Bipolar disorder (BD) is a chronic and often progressive illness that has a significant impact on quality of life and functioning. Pharmacological treatments are not effective for all patients, emphasizing the need to better understand the pathophysiology of the disorder. It is well known that patients with BD present with increased levels of inflammatory markers during mood episodes and often exhibit chronic low grade inflammation, implicating the immune system in the etiology of the disorder. Furthermore, patients with BD show deficits in neurotrophic factors suggesting that alterations in neurogenesis may precipitate clinical features. Recent evidence indicates that accelerated aging processes may underlie the pathophysiological changes observed in BD, implicating biomarkers related to aging. The chemokine C-C motif chemokine 11 (CCL11) and the cytokine growth differentiation factor 11 (GDF11) have been identified as proteins that increase and decrease with age, respectively. As such, this thesis presents research examining serum levels of these proteins in drug-naive young adults with BD and a matched healthy control group. We analyzed serum levels of CCL11 and GDF11 using enzyme linked immunosorbent assay (ELISA). Our results indicate that serum levels of CCL11 and GDF11 do not differ between the BD group and the healthy control group, however CCL11 levels were elevated in males and in individuals with tobacco abuse/dependence when considering the entire sample. Our results suggest that serum levels of these proteins do not differ between drug-naive young adults with BD and healthy controls, but that alterations may be due to demographic and lifestyle factors. Small sample size and low power should be considered when interpreting these results. / Thesis / Master of Science (MSc)

Introdução: São vistos muitos efeitos benéficos do aleitamento materno. Estudos demonstram que crianças amamentadas com leite materno quando comparadas às alimentadas com fórmulas lácteas artificiais, apresentam melhor desenvolvimento cognitivo. Em pacientes com esquizofrenia, o aleitamento materno vem sendo avaliado como fator de proteção. Os níveis de quimiocinas CCL11, marcador biológico relacionado principalmente com o envelhecimento precoce, também tem sido associado ao desempenho cognitivo nesses pacientes. Estudos mostram uma correlação negativa deste marcador com o desempenho em testes de memória de trabalho e com a tarefa de flexibilidade cognitiva. Sendo assim, surge o interesse em estudar as diferenças entre níveis plasmáticos da quimiocina CCL11, quociente de inteligência e história do aleitamento materno (no peito) em pacientes com esquizofrenia e controles. Métodos: Estudo caso-controle com 56 indivíduos, sendo 30 pacientes com esquizofrenia e 26 controles saudáveis, divididos em grupos que foram aleitados e grupos que não foram. Foi aplicado questionário com dados sócio-econômicos, história ao nascer, dados clínicos e alimentação ao nascer. Foi dosado a quimiocina CCL11 e aplicado testes psicológicos para avaliarem quociente de inteligência, funcionalidade, sintomas psiquiátricos, curso da doença e diagnóstico. Para os controles foi utilizada uma escala para descartar doença psiquiátrica. Resultados: A quimiocina CCL11 apresentou valores significativamente mais altos (>0,5) em pacientes com esquizofrenia quando comparados aos controles e no grupo de amamentados, os esquizofrênicos também apresentaram valores significativamente mais altos, mas em nível intermediário (entre 0.106 e 0.5). Não houve correlação da CCL11 com o número de hospitalizações, idade, tempo de diagnóstico e escolaridade. Também não foi evidenciada correlação entre tempo de aleitamento materno em relação aos fatores do Brief Psychiatric Rating Scale. Houve uma tendência de correlação entre a idade de início da doença e o aleitamento materno. Foi encontrada correlação positiva do CCL11 com o tempo de aleitamento materno. Ao comparar os pacientes esquizofrênicos que foram aleitados com os que não foram, foi encontrada diferença estatisticamente significativa apenas para o quociente de inteligência. Conclusão: O aleitamento materno está associado a níveis mais baixos de CCL11, escores mais altos de quociente de inteligência e com a esquizofrenia. A quimiocina CCL11 é mais alta em quem não amamentou, especialmente nos esquizofrênicos. / Introduction: Are seen many beneficial effects of breastfeeding. Studies show that children breastfed, compared to fed artificial milk formulas, have better cognitive development. In patients with schizophrenia, breastfeeding has been evaluated as a protective factor. The levels of CCL11 chemokine, biomarker related mainly to premature aging, have also been associated with cognitive performance in these patients. Studies show a negative correlation of this marker with the performance of working memory tests and with cognitive flexibility task. Thus, there is the interest in studying the differences between plasma levels of CCL11 chemokine, intelligence quotient and history of breastfeeding in patients with schizophrenia and controls. Methods: Case-control study with 56 subjects, 30 patients with schizophrenia and 26 healthy individuals divided into groups that were breastfed group and those who were not. A socio-economic survey, birth history, clinical data and power at birth was applied. It was dosed to CCL11 chemokine and applied psychological tests to assess intelligence quotient, functionality, psychiatric symptoms, course of the disease and diagnosis. For the controls we used a scale to rule out psychiatric illness. Results: The chemokine CCL11 had significantly higher values (> 0.5) in patients with schizophrenia compared with controls and the breastfed group, schizophrenics also had significantly higher values, but on intermediate level (between 0.106 and 0.5). There was no correlation of CCL11 to the number of hospitalizations, age, time of diagnosis and education. It was also not evident correlation between duration of breastfeeding in relation to factors of Brief Psychiatric Rating Scale. There was a trend of correlation between the age of onset of the disease and breastfeeding. There was a positive correlation between CCL11 with the duration of breastfeeding. By comparing the schizophrenic patients who were breastfed with those who were not, there was a statistically significant difference only for the intelligence quotient. Conclusion: Breastfeeding is associated with lower levels of CCL11, higher intelligence quotient scores and schizophrenia. The CCL11 chemokine is higher in those who did not breastfeed, especially in schizophrenic.

Introdução: São vistos muitos efeitos benéficos do aleitamento materno. Estudos demonstram que crianças amamentadas com leite materno quando comparadas às alimentadas com fórmulas lácteas artificiais, apresentam melhor desenvolvimento cognitivo. Em pacientes com esquizofrenia, o aleitamento materno vem sendo avaliado como fator de proteção. Os níveis de quimiocinas CCL11, marcador biológico relacionado principalmente com o envelhecimento precoce, também tem sido associado ao desempenho cognitivo nesses pacientes. Estudos mostram uma correlação negativa deste marcador com o desempenho em testes de memória de trabalho e com a tarefa de flexibilidade cognitiva. Sendo assim, surge o interesse em estudar as diferenças entre níveis plasmáticos da quimiocina CCL11, quociente de inteligência e história do aleitamento materno (no peito) em pacientes com esquizofrenia e controles. Métodos: Estudo caso-controle com 56 indivíduos, sendo 30 pacientes com esquizofrenia e 26 controles saudáveis, divididos em grupos que foram aleitados e grupos que não foram. Foi aplicado questionário com dados sócio-econômicos, história ao nascer, dados clínicos e alimentação ao nascer. Foi dosado a quimiocina CCL11 e aplicado testes psicológicos para avaliarem quociente de inteligência, funcionalidade, sintomas psiquiátricos, curso da doença e diagnóstico. Para os controles foi utilizada uma escala para descartar doença psiquiátrica. Resultados: A quimiocina CCL11 apresentou valores significativamente mais altos (>0,5) em pacientes com esquizofrenia quando comparados aos controles e no grupo de amamentados, os esquizofrênicos também apresentaram valores significativamente mais altos, mas em nível intermediário (entre 0.106 e 0.5). Não houve correlação da CCL11 com o número de hospitalizações, idade, tempo de diagnóstico e escolaridade. Também não foi evidenciada correlação entre tempo de aleitamento materno em relação aos fatores do Brief Psychiatric Rating Scale. Houve uma tendência de correlação entre a idade de início da doença e o aleitamento materno. Foi encontrada correlação positiva do CCL11 com o tempo de aleitamento materno. Ao comparar os pacientes esquizofrênicos que foram aleitados com os que não foram, foi encontrada diferença estatisticamente significativa apenas para o quociente de inteligência. Conclusão: O aleitamento materno está associado a níveis mais baixos de CCL11, escores mais altos de quociente de inteligência e com a esquizofrenia. A quimiocina CCL11 é mais alta em quem não amamentou, especialmente nos esquizofrênicos. / Introduction: Are seen many beneficial effects of breastfeeding. Studies show that children breastfed, compared to fed artificial milk formulas, have better cognitive development. In patients with schizophrenia, breastfeeding has been evaluated as a protective factor. The levels of CCL11 chemokine, biomarker related mainly to premature aging, have also been associated with cognitive performance in these patients. Studies show a negative correlation of this marker with the performance of working memory tests and with cognitive flexibility task. Thus, there is the interest in studying the differences between plasma levels of CCL11 chemokine, intelligence quotient and history of breastfeeding in patients with schizophrenia and controls. Methods: Case-control study with 56 subjects, 30 patients with schizophrenia and 26 healthy individuals divided into groups that were breastfed group and those who were not. A socio-economic survey, birth history, clinical data and power at birth was applied. It was dosed to CCL11 chemokine and applied psychological tests to assess intelligence quotient, functionality, psychiatric symptoms, course of the disease and diagnosis. For the controls we used a scale to rule out psychiatric illness. Results: The chemokine CCL11 had significantly higher values (> 0.5) in patients with schizophrenia compared with controls and the breastfed group, schizophrenics also had significantly higher values, but on intermediate level (between 0.106 and 0.5). There was no correlation of CCL11 to the number of hospitalizations, age, time of diagnosis and education. It was also not evident correlation between duration of breastfeeding in relation to factors of Brief Psychiatric Rating Scale. There was a trend of correlation between the age of onset of the disease and breastfeeding. There was a positive correlation between CCL11 with the duration of breastfeeding. By comparing the schizophrenic patients who were breastfed with those who were not, there was a statistically significant difference only for the intelligence quotient. Conclusion: Breastfeeding is associated with lower levels of CCL11, higher intelligence quotient scores and schizophrenia. The CCL11 chemokine is higher in those who did not breastfeed, especially in schizophrenic.

Introdução: São vistos muitos efeitos benéficos do aleitamento materno. Estudos demonstram que crianças amamentadas com leite materno quando comparadas às alimentadas com fórmulas lácteas artificiais, apresentam melhor desenvolvimento cognitivo. Em pacientes com esquizofrenia, o aleitamento materno vem sendo avaliado como fator de proteção. Os níveis de quimiocinas CCL11, marcador biológico relacionado principalmente com o envelhecimento precoce, também tem sido associado ao desempenho cognitivo nesses pacientes. Estudos mostram uma correlação negativa deste marcador com o desempenho em testes de memória de trabalho e com a tarefa de flexibilidade cognitiva. Sendo assim, surge o interesse em estudar as diferenças entre níveis plasmáticos da quimiocina CCL11, quociente de inteligência e história do aleitamento materno (no peito) em pacientes com esquizofrenia e controles. Métodos: Estudo caso-controle com 56 indivíduos, sendo 30 pacientes com esquizofrenia e 26 controles saudáveis, divididos em grupos que foram aleitados e grupos que não foram. Foi aplicado questionário com dados sócio-econômicos, história ao nascer, dados clínicos e alimentação ao nascer. Foi dosado a quimiocina CCL11 e aplicado testes psicológicos para avaliarem quociente de inteligência, funcionalidade, sintomas psiquiátricos, curso da doença e diagnóstico. Para os controles foi utilizada uma escala para descartar doença psiquiátrica. Resultados: A quimiocina CCL11 apresentou valores significativamente mais altos (>0,5) em pacientes com esquizofrenia quando comparados aos controles e no grupo de amamentados, os esquizofrênicos também apresentaram valores significativamente mais altos, mas em nível intermediário (entre 0.106 e 0.5). Não houve correlação da CCL11 com o número de hospitalizações, idade, tempo de diagnóstico e escolaridade. Também não foi evidenciada correlação entre tempo de aleitamento materno em relação aos fatores do Brief Psychiatric Rating Scale. Houve uma tendência de correlação entre a idade de início da doença e o aleitamento materno. Foi encontrada correlação positiva do CCL11 com o tempo de aleitamento materno. Ao comparar os pacientes esquizofrênicos que foram aleitados com os que não foram, foi encontrada diferença estatisticamente significativa apenas para o quociente de inteligência. Conclusão: O aleitamento materno está associado a níveis mais baixos de CCL11, escores mais altos de quociente de inteligência e com a esquizofrenia. A quimiocina CCL11 é mais alta em quem não amamentou, especialmente nos esquizofrênicos. / Introduction: Are seen many beneficial effects of breastfeeding. Studies show that children breastfed, compared to fed artificial milk formulas, have better cognitive development. In patients with schizophrenia, breastfeeding has been evaluated as a protective factor. The levels of CCL11 chemokine, biomarker related mainly to premature aging, have also been associated with cognitive performance in these patients. Studies show a negative correlation of this marker with the performance of working memory tests and with cognitive flexibility task. Thus, there is the interest in studying the differences between plasma levels of CCL11 chemokine, intelligence quotient and history of breastfeeding in patients with schizophrenia and controls. Methods: Case-control study with 56 subjects, 30 patients with schizophrenia and 26 healthy individuals divided into groups that were breastfed group and those who were not. A socio-economic survey, birth history, clinical data and power at birth was applied. It was dosed to CCL11 chemokine and applied psychological tests to assess intelligence quotient, functionality, psychiatric symptoms, course of the disease and diagnosis. For the controls we used a scale to rule out psychiatric illness. Results: The chemokine CCL11 had significantly higher values (> 0.5) in patients with schizophrenia compared with controls and the breastfed group, schizophrenics also had significantly higher values, but on intermediate level (between 0.106 and 0.5). There was no correlation of CCL11 to the number of hospitalizations, age, time of diagnosis and education. It was also not evident correlation between duration of breastfeeding in relation to factors of Brief Psychiatric Rating Scale. There was a trend of correlation between the age of onset of the disease and breastfeeding. There was a positive correlation between CCL11 with the duration of breastfeeding. By comparing the schizophrenic patients who were breastfed with those who were not, there was a statistically significant difference only for the intelligence quotient. Conclusion: Breastfeeding is associated with lower levels of CCL11, higher intelligence quotient scores and schizophrenia. The CCL11 chemokine is higher in those who did not breastfeed, especially in schizophrenic.

Chronic traumatic encephalopathy is the neurodegenerative disease that is ascribed to the long term development of cognitive, behavioral, emotional, and motor deficits as a result of the exposure to high amounts of sub concussive traumatic brain injuries. The disease has gained recent popularity in the media for its prevalence in American football as a response to recent research that has suggested the prominence of the disease in nearly every NFL player that is examined post mortem. This has produced a growing concern for the consequences of head impact and participation in contact sports. Despite media attention, little is currently known about the specific causes of the disease and an in life diagnosis is still nonexistent. The present study proposes that the chemokine, CCL11, could prove to be a viable biomarker for recognizing the onset and progression of chronic traumatic encephalopathy. The results of our study suggest that football players who are clinically suspicious of CTE show significantly higher levels of CCL11 in their cerebrospinal fluid than do sedentary controls and noncontact athletes. Our results demonstrate that this increase in CCL11 is correlated with the number of years that a football player had participated in. We also suggest that this increase in CCL11 is associated with a unique immune response through results showing that the CCL11 expression increase is correlated with an increase in the expression of the cytokine IL-4 and substantial decrease in IFN-gamma. The analysis of CCL11 expression levels in the cerebrospinal fluid may prove to be a viable method of diagnosing and providing treatment for patients who may be at risk of chronic traumatic encephalopathy.

Orientador: Edson Arantes / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-11T19:45:11Z (GMT). No. of bitstreams: 1 Lintomen_Leticia_D.pdf: 1121941 bytes, checksum: 2c92a8ddfce83c01ed6c71e524096e0b (MD5) Previous issue date: 2008 / Resumo: Os eosinófilos participam da patogênese de várias doenças inflamatórias, incluindo infecções parasíticas e doenças alérgicas. Dentre as doenças alérgicas, a eosinofilia tem presença marcante na asma. Atualmente, a asma afeta 300 milhões de indivíduos no mundo, sendo considerada um problema de saúde pública mundial. A asma é uma doença inflamatória crônica que envolve interações entre fatores externos e genéticos, e tem como características principais a inflamação pulmonar e a hiperresponsividade brônquica. O recrutamento de eosinófilos para as vias aeríferas contribui para o caráter crônico da asma. A migração de eosinófilos para o tecido inflamado é um processo complexo, que é regulado por numerosos fatores, incluindo citocinas, quimiocinas, óxido nítrico (NO) e interações de moléculas de adesão. Estudos prévios investigaram as interações funcionais entre NO e CC-quimiocinas. Young e colaboradores (1999) mostraram que o número de eosinófilos no lavado broncoalveolar (LBA) de macacos desafiados está marcadamente aumentado 24 horas após o desafio, e que este aumento é acompanhado de altos níveis de NO no ar exalado e de eotaxina no LBA. Em pacientes com rinite, a eotaxina aumentou o número de eosinófilos no fluido do lavado nasal e também os níveis de NO nasal (Hanazawa et al., 1999). Em contraste, em modelos murinos de asma a inibição seletiva da NOS induzível resultou na redução da migração eosinofílica para os pulmões (Feder et al., 1997; Iijima et al., 2001), que estava associada com o aumento da expressão da CC-quimiocina proteína quimiotática para monócito-1 (MCP-1) no tecido pulmonar (Trifilieff et al., 2000). Além disso, NO (ou doadores de NO) também são capazes de inibir a produção de RANTES (Frank et al., 2000). O NO via formação de peroxinitrito (ONOO-) também pode reduzir a migração de eosinófilos induzida por eotaxina (Sato et al., 2000). Entretanto, o papel modulatório do NO nas funções do eosinófilo mediadas por CC-quimiocinas ainda permanece contraditório. Portanto, o presente trabalho investigou o efeito modulatório do NO na adesão aumentada, quimiotaxia e desgranulação do eosinófilo induzidas pelas Ccquimiocinas eotaxina e RANTES in vitro, e a expressão de VLA-4 e Mac-1 na superfície do eosinófilo. Nós realizamos ensaios funcionais (adesão e desgranulação), análise da expressão de moléculas de adesão por citometria de fluxo (VLA-4 e Mac-1) e a investigação de resíduos de tirosina nitrada para avaliar interações do NO com CC-quimiocinas em eosinófilos humanos. Os ensaios de MTT mostraram que as incubações de eosinófilos por 2, 3, ou 4 horas com eotaxina (10, 100 e 1000 ng/ml) ou RANTES (10, 100 e 1000 ng/ml) não afetam a viabilidade celular e, em determinadas condições, até promovem a ativação das células. Os resultados de adesão à fibronectina mostraram que a eotaxina (10, 100 e 1000 ng/ml) ou RANTES (10, 100 e 1000 ng/ml) não aumentam a adesão de eosinófilos em períodos de incubação de 2 e 3 horas. Entretanto, a incubação de eosinófilos por 4 horas com eotaxina ou RANTES aumentou significativamente a adesão à fibronectina. O L-NAME (0.1 mM), individualmente, aumentou significativamente a adesão de eosinófilos à fibronectina; porém a co-incubação de L-NAME com eotaxina (ou RANTES) não afetou a adesão observada com cada agente isoladamente. Além disso, a expressão de VLA-4 e de Mac-1 não foi modificada em nenhuma das condições experimentais testadas. Eotaxina e RANTES também não foram capazes de aumentar os níveis de GMPc nos eosinófilos, em condições onde o SNP (0.1 mM), usado como controle positivo, aumentou significativamente os níveis desse segundo mensageiro. Além disso, eosinófilos tratados com L-NAME, eotaxina e RANTES, individualmente, foram capazes de desgranular estas células, mas a co-incubação de LNAME com eotaxina (ou RANTES), não alterou esta resposta. Os resultados de quimiotaxia mostraram migração significativa de eosinófilos (tratados ou não com LNAME) em resposta à eotaxina ou RANTES. Os resultados obtidos de Western blotting para 3-nitrotirosina mostraram ausência de proteínas nitradas nos eosinófilos de indivíduos sadios ou asmáticos. No conjunto, nossos resultados mostram que, nas condições experimentais estabelecidas, o NO não modula adesão, migração e desgranulação em eosinófilos estimulados com eotaxina ou RANTES / Abstract: Eosinophils participate in the pathogenesis of many inflammatory diseases, including parasitic infections and allergic diseases. Of the allergic diseases, asthma is characterized by eosinophilia. Currently, asthma affects 300 million people in world, and is an important public health problem. Asthma is an inflammatory chronic disease that involves interactions between external and genetic factors, and has lung inflammation and bronchial hyperresponsiveness as major features. The recruitment of eosinophils into airways contributes to the asthma chronic character. The eosinophil migration to inflamed tissue is a complex process regulated by several factors, including cytokines, chemokines, nitric oxide (NO) and adhesion molecule interactions. Previous studies have investigated the functional interactions between NO and CC-chemokines. Young et al. (1999) showed that the number of eosinophils in the bronchoalveolar lavage (BAL) fluid of challenged monkeys is markedly increased at 24 h post-challenge, accompanied by higher levels of both exhaled NO and eotaxin in BAL fluid. In rhinitis patients, eotaxin increased the number of eosinophils in the nasal lavage fluid, and that was also accompanied by elevated nasal NO levels (Hanazawa et al., 1999). In contrast, in murine models of asthma, selective inhibition of inducible NOS resulted in a reduction in pulmonary eosinophil migration (Feder et al., 1997; Iijima et al., 2001), with an increased expression of the CC-chemokine monocyte chemoattractant protein-1 (MCP-1) in the lung tissue (Trifilieff et al., 2000). Moreover, NO (or NO donors) have also been shown to inhibit the production of RANTES (Frank et al., 2000). Nitric oxide via peroxynitrite (ONOO-) formation is reported to reduce eotaxin-induced eosinophil migration (Sato et al., 2000). However, the modulatory role of NO in the CC-chemokines-mediated eosinophil functions is still not well understood. Therefore, the present study was designed to investigate the modulatory effect of NO in the enhanced eosinophil adhesion, chemotaxis and degranulation induced by the Ccchemokines eotaxin and RANTES in vitro, and the expression of VLA-4 and Mac-1 on the eosinophil surface. We therefore carried out functional assays (adhesion and degranulation), flow cytometry analysis of adhesion molecules (VLA-4 and Mac-1 expression) and investigation of tyrosine nitration to evaluate the interactions between NO and CC-chemokines in human eosinophils. MTT assays showed that incubation of eosinophils for 2, 3 or 4 hours with eotaxin (10, 100 and 1000 ng/ml) or RANTES (10, 100 and 1000 ng/ml) did not affect cellular viability and, in some conditions, even caused cellular activation. The results of adhesion to fibronectin showed that eotaxin (10, 100 and 1000 ng/ml) or RANTES (10, 100 and 1000 ng/ml) did not increase eosinophil adhesion at 2 or 3 hours of incubation. Nevertheless, the incubation of eosinophils for 4 hours with eotaxin or RANTES significantly increased adhesion to fibronectin. L-NAME (0.1 mM), alone, significantly increased eosinophil adhesion to fibronectin; however the co-incubation of L-NAME with eotaxin (or RANTES) did not affect the adhesion of each agent. Moreover, expression of VLA-4 and Mac-1 were not modified by any of the experimental conditions. Eotaxin and RANTES did not increase eosinophil cGMP levels under the same conditions in which SNP (0.1mM), used as a positive control, significantly increased the levels of this second messenger. Furthermore, eosinophils treated with L-NAME, eotaxin and RANTES, alone, demonstrated degranulation, however the co-incubation of eosinophils with L-NAME and eotaxin (or RANTES) did not alter this response. Chemotaxis assays showed a significant eosinophil (treated or not with L-NAME) migration in response to eotaxin or RANTES. Western blotting for 3-nitrotyrosine-3 showed a lack of nitrated proteins in eosinophils from healthy or asthmatic donors. Taken together, results show that, under the experimental conditions established, NO does not modulate adhesion, migration and degranulation in eotaxin or RANTES-stimulated eosinophils / Doutorado / Doutor em Farmacologia

Bone is a dynamic tissue that is continuously remodelled, a process that requires equal amounts of osteoclastic bone resorption and osteoblastic bone formation. Inflammation may disturb the equilibrium and result in local and/or systemic bone loss. Negative bone mass balance occurs in several chronic inflammatory diseases, e.g. periodontitis and rheumatoid arthritis (RA). The aetiology of periodontitis is infectious, while RA is an autoimmune disease. Despite aetiological differences, an association between the two diseases has been established but it is not known if they are causally related. Periodontitis may develop when the inflammatory process, initially restricted to the gingiva (gingivitis), further invades the periodontium and causes bone resorption. The cellular and molecular mechanisms underlying the transition from gingivitis to periodontitis are not fully elucidated. Osteoclast formation is dependent on receptor activator of nuclear factor kappa B ligand (RANKL), but how osteoclast precursors are recruited to the jawbone is poorly understood. A family of cytokines named chemokines has been reported to possess such properties and increasing evidence points towards their involvement in the pathogenesis of chronic inflammatory diseases. The overall aim of this thesis was to gain extended knowledge about the role of chemokines and a newly discovered family of leukocytes named innate lymphoid cells (ILCs) in periodontitis and concomitant inflammatory disturbed bone remodelling. Furthermore, the aim was also to study the association between periodontitis and RA. We identified increased serum levels of monocyte chemoattractant protein (MCP)-1 and CCL11 in individuals with periodontitis. Moreover, a robust correlation between the two chemokines and periodontitis was detected in a weighted analysis of inflammatory markers, subject characteristics and periodontitis parameters. We detected higher MCP-1 levels in periodontitis tissue compared to non-inflamed. Furthermore we demonstrated that human gingival fibroblasts express MCP-1 and CCL11 in response to pro-inflammatory cytokines through NF-κB signalling. Using an inflammatory bone lesion model and primary cell cultures, we discovered that osteoblasts express CCL11 in vivo and in vitro and that the expression increased under inflammatory conditions. Osteoclasts did not express CCL11, but its high affinity receptor CCR3 was upregulated during osteoclast differentiation and found to co-localise with CCL11 on the surface of osteoclasts. Exogenous CCL11 was internalised in osteoclasts, stimulated the migration of osteoclast precursors and increased bone resorption in vitro. To analyse if periodontitis precedes RA we analysed marginal jawbone loss in dental radiographs taken in pre-symptomatic RA cases and matched controls. The prevalence of jawbone loss was higher among cases, and the amount of jawbone loss correlated with plasma levels of RANKL. In the search of the newly discovered ILCs, we performed flow cytometry analyses on gingivitis and periodontitis tissue samples. We detected twice as many ILCs in periodontitis as in gingivitis. In addition we found RANKL expression on ILC1s (an ILC subset). In conclusion, we demonstrated that CCL11 is systemically and locally increased in periodontitis and that the CCL11/CCR3 axis may be activated in inflammatory disturbed bone remodelling. We also found that marginal jawbone loss correlated with plasma levels of RANKL and preceded clinical onset of symptoms of RA. Furthermore, we demonstrated that ILCs are present in periodontitis and represent a previously unknown source of RANKL. / Skelettet har flera viktiga funktioner i kroppen såsom att möjliggöra en upprätt hållning, utgöra fäste för muskler och mediera rörelse, skydda benmärgen och de inre organen samt reglera mängden av lösligt mineral i blodet. Med tiden uppstår mikroskador i skelettet vilket innebär att benvävnaden måste byggas om för att vara fortsatt funktionell. Ombyggnaden kallas remodellering och är en kontinuerlig process som huvudsakligen utförs av benbildande celler kallade osteoblaster och bennedbrytande celler kallade osteoklaster. Remodelleringen är strikt reglerad av olika signalmolekyler och under friska förhållanden råder jämvikt mellan mängden ben som bryts ner och mängden ben som bildas, vilket innebär att benmassan hålls konstant. Vid sjukdomar som medför långvariga inflammationsprocesser i benvävnad eller i närheten av benvävnad, exempelvis parodontit (tandlossningssjukdom) och ledssjukdomen reumatoid artrit (RA), kan den rådande jämvikten rubbas, vilket oftast resulterar i minskad benmängd. Vid parodontit är den bakomliggande orsaken till inflammationen bakterier som finns i placket på tänderna, men vid RA tros anledningen vara att immunförsvaret attackerar kroppsegna celler. Trots olikheterna delar de två sjukdomarna flera gemensamma drag med avseende på riskfaktorer, vilka signalmolekyler som återfinns i blodet samt hur inflammationsprocessen fortskrider. Parodontit föregås av gingivit (tandköttsinflammation). Hos vissa individer övergår gingivit till parodontit, en process som inkluderar nedbrytning av tandens stödjevävnader inklusive käkben. Det är inte helt klarlagt vilka celler och molekyler som finns närvarande vid gingivit respektive parodontit eller vilka mekanismer som ligger bakom skiftet mellan de två tillstånden. Det är sedan tidigare känt att molekylen RANKL är viktig för osteoklastbildning, men det är delvis okänt hur osteoklastförstadieceller rekryteras från blodcirkulationen till käkbenet. En grupp av molekyler kallade kemokiner, som även finns i förhöjda nivåer i blod vid parodontit och RA, har visat sig ha sådana egenskaper. För att finna läkemedel som kan förhindra bennedbrytning till följd av den inflammatoriskt störda benremodellering som sker vid både parodontit och RA är det viktigt att studera sambandet mellan sjukdomarna och få en tydlig bild av vilka celler som är närvarande vid inflammationsprocessen. Det är även av betydelse att kartlägga vilka celler och molekyler som främjar rekrytering av osteoklastförstadieceller och bidrar till bennedbrytning. Syftet med den här avhandlingen var att undersöka betydelsen av kemokiner vid inflammatoriskt störd benremodellering och vid parodontit samt att undersöka sambandet mellan parodontit och RA. För att skapa en tydligare bild av vilka cellertyper som är närvarande vid inflammationsprocessen vid parodontit undersöktes även förekomsten av en nyligen upptäckt celltyp vid namn ILCimmunceller (ILCs) samt om dessa celler uttrycker RANKL. Först analyserades förekomsten av olika inflammatoriska signalmolekyler i blod från individer med parodontit samt från friska kontroller. Individer med parodontit hade förhöjda nivåer av kemokinerna MCP-1 och CCL11. Genom att använda en statistisk analysmetod som utöver inflammatoriska signalmolekyler även inkluderade kliniska variabler kunde ett samband mellan de två kemokinerna och parodontit påvisas. Vidare undersöktes möjliga ursprung till de i blodet förhöjda kemokinnivåerna genom att analysera tandkött från tänder med parodontit samt friskt tandkött. Vid parodontit uppmättes högre nivåer av MCP-1. Gingivala fibroblaster (en celltyp som producerar bindväv och ansvarar för tandköttets uppbyggnad) från människa bildade MCP-1 och CCL11 när de stimulerats med inflammationsfrämjande substanser, vilket krävde aktivering en intracellulär signaleringsväg kallad NF-κB. För att utreda betydelsen av CCL11 vid inflammatoriskt störd benremodellering analyserades bencellers bildning av CCL11 in vivo i skalltak från möss samt in vitro i cellodlingar. Osteoblaster bildade CCL11 in vivo och in vitro och bildningen ökade under inflammatoriska förhållanden. Osteoklaster bildade inte CCL11, men däremot fanns ett uttryck av receptorn CCR3, vilket är en mottagarmolekyl till CCL11. I vävnadssnitt från skalltak visades att CCL11 och CCR3 ser ut att binda till varandra på osteoklasternas yta. Dessutom hade CCL11 en positiv effekt på rekrytering av osteoklastförstadieceller och CCL11 som tillsattes till cellodlingar togs upp av osteoklaster och stimulerade benresorption. För att studera sambandet mellan parodontit och RA analyserades käkbensförlust vid tänder med hjälp av röntgenbilder tagna på individer som senare utvecklade RA (pre-symptomatiska) samt matchade kontroller. De presymptomatiska individerna hade en högre grad av käkbenförlust och det fanns också ett samband mellan käkbensförlust och nivåer av RANKL i blodet. Förekomsten av ILCs i tandkött från tänder med gingivit respektive parodontit analyserades med flödescytometri. Dubbelt så många ILCs återfanns vid parodontit än vid gingivit, varav majoriteten bestod av ILC1 (en undergrupp till ILCs). Vidare analyser visade att ILC1 cellerna bildar RANKL. Sammanfattningsvis, vid parodontit finns förhöjda nivåer av CCL11 i vävnaden och i blodet, och interaktionen mellan CCL11 CCR3 kan vara av betydelse vid inflammatoriskt störd benremodellering. Käkbensförlust föregår RA och korrelerar med nivåer av den osteoklaststimulerande molekylen RANKL i blodet, vilket stödjer teorin om att det finns ett samband mellan de två sjukdomarna. De nyligen upptäckta ILCs återfinns vid både gingivit och parodontit och utgör dessutom en tidigare okänd källa till RANKL.

L'encéphalomyélite myalgique (EM) aussi connue sous le nom de syndrome de fatigue chronique est une maladie multi-systémique caractérisée par une fatigue extrême et un malaise post-effort, associés à d’autres symptômes débilitants comme l’intolérance orthostatique et des troubles du sommeil. L’EM se caractérise également par des altérations au niveau du système immunitaire et des perturbations du métabolisme énergétique affectant également le métabolisme des lipides. Dans ce contexte, nous avons exploré la contribution possible de la sphingomyéline phosphodiesterase acide 3b (SMPDL3B), produite par le gène SMPDL3B, dans la pathogénèse de l’EM. Celle-ci est une protéine multifonctionnelle ancrée par un groupement glycophosphatidylinositol (GPI) au niveau de la membrane des cellules. Cette enzyme a suscité notre intérêt compte tenu de son rôle dans la régulation de l'immunité innée et dans la conversion métabolique des sphingolipides en céramides. En effet, des études métabolomiques antérieures ont rapporté une réduction drastique des taux plasmatiques de céramides de 50% chez les hommes et de 86% chez les femmes souffrant d'EM. Nous proposons que l’élévation des niveaux circulants en SMPDL3B contribue à la sévérité de plusieurs symptômes chez les personnes atteintes d’EM (PAEM) via différents mécanismes. Les niveaux plasmatiques en SMPDL3B ont été mesurés par ELISA au niveau d’une cohorte prospective québécoise composée de PAEM (n=147) et de témoins sédentaires appariés pour le sexe et l’âge (n=62) n’ayant aucun antécédent familial d’EM. Nous avons également testé, par la même approche, des échantillons de plasma d’une cohorte norvégienne composée de PAEM (n=141). L’analyse de ces deux cohortes indépendantes a permis de mettre en évidence une corrélation positive entre les taux circulants en SMPDL3B et la sévérité des symptômes des PAEM. Nous avons également observé des niveaux plasmatiques plus élevés chez les PAEM atteints d’intolérance orthostatique lorsque comparés aux PAEM ne présentant pas ce symptôme. Finalement, nous avons confirmé à l’aide de la spectrométrie cellulaire diélectrique que la forme soluble de la protéine SMPDL3B peut se lier avec une haute affinité au récepteur de chimiokines CCR3 présent chez les cellules Jurkat et mis en évidence que l’occupation de ce récepteur par la chimiokine CCL11 ou un antagoniste pharmacologique pouvait augmenter la liaison de la forme soluble de la protéine SMPDL3B vers un autre récepteur membranaire qui demeure pour l’instant inconnu. Ce projet de maîtrise a permis une meilleure compréhension de la pathophysiologie de l’EM et de la contribution de la protéine SMPDL3B (forme ancrée et soluble) dans sa pathogénèse. / Myalgic encephalomyelitis (ME) also known as chronic fatigue syndrome is a multi-systemic disease characterized by extreme fatigue, post-exercise malaise, orthostatic intolerance, and sleep disturbances. ME is also characterized by alterations in the immune system and disturbances in energy metabolism that also affect lipid metabolism. In this context, we explored the possible contribution of sphingomyelin acid phosphodiesterase 3b, produced by the SMPDL3B gene, in the pathogenesis of ME. This is a multifunctional protein anchored by a glycophosphatidylinositol (GPI) group at the cell membrane. This enzyme has intrigued our interest given its role in the regulation of innate immunity and in the metabolic conversion of sphingolipids into ceramides. Indeed, previous metabolomics studies have reported a drastic reduction in plasma ceramide levels by 50% in men and 86% in women with ME. We propose that elevation of circulating levels of SMPDL3B increases the severity of several symptoms in persons with ME (PwME) via different mechanisms. Plasma levels of SMPDL3B were measured by ELISA in a Quebec cohort composed of PwME (n=147) and sedentary controls matched for sex and age (n=62) with no family history of ME. We also tested, by the same method, plasma samples from a Norwegian cohort composed of PwME (n=141). The analysis of these two independent cohorts revealed a positive correlation between SMPDL3B and the severity of PwME symptoms. We also observed higher plasma levels in PwMEs with orthostatic instability when compared to PwMEs without this symptom. Finally, we confirmed using dielectric cell spectrometry that the soluble form of the SMPDL3B protein can bind with high affinity to the CCR3 chemokine receptor present in Jurkat cells and demonstrated that the occupation of this receptor by the chemokine CCL11 or a pharmacological antagonist could increase the binding to another membrane receptor which remains unknown for the moment. This master's project allowed a better understanding of the pathophysiology of ME and the contribution of the SMPDL3B protein in its pathogenesis.