No description available.

Activated neutrophils release a neutrophil serine proteinase (NSP) called Proteinase 3 (PR3). In granulomatosis with polyangiitis (GPA), an autoimmune vasculitis, enhanced PR3 release results in endothelial damage. Serine proteinase inhibitors (serpins) such as α-1 proteinase inhibitor (API) inhibit NSPs through the serpin’s reactive center loop (RCL). However, API is known to bind PR3 with a low specificity, compared to its main inhibitory target Human Neutrophil Elastase (HNE). The current treatment for GPA is immunosuppression, which leaves patients immunocompromised. Thus, the overall aim of this study was to engineer an API variant with a higher specificity to PR3 than HNE, which could serve as a possible novel therapeutic strategy for GPA. We created an API expression library, hypervariable at RCL residues A355-I356-P357-M358-S359, and expressed it in a T7 bacteriophage display system. This phage library was then biopanned for PR3 binding. Two conditions were used for each round of biopanning: experimental, with PR3, and the negative control, without PR3. The library was biopanned for a total of five consecutive rounds, with the product of one screen serving as the starting material for the next. A bacterial mass lysate screen was also employed to further probe the library with PR3. The phage-display and bacterial lysate screens resulted in the selection of two novel variants API-DA (D357/A358) and API-N (N359). Serpin-proteinase gel complexing assays indicated that API-N formed complex with PR3 similar to API-WT (wild-type), while API-DA was mainly cleaved as a substrate. There was no significant difference between the second order rate constants of API-N and API-WT reactions with PR3. Rate constants for API-DA binding to PR3 or for API-HNE reactions were not completed due to novel coronavirus (COVID-19) restrictions. However, this project successfully demonstrated the ability to screen a hypervariable API phage library with PR3, yielding two new novel API variants. / Thesis / Master of Science in Medical Sciences (MSMS) / When harmful substances enter our body such as bacteria or viruses, we have ways of protecting ourselves from them. One of those ways is through a cell called the neutrophil. This is an immune cell that can release “fighting tools” into our blood to combat the harm. Some of these tools are called proteins. One of those proteins is Proteinase 3. However, sometimes our neutrophils can be activated without the presence of viruses or bacteria by products made in our bodies called autoantibodies. When this happens, too many of the “fighting tool” Proteinase 3 is released leading to damage to the tubes or vessels that our blood flows through. This project aimed to find a new possible way to stop these extra fighting tools from doing harm to our body. We did this by creating a library of different proteins that can stop Proteinase 3 once it is released by the neutrophil.

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

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

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%

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%