Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 4 uppsatser.

Background Idiopathic pulmonary fibrosis (IPF) is one of a number of interstitial lung diseases (ILDs) that result in extensive and chronic pulmonary fibrosis. In IPF pathology, immunological dysfunction has been identified as a contributing factor to the ongoing fibrotic process, implicating cells and mechanisms of both the innate and humoral immune response. Due to the complex and diverse range of cells and mediators involved in IPF, the pathology is still poorly understood. Evidence of complement activation through the classical pathway in IPF lungs implies a role for IgG in the pathology. The active IgG in IPF may be autoreactive in nature, as IgG that target antigens of alveolar epithelial cells have been. Two autoantibodies in IPF, anti-periplakin IgG and anti-Hsp72 IgG, have been associated with poorer prognoses in IPF patients. The association of anti-Hsp72 IgG with IPF patient outcomes has not been validated and little work has been done to study the underlying mechanisms of autoantibodies in IPF pathogenesis. Hypothesis Anti-Hsp72 IgG is associated with poorer outcomes in IPF, and may induce alveolar macrophages to exhibit a pro-fibrotic phenotype. Aims The aims were to:  Optimise an ELISA for anti-Hsp72 IgG detection and determine any association of anti-Hsp72 IgG with IPF patient outcomes  Determine the location of anti-Hsp72 IgG producing cells and detect if Hsp72-IgG complexes are present in IPF patients’ lungs  Explore a potential underlying pro-fibrotic mechanism through which anti-Hps72 IgG modulates macrophage function. Results The presence of anti-Hsp72 IgG was determined in ILD patient and healthy control bronchoalveolar lavage fluid (BALf) and serum. A novel anti-Hsp72 IgG ELISA was developed and optimised and then compared against a commercial anti-Hsp72 IgGAM ELISA which became available during the PhD. Progression in IPF was defined by a decrease of ≥10% vital capacity (VC) over twelve months. Serum anti-Hsp72 IgG(AM) did not associate with changes in VC over 12 months. In contrast, BALf anti-Hsp72 IgG(AM) concentrations were elevated in IPF non-progressors. Patients with high BALf anti-Hsp72 IgGAM, had improved survival compared patient with low anti-Hsp72 IgGAM (adjusted HR 0.39, 95% CI 0.16-0.92; p=0.032) In contrast there was no association between anti-Hsp72 IgG and survival. Detection of anti-Hsp72 IgG subtypes in the serum and BALf of IPF patients revealed no significant difference in anti-Hsp72 IgG subtype detection levels between progressors and non-progressors. BALf anti-Hsp72 IgG1 levels were associated with a significantly lower rate of decline in VC over twelve months than patients with no detectable anti-Hsp72 IgG1. The presence of Hsp72-IgG complexes was confirmed by detection in purified IgG from IPF patient BALf. Immuno-histological detection of C4d deposition in the lungs of IPF patients coincided in areas of Hsp72 expression in alveolar epithelium. Summary These findings do not validate serum and-Hsp72 IgG as a biomarker for IPF. They support a role for anti-Hsp72 IgG in IPF, but associate with decreased rates of lung function decline and increased patient survival. Data also suggests that the decreased rate of decline may be related to specific anti-Hsp72 IgG subtype expression. The immune-histological data further suggests that anti-Hsp72 IgG may be targeting Hsp72 expressed by lung epithelium. Therefore these findings support a role for immunological dysfunction in IPF, but further work is required to determine the underlying mechanism.

Habilitacijos procedūrai teikiamoje darbų apžvalgoje apibendrinti po daktaro disertacijos apgynimo (1999 m.) atliktų mokslinių tyrimų, skirtų intersticinėms plaučių ligoms, darbų rezultatai. Šioje apžvalgoje remiamasi autoriaus atliktų ir su bendraautoriais vykdytų intersticinių plaučių ligų tyrimų nuo 1999 m. iki 2008 m. Vilniaus universiteto Medicinos fakulteto Krūtinės ligų, alergologijos ir radiologijos klinikoje, Viešojoje įstaigoje Vilniaus universiteto ligoninės Santariškių klinikose, rezultatais. Svarbiausias darbo tikslas buvo nustatyti optimalią intersticinių plaučių ligų diagnostikos ir diferencinės diagnostikos seką kasdienėje klinikinėje praktikoje, pateikti praktines dažniausių intersticinių plaučių ligų diagnostikos rekomendacijas. Darbo uždaviniai buvo nustatyti klinikinių simptomų, radiologinių požymių, plaučių funkcijos rodiklių, bronchoalveolinio lavažo skysčio tyrimo ir bronchoskopinės plaučių biopsijos histologinio tyrimo diagnostinę vertę. Šioje apžvalgoje apibendrinamas 30 mokslinių straipsnių, paskelbtų 1999–2009 m. Dauguma straipsnių parengta kartu su kitais bendraautoriais (23 iš jų apžvalgos autorius buvo pirmasis autorius). Darbo rezultatų pagrindu perskaityta 14 pranešimų nacionalinėse ir 19 pranešimų tarptautinėse konferencijose (14 iš jų užsienio šalyse). / The results of scientific research published since acquisition of PhD degree in 1999 are reviewed in this survey for habilitation procedure. The interstitial lung diseases research was conducted and performed by author together with other researchers at Clinic of Chest Diseases, Allergology and Radiology of Vilnius Univesity and Vilnius University Hospital Santariškių klinikos in 1999–2008. The aim of this research was to determine optimal diagnostic pathway for interstitial lung diseases in daily clinical practice, to propose practical recommendations for diagnostic of the most common interstitial lung disorders. The tasks of the research were to evaluate diagnostic value of clinical symptoms, radiological signs, indices of respiratory function, examination of bronchoalveolar lavage fluid and bronchoscopic lung biopsy material. The review material covers 30 scientific papers published during 1999–2009. The most of papers were prepared together with co-authors, but in 23 of them the author of this survey was the first author. On the base of obtained scientific material 14 presentations at national conferences and 19 presentations at international conferences (14 of them in foreign countries) were done.

An estimated 45% of all deaths can be attributed to various chronic fibroproliferative diseases. Idiopathic pulmonary fibrosis (IPF) is the most common form of interstitial lung disease which is characterized by progressive decline in lung function. While the pathogenesis of IPF is not fully understood, alternatively activated macrophages (M2) have been implicated as a key contributor to the fibrotic process. The plasticity of macrophages in vivo challenges the ability to specifically target the M2 macrophage phenotype across species. Previous bioinformatic analysis from our lab identified Dectin-1/Clec7a as a unique marker of M2 macrophages in both human and murine model systems. The expression of the transmembrane receptor Dectin-1 has not been elucidated in the context of pulmonary fibrosis. To prevent the progression of fibrosis by targeting alternatively activated macrophages, we investigated the expression of Dectin-1 in IPF and an experimental model of fibrotic lung disease. Our data demonstrated that while protein expression of Dectin-1 was increased in archived lung tissues of patients with IPF, mRNA expression of this receptor was downregulated in the tissues of these IPF patients. Gene expression of Dectin-1 was shown to be increased in monocyte-derived macrophages, further suggesting a circulatory component contributing to lung fibrosis. As expected, we confirmed that Dectin-1 was highly expressed past the injury phase of the bleomycin-model of induced pulmonary fibrosis which aligns with the increased immune infiltrates at this time point. Preliminary work into the time dependency of the resolution phase of the bleomycin-induced model of lung fibrosis was shown. All in all, our data suggests that Dectin-1 may be a useful marker in characterizing and differentiating phenotypes of macrophages implicated in the fibrotic process. Future efforts aim to gain insight into the functional requirement of Dectin-1 in the alternative activation of profibrotic macrophages to identify novel therapeutic targets for fibrotic lung disease. / Thesis / Master of Science (MSc)

Nos últimos tempos, observa-se um grande crescimento na utilização de computadores como ferramenta de auxílio nas mais diversas áreas, sendo a Medicina uma das principais áreas dessa utilização. Inúmeras aplicações da Informática podem ser encontradas na área médica, citando-se os sistemas de apoio ao diagnóstico como uma das mais marcantes. Esses sistemas têm como principal objetivo auxiliar o profissional da área no processo de tomada de decisões, o qual pode ocorrer em diferentes etapas de sua atividade. Esse processo normalmente envolve uma grande quantidade de dados e informações, que podem ser armazenadas e processadas muito mais rapidamente por computador. O objetivo principal desse trabalho é o desenvolvimento de um sistema computacional baseado na técnica de redes neurais artificiais, para auxílio ao médico radiologista na confirmação de diagnóstico das chamadas lesões intersticiais pulmonares. O exame radiológico para esse grupo é de difícil interpretação mesmo para profissionais bem treinados, visto o grande número de patologias assim classificadas e a semelhança entre elas. Os dados para treinamento da rede neural são obtidos por protocolo gerado por especialistas, através da revisão de casos com diagnóstico confirmado para determinadas patologias. O sistema desenvolvido é baseado em uma rede neural do tipo perceptron multicamadas, que funciona como um classificador de padrões – dado um conjunto de dados de entrada, a saída é classificada entre determinadas patologias. Nessa pesquisa, são levantados elementos para justificar a utilização de redes neurais artificiais em sistemas de apoio ao diagnóstico, objetivando uma ferramenta confiável para o auxílio ao profissional no seu dia-a-dia, e também uma ferramenta educacional de auxílio ao treinamento e qualificação para os estudantes da área médica. / It is observed that a big growing in the use of computers as a tool to help in several areas, specially in medicine, happened in the past years. A big number of applications of these computers can be found in Medicine, such as the Diagnosis Support System, which is one of the most remarkable. These systems have as its main objective to help in the phases its activity. This process usually involves a lot of data and information, which can be stored by a computer very quickly. The most important objective of this project is the development of a computer system based in artificial neural network to help the Radiologist in the confirmation of the diagnosis of the so-called Interstitial Lung Disease. The radiological examination for this group has a difficult interpretation even to well-trained professionals, due to the big number of classified as well as similar pathologies in this area. The data for the neural net are obtained through a protocol generated by specialists, through the review of cases with confirmed diagnosis for certain pathologies. The developed system is based in a kind of multilayer perceptron neural net, which run as a classifier of patterns. A number of data is given as entry data, afterwards the exit is classified among certain pathologies. In this research, elements to justify the use of the artificial neural net in diagnosis support systems are raised, objectifying a reliable tool to help professionals who use it day by day and also as an educational tool to help in the training and qualification of medical school students.

Nos últimos tempos, observa-se um grande crescimento na utilização de computadores como ferramenta de auxílio nas mais diversas áreas, sendo a Medicina uma das principais áreas dessa utilização. Inúmeras aplicações da Informática podem ser encontradas na área médica, citando-se os sistemas de apoio ao diagnóstico como uma das mais marcantes. Esses sistemas têm como principal objetivo auxiliar o profissional da área no processo de tomada de decisões, o qual pode ocorrer em diferentes etapas de sua atividade. Esse processo normalmente envolve uma grande quantidade de dados e informações, que podem ser armazenadas e processadas muito mais rapidamente por computador. O objetivo principal desse trabalho é o desenvolvimento de um sistema computacional baseado na técnica de redes neurais artificiais, para auxílio ao médico radiologista na confirmação de diagnóstico das chamadas lesões intersticiais pulmonares. O exame radiológico para esse grupo é de difícil interpretação mesmo para profissionais bem treinados, visto o grande número de patologias assim classificadas e a semelhança entre elas. Os dados para treinamento da rede neural são obtidos por protocolo gerado por especialistas, através da revisão de casos com diagnóstico confirmado para determinadas patologias. O sistema desenvolvido é baseado em uma rede neural do tipo perceptron multicamadas, que funciona como um classificador de padrões – dado um conjunto de dados de entrada, a saída é classificada entre determinadas patologias. Nessa pesquisa, são levantados elementos para justificar a utilização de redes neurais artificiais em sistemas de apoio ao diagnóstico, objetivando uma ferramenta confiável para o auxílio ao profissional no seu dia-a-dia, e também uma ferramenta educacional de auxílio ao treinamento e qualificação para os estudantes da área médica. / It is observed that a big growing in the use of computers as a tool to help in several areas, specially in medicine, happened in the past years. A big number of applications of these computers can be found in Medicine, such as the Diagnosis Support System, which is one of the most remarkable. These systems have as its main objective to help in the phases its activity. This process usually involves a lot of data and information, which can be stored by a computer very quickly. The most important objective of this project is the development of a computer system based in artificial neural network to help the Radiologist in the confirmation of the diagnosis of the so-called Interstitial Lung Disease. The radiological examination for this group has a difficult interpretation even to well-trained professionals, due to the big number of classified as well as similar pathologies in this area. The data for the neural net are obtained through a protocol generated by specialists, through the review of cases with confirmed diagnosis for certain pathologies. The developed system is based in a kind of multilayer perceptron neural net, which run as a classifier of patterns. A number of data is given as entry data, afterwards the exit is classified among certain pathologies. In this research, elements to justify the use of the artificial neural net in diagnosis support systems are raised, objectifying a reliable tool to help professionals who use it day by day and also as an educational tool to help in the training and qualification of medical school students.

O objetivo desse estudo foi avaliar o comprometimento de pequenas vias aéreas na Fibrose Pulmonar Idiopática (FPI) analisando curvas Pressão- Volume (PV) do sistema respiratório. Coletamos curvas PV de doze pacientes antes da biopsia pulmonar, que confirmou FPI em sete pacientes e Pneumonite de Hipersensibilidade em cinco. Todas as curvas foram ajustadas com modelo sigmóide, V = a + b / (1 + e -(P-c/d)), e exponencial V = A - B . e -k.P (aplicado apenas à parte superior). O modelo exponencial, apesar do bom ajuste à parte superior, não representou a parte inicial da curva, gerando parâmetros sem significado. O modelo sigmóide ajustou bem toda a curva e gerou parâmetros com significado fisiológico, que sugerem a presença de colapso de pequenas vias aéreas na FPI. / The objective of this study was to evaluate small airways compromise in Idiopathic Pulmonary Fibrosis (IPF) using pressure-volume (PV) curves of the respiratory system. We collected PV curves from twelve patients before lung biopsy, which confirmed IPF in seven patients and Hipersensitivity Pneumonia in five. All curves were fitted with a sigmoid model, V = a + b / (1 + e -(P-c/d)), and an exponential model, V = A - B . e -k.P (applied only to the superior part of the curve). The exponential model, despite having a good fit to the superior part of the curve, did not represent the initial part, and yielded parameters with no physiological meaning. The sigmoid model had a good fit to the entire curve and yielded parameters with physiological meaning, suggesting the presence of small airways collapse in IPF.

O objetivo desse estudo foi avaliar o comprometimento de pequenas vias aéreas na Fibrose Pulmonar Idiopática (FPI) analisando curvas Pressão- Volume (PV) do sistema respiratório. Coletamos curvas PV de doze pacientes antes da biopsia pulmonar, que confirmou FPI em sete pacientes e Pneumonite de Hipersensibilidade em cinco. Todas as curvas foram ajustadas com modelo sigmóide, V = a + b / (1 + e -(P-c/d)), e exponencial V = A - B . e -k.P (aplicado apenas à parte superior). O modelo exponencial, apesar do bom ajuste à parte superior, não representou a parte inicial da curva, gerando parâmetros sem significado. O modelo sigmóide ajustou bem toda a curva e gerou parâmetros com significado fisiológico, que sugerem a presença de colapso de pequenas vias aéreas na FPI. / The objective of this study was to evaluate small airways compromise in Idiopathic Pulmonary Fibrosis (IPF) using pressure-volume (PV) curves of the respiratory system. We collected PV curves from twelve patients before lung biopsy, which confirmed IPF in seven patients and Hipersensitivity Pneumonia in five. All curves were fitted with a sigmoid model, V = a + b / (1 + e -(P-c/d)), and an exponential model, V = A - B . e -k.P (applied only to the superior part of the curve). The exponential model, despite having a good fit to the superior part of the curve, did not represent the initial part, and yielded parameters with no physiological meaning. The sigmoid model had a good fit to the entire curve and yielded parameters with physiological meaning, suggesting the presence of small airways collapse in IPF.

INTRODUÇÃO: A pneumonite de hipersensibilidade (PH) é uma doença intersticial causada pela inalação de antígenos orgânicos específicos ou substâncias de baixo peso molecular em indivíduos geneticamente suscetíveis. A PH crônica representa seu estágio final, na qual a exposição antigênica prolongada leva à fibrose. Na PH crônica, o envolvimento das pequenas vias aéreas (PVA) é proeminente; entretanto, uma avaliação detalhada através de métodos funcionais e de avaliação quantitativa e automatizada pela tomografia computadorizada (TC) não foi realizada previamente. MÉTODOS: estudo transversal de 28 pacientes com PH crônica, com avaliação através de provas de função pulmonar (PFPs); oscilometria forçada (FOT); análise automatizada do volume pulmonar através da TC, incluindo quantificação de aprisionamento aéreo; e teste cardiopulmonar de exercício (TCPE) incremental em cicloergômetro para avaliar performance ao exercício, incluindo medidas seriadas da capacidade inspiratória e hiperinsuflação dinâmica (HD). Foram incluídos pacientes entre 18 a 75 anos, com diagnóstico confirmado pela combinação de achados tomográficos, exposição antigênica e biópsia compatível e/ou LBA com linfocitose. Foram excluídos pacientes com CVF e/ou VEF1 < 30% predito, tabagismo > 20 anos-maço, uso de oxigênio suplementar; diagnóstico prévio de asma ou DPOC, diagnóstico de hipertensão arterial pulmonar ou impossibilidade de realizar TCPE. Os dados foram comparados com controles saudáveis. RESULTADOS: 28 pacientes (16 mulheres; idade média 56 +- 11 anos; CVF 57 +- 17% predito) foram avaliados, e todos apresentavam padrão ventilatório restritivo sem resposta broncodilatadora. Na FOT, 4 pacientes apresentaram resistência aumentada a 5 Hz (R5), enquanto todos apresentaram baixa reactância (X5), sendo que nenhum apresentou resposta broncodilatadora significativa. Pacientes com PH crônica tiveram menor capacidade de exercício com menor O2 de pico, diminuição da reserva ventilatória, hiperventilação, dessaturação de oxigênio e escores de dispneia (Borg) aumentados quando comparado aos controles. A prevalência de HD foi encontrada em apenas 18% da coorte. Ao comparar pacientes com PH crônica com O2 normal e baixo (< 84% predito, LIN), o último grupo apresentou maior hiperventilação (slope E/CO2), um menor volume corrente e menores escores de capacidade física na avaliação do questionário de qualidade de vida (SF-36). A análise da curva ROC mostrou que volumes pulmonares reduzidos (CVF%, CPT% e DLCO%) foram preditores de baixa capacidade ao exercício. Na TC, a PH crônica teve aumento de áreas com alta densidade em unidades Hounsfield, inferindo maior extensão de opacidades em vidro fosco e fibrose em relação aos controles saudáveis. A extensão das áreas de atenuação reduzida (AAR) e aprisionamento aéreo em relação ao volume pulmonar total é pequena, e não se correlaciona com índices funcionais obstrutivos; entretanto, pacientes com maior percentual dessas áreas apresentam menos fibrose e função pulmonar mais preservada. CONCLUSÃO: a PH crônica se caracterizou por um acometimento eminentemente restritivo, e não de obstrução de vias aéreas, nos diferentes métodos diagnósticos aplicados. A redução da capacidade de exercício foi prevalente devido à limitação ventilatória e de troca gasosa, a exemplo de outras doenças intersticiais pulmonares, e não pela HD. Redução dos volumes pulmonares foram bons preditores das respostas ventilatórias durante o exercício / INTRODUCTION: Hypersensitivity pneumonitis (HP) is an interstitial lung disease caused by the inhalation of specific organic antigens or low molecular weight substances in genetically susceptible individuals. Chronic HP represents its final stage, in which prolonged antigenic exposure causes fibrosis. In chronic HP, small airway involvement is prominent; however, a detailed characterization through functional evaluation and through automatic quantitative evaluation of computed tomography (CT) has not been previously assessed. METHODS: Cross-sectional study with 28 chronic HP patients, with evaluation by pulmonary function tests (PFTs), forced oscillometry (FOT), automated lung volume analysis through CT, including quantification of air trapping (AT); and incremental cardiopulmonary exercise testing (CPET) on a cycle ergometer to evaluate exercise performance, including serial measurements of inspiratory capacity to establish dynamic hyperinflation (DH). Inclusion criteria: patients aged 18 to 75 years, with a chronic HP diagnosis confirmed by the combination of CT findings, known antigenic exposure and compatible biopsy and / or BAL with lymphocytosis. Exclusion criteria: FVC and / or FEV1 < 30% predicted, smoking > 20 pack-years, supplemental oxygen use; previous diagnosis of asthma or COPD; pulmonary arterial hypertension, or medical conditions that could interfere with CPET. Data were compared with healthy controls. RESULTS: All patients (16 women; mean age 56 +- 11 years; FVC 57 +- 17% predicted) had restrictive ventilatory pattern without bronchodilator response. In FOT, 4 patients had increased resistance at 5 Hz (R5), all patients presented low reactance (X5) values, and none presented a significant bronchodilator response. Chronic HP patients had reduced exercise performance with lower peak V?O2, diminished breathing reserve, hyperventilation, oxygen desaturation and augmented Borg dyspnea scores when compared with controls. The prevalence of DH was only found in 18% of patients. When comparing chronic HP patients with normal and low peak VO2 (< 84%predicted, LLN), the later exhibited higher hyperventilation (VE/VCO2 slope), lower tidal volumes, and poorer physical functioning scores on Short-form-36 health survey. ROC curve analysis showed that reduced lung volumes (FVC%, TLC% and DLCO%) were high predictors of poor exercise capacity. On CT, chronic HP is characterized by increased pulmonary densities (Hounsfield Units) inferring the extension of ground glass opacities and fibrosis when compared with healthy subjects. The extension of low attenuation areas (LAA) and AT in relation to the hole lung volume is low and does not correlate with PFT indexes of obstruction; however, patients with greater extension of these areas had less fibrosis and more preserved PFTs. CONCLUSIONS: Chronic HP was characterized by an imminently restrictive lung disorder, and not by airway obstruction, according to the different diagnostic methods applied in this study. Reduction of exercise capacity was prevalent due to ventilatory and gas exchange limitation, similarly to other fibrotic interstitial lung diseases, rather than due to DH. Reduced lung volumes were good predictors of ventilatory responses during exercise

Introdução: A área do músculo peitoral (AMP) é uma avaliação facilmente derivada da tomografia computadorizada do tórax com potencial de fornecer informações relevantes sobre outros músculos esqueléticos. A Disfunção muscular respiratória e periférica é cada vez mais reconhecida em pacientes com doença pulmonar intersticial (DPI). A sua relação com a capacidade de exercício tem sido controversa. Nosso objetivo foi investigar se AMP está relacionada com a força muscular esquelética respiratória e periférica em pacientes com DPI, e se a função dos músculos esqueléticos estaria reduzida e independentemente relacionada com a capacidade de exercício e dispneia nesses pacientes. Métodos: Estudo transversal onde foi realizado teste de exercício cardiopulmonar incremental em cicloergômetro com mensuração de pressão inspiratória máxima (PImax), pressão expiratória máxima (PEmax) e contração voluntária máxima (CVM) do quadríceps, antes e após o exercício. Os testes de função pulmonar em repouso e TC de tórax foram obtidos da rotina assistencial dos pacientes. / Background: Pectoralis muscle area (PMA) is an easily derived computed tomography-based assessment that can provide insight into clinical features of other skeletal muscles. Respiratory and locomotor muscle dysfunction has been increasingly recognized in patients with interstitial lung disease (ILD). Its contribution to exercise performance has been controversial. We aimed investigate if PMA is related with respiratory and locomotor skeletal muscle strength in ILD patients, and if skeletal muscle function is compromised and independently related with exercise capacity and dyspnea in these patients. Methods: Cross-sectional study where subjects performed incremental cycling cardiopulmonary exercise testing with maximal inspiratory pressure (MIP), maximal expiratory pressure (MEP), and quadriceps maximal voluntary contraction (MVC) before and after exercise.