Equine arteritis virus (EAV) establishes persistent infection in the stallion reproductive tract, and the carrier stallion continues to shed virus in semen for weeks to years or lifelong. The objective of this study was to elucidate the intra-host evolution of EAV during persistent infection in stallions. Seven EAV seronegative stallions were experimentally infected with EAV KY84 strain and followed for 726 days post-infection, and sequential clinical samples including semen were collected for virus isolation and next-generation sequencing (NGS). In addition, archived sequential semen samples from two stallions that were naturally infected with EAV KY84 for a long-period (up to 10 years) were also sequenced by NGS. The data demonstrated genetic bottleneck event and selection during acute infection followed by intra-host quasispecies diversification during persistent infection in the stallion reproductive tract. Also, the full-length genome of a novel EAV donkey strain from Chile and a noncytopathic bovine viral diarrhea virus-1 (ncpBVDV-1) strain contaminating rabbit kidney-13 cells were also sequenced by NGS. The EAV donkey strain was genetically distinct but antigenically cross-reacted with EAV antisera, and it was phylogenetically closely related to the South African donkey strain of EAV. Genetic and phylogenetic analyses demonstrated that ncpBVDV-1 belongs to BVDV-1b group.

Desinfektionsmittel sind ein elementarer Bestandteil der Tierseuchenbekämpfung und damit auch der Lebensmittelsicherheit. Die Prüfung chemischer Desinfektionsmittel ist Voraussetzung für deren zuverlässige Wirksamkeit und zielgerichteten Einsatz. In Deutschland geschieht dies nach den Richtlinien der Deutschen Veterinärmedizinischen Gesellschaft e.V. (DVG). Seit der ersten Fassung sind die Richtlinien einem ständigen Anpassungsprozess unterworfen. Im Zuge der europäischen Harmonisierung gilt es nun, sich gesamteuropäischen Richtlinien, verfasst durch das europäische Komitee für Normung (Comité Européen de Normalisation) (CEN) anzupassen. Das Thema dieser Arbeit entwickelte sich im Kontext der derzeitigen Diskussion über Verbesserungsvorschläge zu den bestehenden Richtlinien und deren Anpassung an die europäischen Normen. Es wurden je zwei Testviren für die Bereiche Tierhaltung und tierärztliche Praxis ausgewählt, um sie auf Eignung für die Viruzidieprüfung zu testen und gegebenenfalls zu etablieren. Des Weiteren wurde in einem zweiten Teil, in Anlehnung an die Forderungen der europäischen Normen die Prüfung zu vereinfachen, ein alternatives Zellkulturnachweissystem für das Newcastle-Disease-Virus (NDV) geprüft. Die Prüfung der viruziden Wirksamkeit erfolgte mit fünf verschiedenen Grundsubstanzen, gewählt um ein möglichst breites Spektrum an Desinfektionsmittelwirkstoffen abzudecken. Es wurden Glutaraldehyd, Ethanol, Natronlauge, Natriumhypochlorit und Peressigsäure verwendet. Die Versuche wurden mit einer niedrigen Eiweißbelastung und bei einer Temperatur von 20°C durchgeführt. Um eine praxisnahe Situation zu simulieren wurde auf, bereits in den DVG-Richtlinien, verankerten Stahl- und Holzkeimträgertests zurückgegriffen. Als mögliche Prüfviren für die Tierhaltung wurden das Equine Arteritis-Virus (EAV) und das Bovine Virus Diarrhoe Virus verwendet. Bei beiden Viren handelt es sich um weit verbreitete Tierseuchenerreger mit einer großen epidemiologischen Bedeutung. Die Untersuchung von fünf verschiedenen Desinfektionsmitteln erfolgte im Keimträgertest auf Holz. Sowohl EAV als auch BVD stellen ein weniger geeignetes Prüfvirus dar, da beide Viren enorme Titerverluste im Trocknungsvorgang der Holzkeimträger zeigten. Die Viren ließen sich zwar leicht vermehren, aber die erzielten Ausgangstiter reichten nicht aus um die Trocknungsverluste zu kompensieren und aussagekräftige Ergebnisse zu produzieren. Für den Bereich tierärztliche Praxis wurden das Feline Coronavirus (FCoV) und das Murine Parvovirus (MPV) genutzt. FCoV ist ein weltweit in Hauskatzenpopulationen vorkommendes Virus mit einer hohen Seroprävalenz und wurde daher ausgewählt. MPV wurde als Stellvertreter für die, in der Praxis häufig vorkommenden Parvovirusinfektionen gewählt. Es schien ein ideales Modellvirus aufgrund seiner weiten Verbreitung in der Forschung zu sein. Bei beiden Viren erfolgte die Prüfung auf Stahlkeimträgern. Unter Laborbedingungen konnte FCoV ohne Probleme zu hohen Titern vermehrt werden. Es gab keine nennenswerten Trocknungsverluste. FCoV erwies sich als geeignetes Prüfvirus. MPV hingegen ist bedingt durch die langen Versuchszeiten und schwierig auszuwertenden Zellkulturen, sowie wegen der niedrigen Ausgangstiter weniger geeignet als Modellvirus für die Desinfektionsmittelprüfung. Die Anzucht von NDV in Allantoisflüssigkeit von SPF Hühnereiern erschien sehr aufwendig und mit hohem Eiweißfehler belastet. In den Versuchen konnte ein deutlich höherer Eiweißgehalt als in den vergleichend geprüften, in Zellkultur angezogenen Viren nachgewiesen werden. Infolge der Probleme mit der Kultivierung der LMH-Zelllinie und den damit verbundenen langen Wartezeiten bis zur eigentlichen Versuchsdurchführung kann nur eine teilweise Empfehlung, von auf Zellkultur vermehrtem NDV (NDV (ZK)) gegeben werden. Nach Behebung dieser Probleme ist durchaus eine Ablösung, von in Allantoisflüssigkeit angezüchtetem NDV durch NDV (ZK) zu empfehlen. Die Verfälschung der Ergebnisse durch die höheren Eiweißgehalte bei Desinfektionsmitteln mit deutlichem Eiweißfehler könnten so vermieden werden.

There is an urgent need for detection of viral respiratory pathogens to identify the causal agent(s) involved and to prevent the spread of related diseases. The first part of this dissertation focuses on development, optimization and validation of Real-time reverse transcription polymerase chain reaction (rRT-PCR) assays for the detection of several common equine viral pathogens: equine arteritis virus (EAV), equine influenza virus and equine rhinitis viruses A and B. Emphasis of the second part of this dissertation is on studying the role of EAV envelope proteins in virus attachment and entry. Using an infectious cDNA clone of EAV and reverse genetics, a panel of chimeric viruses was generated by swapping the N-terminal ectodomains and full-lengths of the two major envelope proteins (GP5 and M) from porcine reproductive and respiratory syndrome virus (PRRSV). The recombinant viruses expressing the N-terminal ectodomain of PRRSV GP5 or M or both (GP5ecto, Mecto, and GP5&Mecto, respectively) in an EAV backbone were viable and genetically stable. Compared to the parental virus, these three chimeric viruses produced lower titers and smaller plaque sizes indicating that they have a crippled phenotype. Interestingly, the three chimeric viruses could only infect EAV susceptible cell lines but not the PRRSV susceptible cell line. Therefore, the exchange of GP5 and/or M protein N-terminal ectodomains from PRRSV did not alter the cellular tropism of the chimeric viruses. We also investigated the role of one of the minor envelope proteins (E) of EAV in virus attachment and entry. The results showed that EAV infection of equine endothelial cells is heparin-dependent and the Cterminus of the E protein contains a putative heparin-binding domain. We generated a panel of arginine to glycine mutations in the conserved region of both the full-length EAV infectious cDNA clone and individual E protein expression vectors. The triple mutation R52,60,65G construct grew significantly slower and produced much smaller plaques. The double mutant R52,60G completely blocked the interaction between E protein and heparin. Taken together, these data indicated that E protein interacts with heparin to facilitate virus attachment and plays a major role in EAV infection.

Introdução: Os critérios diagnósticos da arterite de Takayasu (AT) se baseiam em alterações do lúmen vascular, detectadas pela angiografia convencional (AC). Além das lesões do lúmen, as alterações na parede da aorta, caracterizadas pela ressonância magnética (RM), estão sempre presentes na AT, não sendo demonstradas pela AC. Objetivos: Descrever as lesões do lúmen da aorta e seus ramos e as alterações na parede da aorta por RM, em pacientes com AT, estabelecendo a associação entre os achados de imagem da aorta e atividade da doença. Métodos: A amostra compreendeu 45 pacientes (43 brancos, 40 mulheres, idade média de 32,5 anos + 15,5 anos), com tempo de história da doença entre três meses e 34 anos, idade de manifestação do primeiro sintoma em torno de 23 anos. Cerca de 40% dos pacientes apresentavam atividade clínica da doença, 57% em uso de corticoterapia e 30% de imunossupressor. Valores de velocidade de hemossedimentação (VHS) e de proteína C-reativa (PCR) estavam elevados em 63,2% e 52,2% dos pacientes, respectivamente. Realizadas seqüências Spin-Eco (SE), dupla inversão e recuperação (DIR) ponderadas em T1 e densidade de prótons (DP) para avaliação do espessamento na parede da aorta. Realizada seqüência DIR ponderada em T2 para pesquisa de edema na parede da aorta. Adquiriu-se seqüência angiográfica Gradiente-Eco (GE) 3D com gadolínio (Gd) para avaliação do lúmen da aorta e ramos. Através de seqüência GE 2D verificou-se a presença de realce tardio pelo Gd na parede da aorta. Resultados: Estenose foi o evento angiográfico da aorta mais encontrado (60%), preferencialmente na aorta abdominal. Dilatação foi mais freqüente na aorta torácica (12%), preferencialmente na ascendente. Irregularidade do lúmen e estenose foram os achados mais freqüentes dos ramos da aorta (69%). Espessamento na parede da aorta foi detectado por RM em todos os pacientes com AT. Edema na parede da aorta demonstrou-se ausente em 56% dos casos e realce tardio esteve presente na maioria (75%) dos casos. Não houve associação dos achados de imagem da aorta por RM com dados clínicos e laboratoriais nos pacientes com AT. Conclusões: Através da análise do lúmen e da parede arterial a RM, de forma não-invasiva, contribuiu ao diagnóstico por imagem da AT. Espessamento na parede da aorta pode ser considerado um marcador diagnóstico da doença. / Introduction: Takayasu arteritis (TA) diagnosis criteria are based on lumen abnormalities detected by conventional angiography (CA). In addition to these lesions, aortic wall abnormalities present in TA, but not clearly demonstrated by CA, can also be characterized by magnetic resonance imaging (MRI). Objectives: Describe lumen lesions in the aorta and its branches and aortic wall abnormalities using MRI, correlating these findings with disease activity in TA patients. Methods: The study comprised 45 patients (43 Caucasians, 40 women, mean age 32.5 years + 15,5 years) with a time course of TA ranging from 3 months to 34 years and first manifestation of the disease with a mean of 23 years of age. Forty percent of the patients presented clinical activity of the disease. Corticosteroid and imunossupressor therapies were used in 57% and 30%, respectively. Elevated erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) were found in 63% and 52% of the patients, respectively. Aortic wall thickening was evaluated using T1 and proton density Spin-Echo (SE) and double inversion recovery (DIR) sequences. Aortic wall edema was evaluated using T2 DIR sequence. Contrast enhanced (CE) gradient-echo (GE) 3D angiography was used for the evaluation of the aortic lumen and its branches. Delayed-enhancement images of the aortic wall were investigated with a GE 2D inversion-recovery sequence after CE angiography. Results: Stenosis was the most frequent angiography finding in the aorta (60%), preferentially in the abdominal segment. Enlargement was more frequent in the thoracic aorta (12%), preferentially in the ascending segment. Minor luminal narrowing and stenosis were the most frequent findings in the aortic branches (69%). MRI detected aortic wall thickening in all patients. Aortic wall edema was absent in 56% of the patients and delayed-enhancement images of the aortic wall were present in the majority (75%) of the patients. No correlation was found between clinical and inflammatory markers and the presence of edema or delayed-enhancement in the aortic wall. Conclusions: Non-invasive MRI can accurately establish the diagnosis of TA with the analysis of the lumen and arterial wall. Aortic wall thickening could be considered a marker of the disease.

Introdução: A arterite de Takayasu (AT) é uma vasculite sistêmica rara caracterizada por oclusão, ectasias, aneurismas e estenose da aorta e seus ramos principais. Consequentemente, pode levar a uma redução no pulso de uma ou mais artérias, diferença nos níveis de pressão sistólica dos membros, presença de sopros (cervicais, cardíacos, axilares e/ou abdominais), além da presença de claudicação vascular (membros e/ou vísceras abdominais) e isquemia periférica, o que por sua vez pode levar a uma maior limitação funcional e consequentemente ao sedentarismo. Esses acometimentos podem, em última análise, levar a uma diminuição da capacidade funcional, da capacidade aeróbia e da força muscular, o que está associado com aumento no risco de mortalidade, assim como já foi demonstrado em outras doenças com manifestações clínicas semelhantes, como a doença arterial periférica (DAP). Nesse sentido, o exercício tem sido apontado como uma estratégia interessante para reduzir esses fatores de risco em pacientes com doenças autoimunes inflamatórias. Entretanto, em pacientes com AT, nenhum estudo até o momento, avaliou a capacidade funcional, a capacidade aeróbia e a força muscular desses pacientes, bem como a segurança da prescrição do exercício por meio da resposta inflamatória a uma sessão aguda de esforço. Da mesma forma, o treinamento aeróbio poderia ser uma potencial estratégia em melhorar essas condições, no entanto, o mesmo ainda foi avaliado nesses pacientes. Objetivos: Avaliar os efeitos agudos e crônicos do exercício físico aeróbio em pacientes com AT em remissão. Métodos: Em um primeiro momento, a capacidade aeróbia, força e função muscular, qualidade de vida, prejuízo de caminhada (WIQ) e função endotelial, foram avaliadas em 11 pacientes com AT e comparados com 10 controles saudáveis (GC) que foram pareados por sexo, idade e IMC. Além disso, foi avaliada e comparada a cinéticas das citocinas (IL1ra, IL-6, IL-10, IL-12p70, TNF-alfa, VEGF e PDGF AA) além dos receptores solúveis de TNF (sTNFRI e sTNFRII) em resposta a uma sessão de exercício aeróbio (~ 60% do VO2 pico). Uma sub-amostra do grupo AT (n = 6) foi submetida a um programa de treinamento aeróbio por 12 semanas (2 vezes por semana, de 30 a 50 minutos, frequência cardíaca (FC) entre os limiares ventilatórios). Antes e depois do treinamento, a sessão de exercício aeróbio agudo foi realizada e as citocinas e os receptores do TNF solúveis foram avaliados como descrito acima. A capacidade aeróbia, força e função muscular, função endotelial, qualidade de vida e o questionário de caminhada foram avaliados. As citocinas e os sTNFRs foram avaliadas por multiplex. Resultados: Pacientes com AT apresentaram uma capacidade cardiorrespiratória, força e função muscular, qualidade de vida e o questionário WIQ prejudicados quando comparados ao grupo controle. Não houve diferença significativa para a função endotelial entre os dois grupos. De forma geral, uma sessão de exercício aeróbio não afeta de forma diferente a cinética de citocinas em pacientes com AT e seus pares saudáveis. O treinamento aeróbio levou a uma melhora da força e função muscular e do questionário WIQ nos pacientes com AT, enquanto a capacidade aeróbia, função endotelial e qualidade de vida permaneceram inalteradas. O programa de treinamento aeróbio não exacerbou as concentrações de citocinas inflamatórias em pacientes com AT; pelo contrário, a citocina pro-inflamatória TNF-alfa foi diminuídatanto em repouso como após uma sessão de exercício aeróbio. Além disso, o treinamento aeróbio aumentou os fatores pró-angiogênicos VEGF (em repouso) e PDGF AA (em repouso e em resposta para a sessão de exercício aeróbico). Conclusões: Pacientes com AT apresentam a capacidade aeróbia, força e função muscular prejudicada comparada a sujeitos saudáveis. Uma sessão de exercício aeróbio não exacerbou a inflamação em pacientes com AT. Além disso, o treinamento aeróbio pode ser uma intervenção bem tolerada, segura e eficiente capaz de induzir efeitos imunomodulatórios e pró-angiogênicos, como também aumentar a força e a função muscular em pacientes com AT / Background: Takayasu arteritis (TA) is a rare systemic vasculitis characterized by occlusion, ectasia, aneurysms and stenosis of the aorta and its main branches. Consequently, it can lead to a reduction in pulse of one or more arteries, a blood pressure difference between arms, bruits presence (neck, heart, axillary and/or abdominal arteries), and the presence of vascular claudication (limbs and/or abdominal arteries) and peripheral ischemia, which in turn can lead to a greater functional limitation and consequently inactivity. These manifestations may cause a decrease in aerobic capacity, muscular strength and muscular function, which is associated with increased risk of mortality, as has been shown in other diseases with similar clinical manifestations, such as peripheral arterial disease (PAD). In this scenario, exercise emerges as a promising therapeutic tool to partially offset these adverse outcomes, similarly as it occurs in many other inflammatory rheumatic diseases. However, in patients with TA, no study to date has evaluated aerobic capacity, muscular strength and function in this patients, as well as the safety of exercise prescription in inflammatory response to an acute session of aerobic exercise. Similarly, aerobic training could be a potential strategy to improve such conditions in these patients, however, until date, no study has evaluated the effects of exercise training program in this patients. Objectives: To evaluate the acute and chronic effects of aerobic exercise in patients with TA in remission. Methods: At first, aerobic capacity, muscle strength and function, quality of life, walking impairment questionnaire (WIQ) and endothelial function were evaluated in 11 patients with TA and compared with 10 healthy controls (HC) that were matched for sex , age and body mass index (BMI). Furthermore, it was evaluated and compared the cytokines kinetics (IL1ra, IL-6, IL-10, IL-12p70, TNF-alfa, VEGF and PDGF) and soluble TNF receptors (sTNFRI and sTNFRII) in response to an acute session of aerobic exercise (~ 60% VO2 peak). A sub-sample from TA group (n=6) underwent a 12-week exercise training program (12 weeks, 2 times a week, 30 to 50 minutes, heart rate (FC) between the ventilatory threshold). Before and after training, the acute session of aerobic exercise was performed and cytokines and soluble TNF receptors were assessed as described above. Muscle strength and function, aerobic capacity, endothelial function, quality of life, and walking impairment scores were evaluated. Cytokines and sTNFRs were assessed by multiplex. Results: TA patients showed impaired aerobic capacity, muscle strength and function, worst quality of life and walking impairment compared to their healthy counterparts. There was no significant difference in endothelial function between the two groups. The acute session of aerobic exercise lead to overall similar responses on cytokine kinetics in TA and HC groups. The exercise training program improved muscle strength and function, whereas aerobic capacity, quality of life, and endothelial function parameters remained generally unchanged. The exercise training program did not exacerbate inflammatory cytokines in TA patients; on the contrary, the pro-inflammatory cytokine TNF-alfa was diminished both at resting and following the acute session of aerobic exercise. In addition, the exercise training program increased the pro-angiogenic factors VEGF (at resting) and PDGF AA (at resting and in response to the acute session of aerobic exercise). Conclusions: Patients with TA has an impaired aerobic capacity, muscle strength and function compared to healthy subjects. An acute session of aerobic exercise does not exacerbate inflammation in these patients. Furthermore, exercise could be a well-tolerable, safe and efficient intervention able to induce immunomodulatory and pro-angiogenic effects as well as to increase muscle strength and function in TA patients

Introdução: A arterite de Takayasu (AT) é uma vasculite sistêmica rara caracterizada por oclusão, ectasias, aneurismas e estenose da aorta e seus ramos principais. Consequentemente, pode levar a uma redução no pulso de uma ou mais artérias, diferença nos níveis de pressão sistólica dos membros, presença de sopros (cervicais, cardíacos, axilares e/ou abdominais), além da presença de claudicação vascular (membros e/ou vísceras abdominais) e isquemia periférica, o que por sua vez pode levar a uma maior limitação funcional e consequentemente ao sedentarismo. Esses acometimentos podem, em última análise, levar a uma diminuição da capacidade funcional, da capacidade aeróbia e da força muscular, o que está associado com aumento no risco de mortalidade, assim como já foi demonstrado em outras doenças com manifestações clínicas semelhantes, como a doença arterial periférica (DAP). Nesse sentido, o exercício tem sido apontado como uma estratégia interessante para reduzir esses fatores de risco em pacientes com doenças autoimunes inflamatórias. Entretanto, em pacientes com AT, nenhum estudo até o momento, avaliou a capacidade funcional, a capacidade aeróbia e a força muscular desses pacientes, bem como a segurança da prescrição do exercício por meio da resposta inflamatória a uma sessão aguda de esforço. Da mesma forma, o treinamento aeróbio poderia ser uma potencial estratégia em melhorar essas condições, no entanto, o mesmo ainda foi avaliado nesses pacientes. Objetivos: Avaliar os efeitos agudos e crônicos do exercício físico aeróbio em pacientes com AT em remissão. Métodos: Em um primeiro momento, a capacidade aeróbia, força e função muscular, qualidade de vida, prejuízo de caminhada (WIQ) e função endotelial, foram avaliadas em 11 pacientes com AT e comparados com 10 controles saudáveis (GC) que foram pareados por sexo, idade e IMC. Além disso, foi avaliada e comparada a cinéticas das citocinas (IL1ra, IL-6, IL-10, IL-12p70, TNF-alfa, VEGF e PDGF AA) além dos receptores solúveis de TNF (sTNFRI e sTNFRII) em resposta a uma sessão de exercício aeróbio (~ 60% do VO2 pico). Uma sub-amostra do grupo AT (n = 6) foi submetida a um programa de treinamento aeróbio por 12 semanas (2 vezes por semana, de 30 a 50 minutos, frequência cardíaca (FC) entre os limiares ventilatórios). Antes e depois do treinamento, a sessão de exercício aeróbio agudo foi realizada e as citocinas e os receptores do TNF solúveis foram avaliados como descrito acima. A capacidade aeróbia, força e função muscular, função endotelial, qualidade de vida e o questionário de caminhada foram avaliados. As citocinas e os sTNFRs foram avaliadas por multiplex. Resultados: Pacientes com AT apresentaram uma capacidade cardiorrespiratória, força e função muscular, qualidade de vida e o questionário WIQ prejudicados quando comparados ao grupo controle. Não houve diferença significativa para a função endotelial entre os dois grupos. De forma geral, uma sessão de exercício aeróbio não afeta de forma diferente a cinética de citocinas em pacientes com AT e seus pares saudáveis. O treinamento aeróbio levou a uma melhora da força e função muscular e do questionário WIQ nos pacientes com AT, enquanto a capacidade aeróbia, função endotelial e qualidade de vida permaneceram inalteradas. O programa de treinamento aeróbio não exacerbou as concentrações de citocinas inflamatórias em pacientes com AT; pelo contrário, a citocina pro-inflamatória TNF-alfa foi diminuídatanto em repouso como após uma sessão de exercício aeróbio. Além disso, o treinamento aeróbio aumentou os fatores pró-angiogênicos VEGF (em repouso) e PDGF AA (em repouso e em resposta para a sessão de exercício aeróbico). Conclusões: Pacientes com AT apresentam a capacidade aeróbia, força e função muscular prejudicada comparada a sujeitos saudáveis. Uma sessão de exercício aeróbio não exacerbou a inflamação em pacientes com AT. Além disso, o treinamento aeróbio pode ser uma intervenção bem tolerada, segura e eficiente capaz de induzir efeitos imunomodulatórios e pró-angiogênicos, como também aumentar a força e a função muscular em pacientes com AT / Background: Takayasu arteritis (TA) is a rare systemic vasculitis characterized by occlusion, ectasia, aneurysms and stenosis of the aorta and its main branches. Consequently, it can lead to a reduction in pulse of one or more arteries, a blood pressure difference between arms, bruits presence (neck, heart, axillary and/or abdominal arteries), and the presence of vascular claudication (limbs and/or abdominal arteries) and peripheral ischemia, which in turn can lead to a greater functional limitation and consequently inactivity. These manifestations may cause a decrease in aerobic capacity, muscular strength and muscular function, which is associated with increased risk of mortality, as has been shown in other diseases with similar clinical manifestations, such as peripheral arterial disease (PAD). In this scenario, exercise emerges as a promising therapeutic tool to partially offset these adverse outcomes, similarly as it occurs in many other inflammatory rheumatic diseases. However, in patients with TA, no study to date has evaluated aerobic capacity, muscular strength and function in this patients, as well as the safety of exercise prescription in inflammatory response to an acute session of aerobic exercise. Similarly, aerobic training could be a potential strategy to improve such conditions in these patients, however, until date, no study has evaluated the effects of exercise training program in this patients. Objectives: To evaluate the acute and chronic effects of aerobic exercise in patients with TA in remission. Methods: At first, aerobic capacity, muscle strength and function, quality of life, walking impairment questionnaire (WIQ) and endothelial function were evaluated in 11 patients with TA and compared with 10 healthy controls (HC) that were matched for sex , age and body mass index (BMI). Furthermore, it was evaluated and compared the cytokines kinetics (IL1ra, IL-6, IL-10, IL-12p70, TNF-alfa, VEGF and PDGF) and soluble TNF receptors (sTNFRI and sTNFRII) in response to an acute session of aerobic exercise (~ 60% VO2 peak). A sub-sample from TA group (n=6) underwent a 12-week exercise training program (12 weeks, 2 times a week, 30 to 50 minutes, heart rate (FC) between the ventilatory threshold). Before and after training, the acute session of aerobic exercise was performed and cytokines and soluble TNF receptors were assessed as described above. Muscle strength and function, aerobic capacity, endothelial function, quality of life, and walking impairment scores were evaluated. Cytokines and sTNFRs were assessed by multiplex. Results: TA patients showed impaired aerobic capacity, muscle strength and function, worst quality of life and walking impairment compared to their healthy counterparts. There was no significant difference in endothelial function between the two groups. The acute session of aerobic exercise lead to overall similar responses on cytokine kinetics in TA and HC groups. The exercise training program improved muscle strength and function, whereas aerobic capacity, quality of life, and endothelial function parameters remained generally unchanged. The exercise training program did not exacerbate inflammatory cytokines in TA patients; on the contrary, the pro-inflammatory cytokine TNF-alfa was diminished both at resting and following the acute session of aerobic exercise. In addition, the exercise training program increased the pro-angiogenic factors VEGF (at resting) and PDGF AA (at resting and in response to the acute session of aerobic exercise). Conclusions: Patients with TA has an impaired aerobic capacity, muscle strength and function compared to healthy subjects. An acute session of aerobic exercise does not exacerbate inflammation in these patients. Furthermore, exercise could be a well-tolerable, safe and efficient intervention able to induce immunomodulatory and pro-angiogenic effects as well as to increase muscle strength and function in TA patients

Amyloid is a form of abnormal protein aggregation within the living body. Massive deposits can lead to organ failure. There is also increasing evidence that smaller pre-amyloid aggregates exert direct toxic effects to cells. To date 25 different proteins are known to occur as amyloid deposition in human tissues, although not all of these conditions are known to be associated with clinical diseases. This thesis deals with the very common form of amyloid localized to the arterial media. The fibril protein called ‘medin’ was identified in 1999. Medin is a 50 amino acid residue internal fragment of the precursor protein lactadherin. Lactadherin, first found in human milk, is expressed in various tissues such as breast epithelium (including carcinomas), macrophages and aorta. The function of the protein is not known but it has several functional domains. There is an EFG like domain, including an RGD-sequence, in the N-terminal part of the molecule. The C-terminal part consists of C1 and C2 coagulation factor V and VIII like domains. Medin is from within the C2 domain. This region is suggested to be involved in phosphatidyl serine binding, important in phagocytosis of apoptotic cells. Medin amyloid was originally described from studies of the aorta. It is shown here that deposits are more widely spread and can be found in many large arteries, particularly within the upper part of the body. The prevalence of medin amyloid increases with age and deposits are found, to a certain degree, in virtually everyone above the age of 60 years. The amyloid is not only found extracellularly but intracellular deposits may also occur. Amyloid is usually associated with elastic lamina or lamellae which often show signs of fragmentation. Given the localization of amyloid to elastic structures of the arterial media, three different vascular diseases were studied: temporal (giant cell) arteritis, thoracic aortic aneurysm and thoracic aortic dissection. Medin amyloid was found in temporal arteries with and without inflammation. In inflamed arteries, amyloid was mainly located along the broken internal elastic lamina. Medin was also demonstrated within giant cells. It is suggested that medin may be an antigen triggering autoimmune giant cell arteritis. In the study of thoracic aortic aneurysms and dissections, we found significant less medin amyloid in diseased aortic tissues compared with a control material. On the other hand, immunoreactive medin, probably in the state of oligomeric aggregates, was regularly found in association with aneurysms and dissections but not in the control material. It is suggested that medin oligomers exert toxic effects on smooth muscle cells which may lead to weakening of the arterial wall with aneurysm or dissection as a consequence.

Introdução: Os critérios diagnósticos da arterite de Takayasu (AT) se baseiam em alterações do lúmen vascular, detectadas pela angiografia convencional (AC). Além das lesões do lúmen, as alterações na parede da aorta, caracterizadas pela ressonância magnética (RM), estão sempre presentes na AT, não sendo demonstradas pela AC. Objetivos: Descrever as lesões do lúmen da aorta e seus ramos e as alterações na parede da aorta por RM, em pacientes com AT, estabelecendo a associação entre os achados de imagem da aorta e atividade da doença. Métodos: A amostra compreendeu 45 pacientes (43 brancos, 40 mulheres, idade média de 32,5 anos + 15,5 anos), com tempo de história da doença entre três meses e 34 anos, idade de manifestação do primeiro sintoma em torno de 23 anos. Cerca de 40% dos pacientes apresentavam atividade clínica da doença, 57% em uso de corticoterapia e 30% de imunossupressor. Valores de velocidade de hemossedimentação (VHS) e de proteína C-reativa (PCR) estavam elevados em 63,2% e 52,2% dos pacientes, respectivamente. Realizadas seqüências Spin-Eco (SE), dupla inversão e recuperação (DIR) ponderadas em T1 e densidade de prótons (DP) para avaliação do espessamento na parede da aorta. Realizada seqüência DIR ponderada em T2 para pesquisa de edema na parede da aorta. Adquiriu-se seqüência angiográfica Gradiente-Eco (GE) 3D com gadolínio (Gd) para avaliação do lúmen da aorta e ramos. Através de seqüência GE 2D verificou-se a presença de realce tardio pelo Gd na parede da aorta. Resultados: Estenose foi o evento angiográfico da aorta mais encontrado (60%), preferencialmente na aorta abdominal. Dilatação foi mais freqüente na aorta torácica (12%), preferencialmente na ascendente. Irregularidade do lúmen e estenose foram os achados mais freqüentes dos ramos da aorta (69%). Espessamento na parede da aorta foi detectado por RM em todos os pacientes com AT. Edema na parede da aorta demonstrou-se ausente em 56% dos casos e realce tardio esteve presente na maioria (75%) dos casos. Não houve associação dos achados de imagem da aorta por RM com dados clínicos e laboratoriais nos pacientes com AT. Conclusões: Através da análise do lúmen e da parede arterial a RM, de forma não-invasiva, contribuiu ao diagnóstico por imagem da AT. Espessamento na parede da aorta pode ser considerado um marcador diagnóstico da doença. / Introduction: Takayasu arteritis (TA) diagnosis criteria are based on lumen abnormalities detected by conventional angiography (CA). In addition to these lesions, aortic wall abnormalities present in TA, but not clearly demonstrated by CA, can also be characterized by magnetic resonance imaging (MRI). Objectives: Describe lumen lesions in the aorta and its branches and aortic wall abnormalities using MRI, correlating these findings with disease activity in TA patients. Methods: The study comprised 45 patients (43 Caucasians, 40 women, mean age 32.5 years + 15,5 years) with a time course of TA ranging from 3 months to 34 years and first manifestation of the disease with a mean of 23 years of age. Forty percent of the patients presented clinical activity of the disease. Corticosteroid and imunossupressor therapies were used in 57% and 30%, respectively. Elevated erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) were found in 63% and 52% of the patients, respectively. Aortic wall thickening was evaluated using T1 and proton density Spin-Echo (SE) and double inversion recovery (DIR) sequences. Aortic wall edema was evaluated using T2 DIR sequence. Contrast enhanced (CE) gradient-echo (GE) 3D angiography was used for the evaluation of the aortic lumen and its branches. Delayed-enhancement images of the aortic wall were investigated with a GE 2D inversion-recovery sequence after CE angiography. Results: Stenosis was the most frequent angiography finding in the aorta (60%), preferentially in the abdominal segment. Enlargement was more frequent in the thoracic aorta (12%), preferentially in the ascending segment. Minor luminal narrowing and stenosis were the most frequent findings in the aortic branches (69%). MRI detected aortic wall thickening in all patients. Aortic wall edema was absent in 56% of the patients and delayed-enhancement images of the aortic wall were present in the majority (75%) of the patients. No correlation was found between clinical and inflammatory markers and the presence of edema or delayed-enhancement in the aortic wall. Conclusions: Non-invasive MRI can accurately establish the diagnosis of TA with the analysis of the lumen and arterial wall. Aortic wall thickening could be considered a marker of the disease.

Ce travail de thèse a été axé sur l’étude de la réponse immunitaire T chez des patients atteints d’artérite à cellules géantes (ACG) et de pseudo-polyarthrite rhizomélique (PPR). Plusieurs études cliniques successives interrégionales ont permis d’inclure de nombreux patients (57 ACG et 27 PPR) des Centres Hospitaliers (CH) Universitaires et des CH de l’interrégion Est. Les échantillons sanguins ont été étudiés dans le laboratoire de l’unité INSERM U1098. Tout d’abord, nous avons confirmé l’implication des lymphocytes Th17 dans la pathogénie de l’ACG et avons montré pour la première fois leur implication au cours de la PPR. De plus, notre étude des lymphocytes T (LT) CD4+CD161+ a permis de mieux comprendre les mécanismes de plasticité entre les réponses Th1 et Th17 au cours de ces deux pathologies. Nous avons complété ces travaux par l’étude de la réponse T régulatrice en montrant qu’il existe un déficit quantitatif en Treg au cours de l’ACG et la PPR. Dans la suite de ce travail, nous avons mis en évidence, chez des patients atteints de polyarthrite rhumatoïde, que le blocage de la voie de signalisation de l’IL-6 par un anticorps monoclonal dirigé contre le récepteur de l’IL-6 permet de corriger le déséquilibre de la balance Th17/Treg, en diminuant la réponse Th17 et en augmentant simultanément la réponse T régulatrice, à l’inverse des corticoïdes qui diminuent le pourcentage de Th17 sans corriger le déficit en Treg. Enfin, dans la dernière partie de ce travail, nous avons montré pour la première fois que les LT CD8+ étaient également impliqués dans la pathogénie de l’ACG et la PPR. Ces résultats ont permis de progresser dans les connaissances physiopathologiques de l’ACG et la PPR en évoluant d’un modèle articulé autour d’un déséquilibre de la balance Th1/Th2 vers celui d’un déséquilibre de la balance Th17/Treg et permettent de proposer des thérapeutiques mieux ciblées pour l’ACG et la PPR. / The aim of this thesis was to investigate the T-cell immune response in the course of giant-cell arteritis (GCA) and polymyalgia rheumatica (PMR). Several studies conducted by our team allowed us to obtain blood samples from many patients affected by GCA (n=57) and PMR (n=28). Immunological studies were performed in INSERM U1098, University Of Burgundy, Dijon, France. We firstly demonstrated the implication of Th17 and CD4+CD161+ T cells in the pathogenesis of these two diseases, thus extending the knowledge in the plasticity mechanisms arising between Th1 and Th17 cell-immune responses in GCA and PMR. Furthermore, we investigated the regulatory T cell immune response in these two affections, demonstrating that although being functional, the percentage of circulating Treg was decreased in GCA and PMR patients. As interleukin-6 (IL-6) had been shown to control the Th17/Treg balance, we studied Th17 and Treg frequencies in rheumatoid arthritis patients treated with an anti-IL-6 receptor antibody (tocilizumab). We showed that the blockade of the IL-6 pathway was able to correct the Th17/Treg imbalance by decreasing the number of Th17 cells and simultaneously increasing that of Treg. Finally, we demonstrated for the first time the implication of CD8+ T cells in the pathogenesis of GCA and PMR. This thesis allowed us to progress in the knowledge of the pathogenesis of GCA making the pathogenesis model progress from a Th1/Th2 to a Th17/Treg imbalance model. Altogether, these data deciphering the immune response in the pathogenesis of GCA and PMR bring new knowledge which will lead to better targeted therapies.

A Arterite de Takayasu (AT) é uma vasculite granulomatosa de aorta e grandes vasos associada a elevado risco cardiovascular. A velocidade de onda de pulso (VOP) é um método de avaliação indireta de diminuição da distensibilidade arterial, e valores elevados de VOP correlacionam-se com maior morbimortalidade cardiovascular. A avaliação da VOP em pacientes com arterite de Takayasu é complexa devido a muitos fatores de confusão. O objetivo do presente estudo foi avaliar a rigidez arterial, por meio da velocidade de onda de pulso carótido-femoral (VOP-CF) em pacientes do sexo feminino com arterite de Takayasu e controles saudáveis com variáveis clínicas e antropométricas comparáveis, e sua possível associação com os parâmetros da doença. Método: Pacientes com arterite de Takayasu (n = 27) foram avaliados consecutivamente e foram selecionados controles saudáveis com idade, pressão arterial, peso e altura comparáveis (n = 27). Os critérios de exclusão foram menopausa, tabagismo, diabetes, insuficiência renal, hipertensão mal controlada, arritmias cardíacas, obesidade, comorbidades inflamatórias, gravidez e história de procedimentos cirúrgicos que envolvessem a aorta. A atividade da doença foi determinada por parâmetros clínicos e laboratoriais. As medições de VOP-CF foram obtidas pelo Sistema Complior. Resultados: A média de VOP-CF foi maior em pacientes com arterite de Takayasu do que em controles (9,77 ± 3,49 vs. 7,83 ± 1.06 m/s, p = 0,009). Apesar dos rigorosos xv critérios de seleção, os pacientes com arterite de Takayasu ainda apresentavam, em média, pressão arterial sistólica de 8 mmHg maior do que os controles (p > 0,05), e os valores de pressão de pulso significativamente mais elevados. O modelo de regressão linear múltipla mostra que 93,8% da variabilidade da VOP é explicada pelas variáveis idade, pressão arterial média (PAM) e pela própria doença (R2 ajustado = 0,938). A análise logística stepwise usando como variável dependente o valor de corte de VOP estabelecido pela curva ROC (> 8,34 m/s) e, como variáveis independentes, os parâmetros com significância na análise univariada, revelou que arterite de Takayasu (OR: 4,69, IC 95% 1,31 - 16,72; p = 0,017) e PAM (OR: 1,06, IC 95% 1,00 - 1,12, p = 0,048) foram independentemente associados a maior VOP. Uma análise mais aprofundada dos parâmetros de doença revelou que os valores de VOP não foram correlacionados com velocidade de hemossedimentação, proteína C-reativa, dose cumulativa de glicocorticoides e fração de ejeção (p > 0,05). Conclusão: Nesta coorte de pacientes do sexo feminino com arterite de Takayasu, a própria doença e a pressão arterial média foram os determinantes mais fortemente associados com elevada rigidez arterial e não houve correlação dos valores de VOP com parâmetros de atividade da doença / Takayasu arteritis (TA) is a granulomatous vasculitis that affects the aorta and large vessels and is associated with higher cardiovascular risk. Pulse wave velocity (PWV) is a method of indirect evaluation of decreased arterial distensibility, and elevated PWV correlates with increased cardiovascular morbidity and mortality. The assessment of PWV in patients with Takayasu arteritis is complex due to many confounding factors. The aim of this study was to evaluate arterial stiffness, assessed by carotid-femoral pulse wave velocity (CF-PWV) in female patients with TA and healthy controls with comparable anthropometric and clinical variables, and the possible association with parameters of the disease. Method: Patients with TA (n = 27) were consecutively evaluated and healthy controls were selected with comparable age, blood pressure, weight and height (n = 27). Exclusion criteria were menopause, smoking, diabetes, renal insufficiency, poorly controlled hypertension, cardiac arrhythmias, obesity, inflammatory comorbidities, pregnancy and history of surgical procedures involving the aorta. Disease activity was determined by clinical and laboratory parameters. The CF-PWV measurements were obtained by the Complior System. Results: The mean CF-PWV was higher in patients with TA than in controls (9.77 ± 3.49 vs. 7.83 ± 6.1 m / s, p = 0.009). Despite the strict selection criteria, TA patients still had, on average, systolic blood pressure of 8 mmHg greater than controls (p > 0.05), and pulse pressure values significantly higher. The multiple linear regression model showed that 93.8% of the variability in PWV is explained by the variables age, mean arterial pressure (MAP) and the disease itself (adjusted R2 = 0.938). A stepwise logistic analysis using as the dependent variable the cutoff value of VOP established by the ROC curve (> 8.34 m/s) and, as independent variables, parameters with significance in the univariate analysis, revealed that Takayasu arteritis (OR: 4.69 95% CI 1.31 - 16.72, p = 0.017) and MAP (OR: 1.06, 95% CI 1.00 - 1.12, p = 0.048) were independently associated with increased PWV. Further analysis of disease parameters revealed that PWV values were not correlated with erythrocyte sedimentation rate, C-reactive protein, cumulative dose of glucocorticoids or ejection fraction (p > 0.05). Conclusion: In this cohort of female patients with Takayasu arteritis, the disease itself and mean arterial pressure were determinants most strongly associated with elevated arterial stiffness and no correlation of PWV values and parameters of disease activity was found