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Biomarcadores respiratórios na abordagem clínica da esclerose lateral amiotrófica
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A Esclerose Lateral Amiotrófica (ELA) é uma doença neurodegenerativa de progressão rápida e fatal, caracterizada pela rápida destruição dos neurónios motores localizados no córtex motor, tronco cerebral e na medula espinhal, associada a um processo neuro-inflamatório cujo papel etiopatogénico não está bem esclarecido. Clinicamente, observa-se uma grave diminuição da força muscular, envolvendo os membros superiores e inferiores, os músculos da deglutição e da fala, e os músculos respiratórios. A insuficiência respiratória é a principal causa de morte, que ocorre, em geral, 3-5 anos após o aparecimento dos primeiros sintomas. A maioria dos casos de ELA, 90-95%, são de etiologia desconhecida (ELA esporádica). Nos restantes 5-10% dos casos há outros membros afectados na família (ELA familiar).
O único fármaco aprovado na Europa, Riluzol, apenas confere um modesto aumento na sobrevida destes pacientes. Actualmente, nenhum tratamento de relevante eficácia está disponível, apesar dos persistentes esforços para encontrar novos medicamentos, resultando em centenas de ensaios clínicos muito dispendiosos. Tal deve-se, sobretudo, ao facto dos mecanismos envolvidos na origem da ELA serem complexos, multifactoriais e não conhecidos. O diagnóstico da doença demora cerca de 9-12 meses, fundamentalmente devido à ausência biomarcadores moleculares de diagnóstico.
Sendo um determinante crítico do prognóstico, a função respiratória deve ser monitorizada no curso da progressão da doença. Tal processo ocorre pela avaliação clínica e execução de provas da função respiratória, eventualmente complementada com métodos de imagem ou neurofisiológicos. Não tem havido um marcador molecular que seja relevante para o diagnóstico precoce ou no prognóstico da insuficiência respiratória. Tal pode ter implicações na implementação de medidas adequadas de reabilitação respiratória, tal como o suporte ventilatório.
Nesta tese, pretendeu-se identificar novos biomarcadores relacionados com a insuficiência respiratória na ELA. Como objectivo secundário explorou-se a associação entre a função respiratória e as propriedades eritrocitárias, de forma a melhor compreender a hipóxia tecidular.
De modo a identificar um biomarcador molecular de disfunção respiratória na ELA, estudaram-se a proteína Club cell secretory protein (CC-16), a interleucina-6 (IL-6), e a creatinina (Cr). Adicionalmente, analisou-se o papel do fibrinogénio ’ na ELA como marcador da disfunção respiratória e da inflamação na ELA.
Relativamente às propriedades eritrocitárias efectuou-se a caracterização biofísica, hemorreológica e morfológica destas células.
Foram incluídos doentes com ELA regularmente seguidos na consulta dedicada a esta doença na Unidade de Neuromuscular do CHULN-H Santa Maria, respeitando os critérios de diagnóstico para esta doença e excluindo doentes com outras patologias médicas. Os valores obtidos foram comparados com os obtidos numa população de controlo com similar idade e distribuição por género. Os doentes tiveram seguimento regular na consulta, com avaliação clínica (escala funcional ALSFRS-R), provas de função respiratória (espirometria, pressões máximas, SNIP, estudo neurofisiológico do nervo frénico) e registo dos eventos, como a data da introdução da ventilação não-invasiva, a morte ou a traqueostomia.
Foram aplicadas diversas técnicas laboratoriais, em particular ELISA (CC-16), tecnologia Multiplex (IL-6), microscopia de força atómica (AFM), medidas do potencial Zeta e técnicas hemorreológicas, para além de outras técnicas e metodologias utilizadas de rotina no nosso centro.
Os resultados mostraram que na população de doentes os valores de CC-16 estavam significativamente mais elevados, sem correlação com a idade, forma de apresentação ou duração da doença, ALSFRS-R, FVC (“Forced vital capacity”) ou com a amplitude da resposta do nervo frénico. No entanto os doentes com níveis mais elevados de CC-16 apresentavam um risco acrescido da necessidade de ventilação não-invasiva nos seis meses seguintes, e tendiam para uma maior mortalidade nos 30 meses seguintes.
Níveis aumentados de IL-6 foram observados doentes com ELA, quando comparados com o grupo controlo. Nestes, os valores tinham correlação positiva com a idade. Na população de doentes, foi encontrada uma correlação negativa independente da idade entre os níveis de IL-6 e os valores da amplitude da resposta do nervo frénico.
Embora a creatinina seja um factor preditivo da perda funcional e da sobrevida, na ELA, na nossa amostra não foi possível confirmar ser este marcador preditor da função respiratória (FVC) ou do seu declínio.
Da avaliação da quantificação plasmática do fibrinogénio ’ observaram-se valores mais elevados nos pacientes de ELA quando comparados com o grupo controlo, níveis mais elevados foram associados a maior sobrevida.
Os eritrócitos de doentes ELA, estavam morfologicamente alterados quando comparados com os eritrócitos do grupo controlo, tendo uma membrana menos rugosa, menor área, menor volume, menor diâmetro, mas sendo mais alongados. Os eritrócitos dos pacientes de ELA tendem a perder a concavidade. Adicionalmente, por quantificação do potencial Zeta, observou-se que a carga superficial da membrana eritrocitária é mais negativa, quando comparada com os controlos. Estudos de espectroscopia de força por AFM para analisar as propriedades elásticas dos eritrócitos revelaram que os eritrócitos dos pacientes de ELA são mais deformáveis, menos rígidos e têm maior profundidade de penetração que os eritrócitos dos indivíduos controlo.
Em suma, os resultados revelam um aumento da resposta inflamatória pulmonar relacionada com a disfunção respiratória. As alterações nas propriedades hemorreológicas e morfológicas dos eritrócitos poderão promover interacções entre moléculas inflamatórias e o eritrócito, eritrócito-fibrinogénio e/ou eritrócito-eritrócito, promovendo um factor de risco adicional para eventos tromboembólicos venosos e de hipoxia tecidual, associados à progressão da doença. Em resumo, os nossos resultados permitiram:
1- Identificar potenciais novos marcadores moleculares de disfunção respiratória na ELA, com valor preditivo no diagnóstico e prognóstico, bem como potencial utilização em futuros ensaios clínicos;
2- Identificar alterações na elasticidade e rigidez dos eritrócitos dos pacientes de ELA bem como alterações morfológicas no eritrócito e hemorreológicas, nos pacientes com ELA.
Os resultados da tese devem incentivar estudos futuros. O papel das alterações da membrana eritrocitária na hipoxia tecidual é uma questão inovadora relevante que merece uma investigação mais profunda.
Amyotrophic Lateral Sclerosis (ALS) is a rapidly progressive and fatal neurodegenerative disease characterized by the rapid destruction of motor neurons in the motor cortex, brain stem and spinal cord, associated with a neuroinflammatory process whose etiopathogenic role is not yet well understood. Clinically, there is a severe weakness involving the upper and lower limbs, swallowing and speech muscles, and the respiratory muscles. Respiratory failure is the leading cause of death, which usually occurs 3-5 years after the onset of the first symptoms. Most cases of ALS are sporadic, meaning no famility history (90-95%), in the remaining 5-10% of cases there are other affected family members (familiar ALS). Riluzole is the only drug approved in Europe, but confers a modest improvement in the survival of these patients. Currently, no effective treatment is available despite a huge international effort in hundreds of very expensive clinical trials to find a new drugs. This insucess history derives from the ignorance of the mechanisms involved in the origin of ALS, which are complex and multifactorial. The diagnostic delay is typically 9-12 months, mainly due to the absence of diagnostic molecular biomarkers. Being a critical predictor of prognosis, respiratory function should be monitored in the course of the disease progression. This includes clinical evaluation and a number of respiratory function tests, possibly complemented with imaging and neurophysiological methods. There has been no respiratory molecular biomarker relevant for early diagnosis or prognosis. This might have implications for the implementation of appropriate respiratory rehabilitation measures, such as ventilatory support. In this thesis, we aimed to identify new biomarkers related to respiratory failure in ALS. As a secondary objective, the association between respiratory function and erythrocyte properties was explored to better understand tissue hypoxia. In order to identify a molecular biomarker of respiratory dysfunction in ALS, Club cell secretory protein (CC-16), interleukin-6 (IL-6), and creatinine (Cr) were studied. Additionally, the role of the ' fibrinogen in ALS as a marker of respiratory dysfunction and inflammation in ALS was analyzed, to study the relation between plasma levels of this protein and disease progression. Regarding the erythrocyte properties, the biophysical, hemoreological and morphological characterization of these cells was performed. In this project, we included ALS patients regularly followed at the out-patient clinic dedicated to this disease, in the Neuromuscular Unit of Santa Maria’s Hospital (“Unidade de Neuromuscular do CHULN-H Santa Maria”), respecting the diagnostic criteria for this disease. Patients with other medical conditions were excluded. The values obtained were compared with those obtained in a control population, matched for age and gender. Patients were assessed by clinical evaluation (ALSFRS-R functional scale), respiratory function tests (spirometry, maximal pressures, SNIP, phrenic nerve study) and by critical events, such as the date non-invasive ventilation initiation, and tracheostomy or death. Several laboratory techniques have been applied, in particular ELISA (CC-16), Multiplex technology (IL-6), atomic force microscopy (AFM), Zeta potential measurements and hemorheological techniques, in addition to other routinely used techniques and methodologies. The results have shown that in the patient population, CC-16 values were significantly higher, with no correlation to age, phenotype, disease duration, ALSFRS-R, forced vital capacity (FVC) or with the amplitude of the phrenic nerve response. However, patients with higher levels of CC-16 were at increased risk of the need for noninvasive ventilation (NIV) over the following six months, and tended to have higher mortality over the subsequent 30 months. Increased levels of IL-6 were observed in ALS patients when compared to the control group. In this control group, IL-6 values had a positive correlation with age. In the patient population, a negative age-independent correlation was found between IL-6 levels and phrenic nerve response amplitude. Although creatinine is a predictor of functional loss and mortality in ALS, it was not possible to confirm in our sample that creatinine was a predictor of respiratory function (FVC) or its decline rate. From the quantification of plasma ’ fibrinogen, we observed higher values in ALS patients when compared to the control group. Higher levels are associated with longer survival in ALS. Erythrocytes in ALS patients were morphologically modified as compared to the control group, having a decreased rough membrane, smaller area, smaller volume, smaller diameter, but being more elongated. Erythrocytes of ALS patients tend to lose their concavity. Moreover, by measuring the Zeta potential, it was observed that the erythrocyte membrane surface charge was more negative in ALS when compared to controls. The AFM force spectroscopy studies performed in order to analyze erythrocyte elastic properties have shown that in ALS patients they were more deformable, less rigid, and with greater penetration depth than erythrocytes from control subjects. Our results support an increased inflammatory response related to pulmonary dysfunction. in Changes in hemorheological and morphological properties of erythrocytes may promote interaction between inflammatory molecules and erythrocyte, erythrocyte-fibrinogen and/or erythrocyte-erythrocyte, causing an increased risk of venous thromboembolic events and tissue hypoxia, associated with disease progression. In summary, our results allowed to: 1- Identify potential new molecular markers of respiratory dysfunction in ALS, with predictive value in diagnosis and prognosis, as well as suggesting its potential use in future clinical trials; 2- Identify alterations in erythrocyte elasticity and stiffness, as well as hemorheological morphological changes, in ALS patients. The results of the thesis should encourage future studies. The role of erythrocyte membrane alterations in tissue hypoxia is a relevant innovative issue that deserves further investigation.
Amyotrophic Lateral Sclerosis (ALS) is a rapidly progressive and fatal neurodegenerative disease characterized by the rapid destruction of motor neurons in the motor cortex, brain stem and spinal cord, associated with a neuroinflammatory process whose etiopathogenic role is not yet well understood. Clinically, there is a severe weakness involving the upper and lower limbs, swallowing and speech muscles, and the respiratory muscles. Respiratory failure is the leading cause of death, which usually occurs 3-5 years after the onset of the first symptoms. Most cases of ALS are sporadic, meaning no famility history (90-95%), in the remaining 5-10% of cases there are other affected family members (familiar ALS). Riluzole is the only drug approved in Europe, but confers a modest improvement in the survival of these patients. Currently, no effective treatment is available despite a huge international effort in hundreds of very expensive clinical trials to find a new drugs. This insucess history derives from the ignorance of the mechanisms involved in the origin of ALS, which are complex and multifactorial. The diagnostic delay is typically 9-12 months, mainly due to the absence of diagnostic molecular biomarkers. Being a critical predictor of prognosis, respiratory function should be monitored in the course of the disease progression. This includes clinical evaluation and a number of respiratory function tests, possibly complemented with imaging and neurophysiological methods. There has been no respiratory molecular biomarker relevant for early diagnosis or prognosis. This might have implications for the implementation of appropriate respiratory rehabilitation measures, such as ventilatory support. In this thesis, we aimed to identify new biomarkers related to respiratory failure in ALS. As a secondary objective, the association between respiratory function and erythrocyte properties was explored to better understand tissue hypoxia. In order to identify a molecular biomarker of respiratory dysfunction in ALS, Club cell secretory protein (CC-16), interleukin-6 (IL-6), and creatinine (Cr) were studied. Additionally, the role of the ' fibrinogen in ALS as a marker of respiratory dysfunction and inflammation in ALS was analyzed, to study the relation between plasma levels of this protein and disease progression. Regarding the erythrocyte properties, the biophysical, hemoreological and morphological characterization of these cells was performed. In this project, we included ALS patients regularly followed at the out-patient clinic dedicated to this disease, in the Neuromuscular Unit of Santa Maria’s Hospital (“Unidade de Neuromuscular do CHULN-H Santa Maria”), respecting the diagnostic criteria for this disease. Patients with other medical conditions were excluded. The values obtained were compared with those obtained in a control population, matched for age and gender. Patients were assessed by clinical evaluation (ALSFRS-R functional scale), respiratory function tests (spirometry, maximal pressures, SNIP, phrenic nerve study) and by critical events, such as the date non-invasive ventilation initiation, and tracheostomy or death. Several laboratory techniques have been applied, in particular ELISA (CC-16), Multiplex technology (IL-6), atomic force microscopy (AFM), Zeta potential measurements and hemorheological techniques, in addition to other routinely used techniques and methodologies. The results have shown that in the patient population, CC-16 values were significantly higher, with no correlation to age, phenotype, disease duration, ALSFRS-R, forced vital capacity (FVC) or with the amplitude of the phrenic nerve response. However, patients with higher levels of CC-16 were at increased risk of the need for noninvasive ventilation (NIV) over the following six months, and tended to have higher mortality over the subsequent 30 months. Increased levels of IL-6 were observed in ALS patients when compared to the control group. In this control group, IL-6 values had a positive correlation with age. In the patient population, a negative age-independent correlation was found between IL-6 levels and phrenic nerve response amplitude. Although creatinine is a predictor of functional loss and mortality in ALS, it was not possible to confirm in our sample that creatinine was a predictor of respiratory function (FVC) or its decline rate. From the quantification of plasma ’ fibrinogen, we observed higher values in ALS patients when compared to the control group. Higher levels are associated with longer survival in ALS. Erythrocytes in ALS patients were morphologically modified as compared to the control group, having a decreased rough membrane, smaller area, smaller volume, smaller diameter, but being more elongated. Erythrocytes of ALS patients tend to lose their concavity. Moreover, by measuring the Zeta potential, it was observed that the erythrocyte membrane surface charge was more negative in ALS when compared to controls. The AFM force spectroscopy studies performed in order to analyze erythrocyte elastic properties have shown that in ALS patients they were more deformable, less rigid, and with greater penetration depth than erythrocytes from control subjects. Our results support an increased inflammatory response related to pulmonary dysfunction. in Changes in hemorheological and morphological properties of erythrocytes may promote interaction between inflammatory molecules and erythrocyte, erythrocyte-fibrinogen and/or erythrocyte-erythrocyte, causing an increased risk of venous thromboembolic events and tissue hypoxia, associated with disease progression. In summary, our results allowed to: 1- Identify potential new molecular markers of respiratory dysfunction in ALS, with predictive value in diagnosis and prognosis, as well as suggesting its potential use in future clinical trials; 2- Identify alterations in erythrocyte elasticity and stiffness, as well as hemorheological morphological changes, in ALS patients. The results of the thesis should encourage future studies. The role of erythrocyte membrane alterations in tissue hypoxia is a relevant innovative issue that deserves further investigation.
Descrição
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Esclerose Lateral Amiotrófica (ELA) Biomarcadores Insuficiência respiratória (IR) Hipóxia Eritrócitos Teses de doutoramento - 2020