScl (p = 0.017) had significantly higher endothelial expression of ICAM-1. VCAM-1: Median endothelial expression of VCAM-1 was significantly enhanced in GPA when compared to SScl (79.5% vs 41.4%; p = 0.012), however the endothelial expression of VCAM-1 in the controls was also moderately enhanced (49.8%) and no statistically significant difference was found between SScl and controls ( p = 0.549) or between GPA and controls (p = 0.242). E-selectin: Median endothelial expression of E-selectin was similarly enhanced in GPA and SScl (100% and 88.2%), respectively; p = 0.272. When compared to controls (13.8%), both GPA (p < 0.001) and SScl (p = 0.045) had significantly higher endothelial expression of E-selectin. CONCLUSION: These observations are evidence of in situ pulmonary endothelial activation in lesions of GPA. The profile of expression of cellular adhesion molecules seems to be particular for each disease state and timing as evidenced by the enhanced expression of VCAM-1 in GPA when compared to SScl. These observations add information to the pathogenetic knowledge of GPA

Les protéases à serine du neutrophile (PSN), maturées par une protéase à cystéine nommée la cathepsine C (CatC), sont des acteurs majeurs dans la dégradation tissulaire et la réponse immunitaire médiées par les polynucléaires neutrophiles (PNN). La protéinase 3 (PR3), l’une des PSN, est l’auto-antigène majeur de la granulomatose avec polyangéite (GPA) qui est une vascularite systémique auto-immune. Le ciblage pharmacologique des PSN par des inhibiteurs est une approche intéressante pour le traitement de la GPA mais également pour toutes les maladies inflammatoires chroniques impliquant les PSN mais reste sans résultat jusqu’à maintenant. L’inactivation génétique de la CatC chez les patients atteints du syndrome de Papillon-Lefèvre est associée à une élimination presque totale des PSN. Au cours de ce travail de thèse, nous avons ciblé la CatC à l’aide d’un inhibiteur de type nitrile dans un modèle de cellules souches différenciées en PNN et nous avons obtenu une élimination presque complète de la PR3 intracellulaire et membranaire. Dans un deuxième temps, nous avons identifié la cathepsine S (CatS) comme la protéase activatrice majeure de la CatC dans les précurseurs neutrophiliques. Nous avons donc ciblé la CatS avec un inhibiteur de type nitrile pendant la différentiation des PNN ce qui a diminué significativement les taux de PSN. L’inhibition pharmacologique de la CatC pourrait donc éliminer l’auto- antigène de la GPA et constituer une nouvelle stratégie thérapeutique innovante pour contrôler l’auto-immunité et l’inflammation associée dans cette pathologie. Une combinaison d’inhibiteurs de CatS et de CatC pourrait s’avérer plus efficace pour éliminer la PR3 ainsi que les PSN pro-inflammatoires dans la GPA et dans d’autres pathologies inflammatoires chroniques
Neutrophil serine proteases (NSP), maturated by cathepsin C (CatC), are the major players in neutrophil-mediated tissue degradation and immune response. Proteinase 3 (PR3) is the main target antigen of anti-neutrophil cytoplasmic auto-antibodies (ANCA) in granulomatosis with polyangiitis (GPA), a systemic small-vessel vasculitis. The pharmacological targeting of NSP by proteinase inhibitors is promising approach for GPA treatment but also for all chronic inflammatory diseases involving NSP but remains unsuccessful until now. The genetic inactivation of CatC in patients with Papillon-Lefèvre syndrome is associated with almost complete elimination of NSP in blood neutrophils. In this work, we targeted CatC using a nitrile inhibitor in neutrophil generated from umbilical cord stem cells and we observed strong reductions in intracellular and membrane- bound PR3. Among the five-proCatC activating proteases, we found CatS in neutrophilic precursor cells and in mature neutrophils. Pharmacological inhibition of CatS in neutrophils generated from stem cells resulted in significant reduction of cellular NSP. The pharmacological inhibition of CatC could help eliminate the auto- antigen of GPA and constitute a novel therapeutic strategy to reduce auto-immune inflammation in this pathology. Pharmacological targeting of both CatS and CatC might help to efficient inhibition and elimination of NSP in GPA and in chronic inflammatory diseases

Os alelos HLA-DPB1*04 e HLA-DRB1*15 estão fortemente associados à Granulomatose com poliangeíte (GPA). Neste estudo, analisamos se os pacientes brasileiros com diagnóstico de GPA apresentam uma base genética na região HLA. Conduzimos um estudo caso-controle, em que analisamos os alelos da região HLA classe I e II em 55 pacientes com diagnóstico de GPA, atendidos no ambulatório de Vasculites Pulmonares do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, e comparamos com os resultados de 110 controles saudáveis. Comparamos também quatro diferentes apresentações clínicas da GPA e a positividade do anticorpo anticitoplasma de neutrófilos (ANCA) com os alelos da região HLA classe I e II. Foi também construída uma árvore de decisões, usando o algoritmo de CART, para a verificação da associação entre os alelos HLA e GPA. Como resultados, observamos que a GPA esteve fortemente associada à presença dos alelos DPB1*04 e DRB1*15 (p = 0,007, odds ratio [OR]: 2,9, 95% intervalo de confiança [IC]: 1,09-3,8; p = 0,006, OR: 2,87, 95% IC: 1,44-4,75, respectivamente) e não à presença do alelo DRB1*04. O alelo DRB1*13 esteve associado com proteção contra GPA (p = 0,042, OR: 0,42, 95% CI: 0,21-0,99). O alelo DPB1*04 esteve significativamente associado a GPA e ANCA-C positivo (OR: 5,47) e à presença de insuficiência renal aguda (p = 0,01037). Concluímos que houve uma interdependência significativa entre os alelos DPB1*0401, DPB1*0402, DRB1*13, C*2 e GPA. Na população estudada, quando o alelo DPB1*04 esteve presente em homozigose, o risco de GPA foi de 81%. Quando o alelo DPB1*0401 esteve ausente ou em heterozigose com o DPB1*0402, como o outro alelo, ou DPB1*0402 esteve em homozigose, o risco da GPA foi de 52,9%. No caso de ausência dos alelos DPB1*0401, DPB1*0402 e DRB1*13, a presença do alelo C*2 aumentou o risco da GPA para 62,5%. Finalmente, na ausência do alelo DPB1*0401 e DPB1*0402 e na presença do alelo DRB1*13, o risco de GPA diminuiu para 0%
The alleles HLA-DPB1*04 and HLA-DRB1*15 are strongly associated with granulomatosis with polyangiitis (GPA). In this study, we examined whether Brazilian patients with GPA had an HLA region genetic background. We conducted a case-control study, in which we analysed alleles of HLA region class I and II from 55 patients with GPA (at the Pulmonary Vasculitis Clinic of the University of São Paulo) and compared the results with those from 110 healthy controls. Comparisons were also performed for 4 different clinical presentations of GPA and anti-neutrophil cytoplasmic antibody (ANCA) positivity and the HLA class I and II region alleles. A tree model decision analysis was conducted using CART algorithm. Our results showed that GPA was strongly associated with alleles DPB1*04 and DRB1*15 (p = 0.007, odds ratio [OR]: 2.9, 95% confidence interval [CI]: 1.09-3.8; p = 0.006, OR: 2.87, 95% CI: 1.44-4.75, respectively) and not with the allele DRB1*04. DRB1*13 allele was associated with protection against GPA (p = 0.042, OR: 0.42, 95% CI: 0.21-0.99). DPB1*04 was significantly associated with GPA plus positive C-ANCA (OR: 5.47) and acute renal failure (p = 0.01037). We concluded that there was a significant interdependence among alleles and GPA. In our population, when allele DPB1*04 was presented in homozygous, the risk of GPA was 81%. When DPB1*0401 allele was absent or heterozygous with DPB1*0402 as the other allele, or DPB1*0402 was homozygous, the risk of disease was 52.9%. If DPB1*0401, DPB1*0402, and DRB1*13 were absent, the presence of C*2 increased the risk of GPA to 62.5%. Finally, in the absence of DPB1*0401 and DPB1*0402 and the presence of DRB1*13, the risk of GPA decreased to 0%

Eosinophilic granulomatosis with polyangiitis (EGPA), formerly called Churg–Strauss syndrome, is a rare systemic vasculitis characterised by peripheral eosinophilia, eosinophil tissue infiltration and disseminated necrotizing vasculitis with extravascular granulomas. In 30% of EGPA patients (especially during active phase of disease) cardiovascular complications represent a major cause of morbidity and mortality. Recent studies explored the role of oxidative stress in EGPA pathogenesis, suggesting that its clinical manifestations could be caused by an imbalance in redox homeostasis. Oxidative stress, a condition characterized by an imbalance between reactive oxygen species (ROS) and/or reactive nitrogen species (RNS) production and antioxidant defences, has been widely implicated in several pathological disorders such as diabetes, cancer, degenerative diseases, chronic inflammatory diseases, cardiovascular diseases and thrombosis. It is now widely accepted that inflammation and oxidative stress display a prominent role in cardiovascular diseases pathogenesis. Proteins are the main targets for ROS, with consequent protein structure and function alterations. Compared with other plasma proteins, fibrinogen, which plays a key role in blood clotting, fibrinolysis, cellular and matrix interactions and in the inflammatory response, is particularly susceptible to oxidation, 20x than albumin. Fibrinogen oxidation can affect fibrinogen function, influencing fibrin formation, clot structure and fibrin susceptibility to lysis. Based on this background, the present project was undertaken to investigate, in EGPA patients, the effect of oxidative stress on fibrinogen structural/functional features. In 35 EGPA patients and 35 age-matched healthy controls we investigated plasma redox status, blood leukocyte ROS production and, in fibrinogen purified fractions, its structure and function. H2DCF-DA fluorescent probe was used to detect leukocyte intracellular ROS levels which were quantified by FACS analysis. Plasma malondialdehyde (MDA) concentration (colorimetric assay) was used as an index of lipid peroxidation and Oxygen Radical Absorbance Capacity (ORAC) for the assessment of plasma total antioxidant capacity. Fibrinogen was purified from patients and controls using the ethanol precipitation method. Furthermore, thrombin-catalyzed fibrin polymerization and plasmin-induced fibrinolysis were investigated in patients and controls. Fibrinogen conformational properties were explored by Intrinsic Fluorescence and Circular Dichroism Spectroscopy (CD). Fibrinogen oxidation was evaluated fluorimetrically by dityrosine content. Overall, the obtained results are indicative of an altered redox status in EGPA patients and suggest its association with impaired structural and functional fibrinogen properties. Our data demonstrate that oxidative-mediated fibrinogen structure modifications such as those observed in EGPA patients are associated with thrombosis tendency, suggesting new potential targets for innovative therapeutic approaches.

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014
Vasculitides are systemic diseases characterised by the presence of vascular inflammation. They can be classified according to the American College of Rheumatology Criteria and to the Chapel Hill Consensus Conference Nomenclature of Vasculitides. These do not enable to make the diagnosis, but to place the patients in diagnostic subgroups. The diagnosis is given by a compatible fenotype, specific serology or imagiology and confirmation is set by biopsy. There are different scores that allow the measurement of the severity and damage caused by vasculitides. Granulomatosis with Polyangiitis is an auto-imune disease characterised by granulomatous inflammation of the respiratory airways and renal and pulmonary small vessel vasculitis. It is more common among caucasians and white male in Europe. The symptoms usually start between the ages of 45 and 65. It is associated with the presence of antineutrophil cytoplasmic antibodies (ANCA) aginst proteinase 3, yet its etiology is still unknown. The treatment is based on stage and disease activity. In severe disease it is administrated cyclophosphamide with glucocorticoids, which are then changed to less potent imunosupressors after the induction of remission. There are new biological therapies under investigation.
As vasculites são doenças sistémicas caracterizadas por inflamação vascular. Podem ser classificadas de acordo com critérios definidos pela American College of Rheumatology e pela Nomenclatura da Chapel Hill Consensus Conference. Estas classificações não permitem fazer o diagnóstico, mas sim agrupar os pacientes com vasculite em subgrupos de diagnóstico. O diagnóstico é feito com base num fenótipo compatível, serologia ou imagiologia específicas e confirmação por biópsia. Existem diversos scores que permitem avaliar a severidade e o dano provocado pelas vasculites. A Granulomatose com Poliangeíte é uma doença auto-imune caracterizada geralmente por inflamação granulomatosa das vias respiratórias e vasculite renal e pulmonar de pequenos vasos. É mais comum em caucasianos e na população europeia em homens. O aparecimento dos sintomas ocorre tipicamente entre os 45 e os 65 anos. É associada a anticorpos contra o citoplasma de neutrófilos (ANCA) contra a proteinase 3, contudo a sua etiologia ainda é desconhecida. O tratamento é feito de acordo com o estadio e actividade da doença. Na doença grave é feita a associação de ciclofosfamida e glicocorticóides, com posterior substituição para imunossupressores menos potentes após a indução da remissão. Existem novas terapias biológicas em estudo.