Thesis (M.D.)—Boston University

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 5 uppsatser.

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

Millionen Menschen weltweit leiden an den verschiedensten Autoimmunerkrankungen. Diese Krankheiten entstehen, wenn das Immunsystem gesundes körpereigenes Gewebe angreift und zerstört. An der Pathogenese sind sowohl Komponenten des angeborenen Immunsystems als auch Bestandteile des adaptiven Immunsystems, wie Lymphozyten und Antikörper, beteiligt. Da die Ursachen und molekularen Mechanismen der Pathogenese dieser Erkrankungen bis heute weitgehend unbekannt sind, wurden in dieser Arbeit autoaggressive Lymphozyten bei den humanen Autoimmunerkrankungen Polymyositis und Multiple Sklerose näher untersucht. Die Polymyositis ist eine chronisch entzündliche Erkrankung der Skelettmuskulatur. Die Muskelfasern werden dabei von zytotoxischen CD8+ gd-T-Lymphozyten infiltriert, attackiert und schließlich zerstört. In einem seltenen Fall der Polymyositis wurden die Muskelzellen hingegen in ähnlicher Weise von CD8- gd-T-Lymphozyten angegriffen. Die gd-T-Lymphozyten waren monoklonal expandiert und ihr Rezeptor, im Folgenden als M88 bezeichnet, wurde als Vg1.3+Vd2+ identifiziert. Frühere Untersuchungen der Antigenspezifität dieser Zellen zeigten, dass M88 mehrere funktionell und strukturell verschiedene Proteine aus unterschiedlichen Spezies erkennt. Die Bindung erfolgt spezifisch durch die Antigenerkennungsregionen beider Rezeptorketten von M88. In dieser Arbeit wurden verschiedene bakterielle und humane Proteine des Translationsapparates als Antigene von M88 identifiziert. Weitere ausführliche Untersuchungen eines paradigmatischen bakteriellen Antigens, dem Translationsinitiationsfaktor EcIF1, zeigten, dass M88 an Oberflächen-exponierte Konformationsepitope von Proteinen bindet. Interessanterweise erkennt M88 mehrere humane Aminoacyl-tRNA-Synthetasen, Antigene, die in anderen Formen der Myositis von Autoantikörpern angegriffen werden. Diese Beobachtung ergibt eine bemerkenswerte Verbindung zwischen T-Zell- und Antikörper-vermittelten B-Zell-Antworten bei der autoimmunen Myositis. Bei der Multiplen Sklerose ist das zentrale Nervensystem betroffen. Autoaggressive Lymphozyten greifen die Myelinschicht der Nervenzellen im Gehirn und Rückenmark an und zerstören sie. Im Liquor cerebrospinalis von Patienten lassen sich klonal expandierte und affinitätsgereifte B-Zellen sowie „oligoklonale Banden“ (OKB) Antikörper nachweisen. Obwohl diese Merkmale auf eine Antigen-induzierte Immunantwort hindeuten, sind die zugrundeliegenden Antigene und die Rolle der OKB bei der Pathogenese bis heute unbekannt. In dieser Arbeit wurde die Antigenspezifität von fünf IgG OKB-Antikörpern aus drei Patienten untersucht. Durch verschiedene proteinbiochemische Methoden konnten intrazelluläre Kandidatenantigene identifiziert werden. Interessanterweise sind darunter mehrere nukleäre Proteine, die an der Transkriptionsregulation oder der RNA-Prozessierung beteiligt sind. Reaktivitäten gegen intrazelluläre Antigene treten auch bei anderen Autoimmunerkrankungen, wie beispielsweise dem systemischen Lupus erythematodes, auf. Diese Ergebnisse könnten auf einen allgemeinen Mechanismus der Entstehung und Funktion von Autoantikörpern bei diesen humanen Autoimmunerkrankungen hindeuten. / Millions of people worldwide suffer from various autoimmune diseases. These disorders occur if the immune system reacts to and destroys healthy body tissue. Pathogenesis is mediated by components of the innate immune system as well as by constituents of the adaptive immune system, like lymphocytes and antibodies. However, the origin and molecular mechanisms of these diseases remain still largely unknown. Therefore we investigated the role of autoaggressive lymphocytes in the human autoimmune diseases polymyositis and multiple sclerosis. Polymyositis is a chronically inflammatory disease of the skeletal muscles. Cytotoxic CD8+ gd-T lymphocytes invade, attack and finally destroy muscle fibers. However, in a rare variant of polymyositis, muscle fibers are similarly attacked by CD8- gd-T lymphocytes. These gd-T lymphocytes were monoclonally expanded and their receptor, termed M88, was identified as Vg1.3+Vd2+. Previous investigations of the antigen specificity of these cells revealed that M88 recognizes several structurally and functionally different proteins from various species. This recognition is specifically mediated by the antigen recognition sites of both receptor chains of M88. In this work we have identified different bacterial and human proteins of the translation apparatus as antigens of M88. Further detailed investigations of one paradigmatic bacterial antigen, the translation initiation factor EcIF1, have shown that M88 binds to surface-exposed conformational protein epitopes. Interestingly, M88 recognizes several human aminoacyl-tRNA-synthetases. These antigens have been described to be also targeted by autoantibodies in other forms of myositis. This observation reveals a remarkable link between T cell and antibody-mediated B cell responses in autoimmune myositis. In multiple sclerosis the central nervous system is affected. Autoaggressive lymphocytes attack and destroy the myelin sheet of nerve cells of the brain and spinal cord. In the cerebrospinal fluid of these patients clonally expanded and affinity-maturated B cells as well as „oligoclonal band“ (OCB) antibodies can be detected. Although these features indicate an antigen-induced immune response, the underlying antigens and the role of the OCB antibodies in the pathogenesis are still unknown. In this work we describe the antigen specificity of five IgG OCB antibodies from three patients. Through various biochemical methods we have identified intracellular candidate antigens. Interestingly, these include several nuclear proteins involved in transcription regulation and RNA processing. Reactivity against intracellular antigens also occurs in other autoimmune diseases such as systemic lupus erythematosus. These findings suggest a general mechanism for the generation and function of autoantibodies in these human autoimmune diseases.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2002. / Härtill 4 uppsatser.

Made available in DSpace on 2014-06-11T19:33:26Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-08-04Bitstream added on 2014-06-13T19:04:11Z : No. of bitstreams: 1 ferraro_gc_dr_jabo_prot.pdf: 1010963 bytes, checksum: c1579c5e7b3fa428f65ff2098235f564 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A leishmaniose visceral canina é uma enfermidade geralmente crônica que pode causar hipertermia, perda progressiva de peso, caquexia e atrofia muscular. Existem na literatura relatos da observação de formas amastigotas do parasita na musculatura estriada esquelética de cães, entretanto a patogenia da miopatia permanece incerta. Dessa forma, o objetivo desse estudo foi avaliar as alterações eletromiográficas e histopatológicas em músculos estriados esqueléticos de cães naturalmente acometidos por leishmaniose visceral. Para tanto, 25 cães adultos, machos ou fêmeas, sem raça definida e com diagnóstico parasitológico e sorológico estabelecido para a doença foram utilizados. Os músculos selecionados foram: cabeça longa do tríceps braquial, extensor carpo radial, bíceps femoral e cabeça lateral do gastrocnêmio. Entre os achados eletromiográficos estão o aumento (24% dos cães) e diminuição (68%) da atividade elétrica insersional; presença de potenciais espontâneos (52%) durante a atividade elétrica de repouso, caracterizados por ondas positivas, fibrilações e descargas complexas repetitivas; e recrutamento alterado (72%) na atividade elétrica voluntária. O exame histopatológico revelou variados graus de degeneração e necrose (80%); presença de infiltrado inflamatório mononuclear (48%); presença de fibrose (56%); variação no diâmetro das miofibras (60%); e presença de células adiposas no endomísio (40%). Estas alterações são consistentes com um quadro de polimiosite em fase crônica para 52% dos animais, em fase aguda para 8% e em fase indefinida em 16%. / Canine visceral leishmaniasis is a cronic disease that can cause fever, progressive weight loss, cachexia and muscle atrophy. There are few reports in literature that describe the observation of amastigote forms of the parasite in muscles, however, the pathogenesis of the miopathy remains unclear. The aim of the present study was evaluate the electromyographic and histopathologic changes in skeletal muscle of dogs naturally infected by visceral leishmaniasis. For this, twenty-five dogs adult mongrels of both sexes with a parasitologically and serologically established diagnosis of leishmaniasis were used. The muscles selected were: triceps brachial, extensor carpi radialis, biceps femoris and gastrocnemius. The electromyograph abnormalities included decreased (68% of dogs) and increased (24%) insertion activity; abnormal spontaneous activity (52%) in the form of positive sharp waves, fibrilation potentials and bizarre high frequency discharges and abnormal recruitment of motor unit action potential (72%) during voluntary activity. Histological examination showed various degrees of degeneration and myonecrosis (80%), perimysial and endomysial lymphocytes infiltrations (48%), variation in myofibers sizes (60%), and replacement of muscle fibres by fibrous tissue (56%) and adipose tissue (40%). These changes were considered to be consistent with a chronic phase of polymyositis in 52% of the dogs, in acute phase for 8% and variable phase for 16% of the dogs.

Orientadora: Mary Marcondes / Banca: Márcia Dalastra Laurenti / Banca: Maria Rita Pacheco / Banca: Rosemeri de Oliveira Vasconcelos / Banca: Raimundo Souza Lopes / Resumo: A leishmaniose visceral canina é uma enfermidade geralmente crônica que pode causar hipertermia, perda progressiva de peso, caquexia e atrofia muscular. Existem na literatura relatos da observação de formas amastigotas do parasita na musculatura estriada esquelética de cães, entretanto a patogenia da miopatia permanece incerta. Dessa forma, o objetivo desse estudo foi avaliar as alterações eletromiográficas e histopatológicas em músculos estriados esqueléticos de cães naturalmente acometidos por leishmaniose visceral. Para tanto, 25 cães adultos, machos ou fêmeas, sem raça definida e com diagnóstico parasitológico e sorológico estabelecido para a doença foram utilizados. Os músculos selecionados foram: cabeça longa do tríceps braquial, extensor carpo radial, bíceps femoral e cabeça lateral do gastrocnêmio. Entre os achados eletromiográficos estão o aumento (24% dos cães) e diminuição (68%) da atividade elétrica insersional; presença de potenciais espontâneos (52%) durante a atividade elétrica de repouso, caracterizados por ondas positivas, fibrilações e descargas complexas repetitivas; e recrutamento alterado (72%) na atividade elétrica voluntária. O exame histopatológico revelou variados graus de degeneração e necrose (80%); presença de infiltrado inflamatório mononuclear (48%); presença de fibrose (56%); variação no diâmetro das miofibras (60%); e presença de células adiposas no endomísio (40%). Estas alterações são consistentes com um quadro de polimiosite em fase crônica para 52% dos animais, em fase aguda para 8% e em fase indefinida em 16%. / Abstract: Canine visceral leishmaniasis is a cronic disease that can cause fever, progressive weight loss, cachexia and muscle atrophy. There are few reports in literature that describe the observation of amastigote forms of the parasite in muscles, however, the pathogenesis of the miopathy remains unclear. The aim of the present study was evaluate the electromyographic and histopathologic changes in skeletal muscle of dogs naturally infected by visceral leishmaniasis. For this, twenty-five dogs adult mongrels of both sexes with a parasitologically and serologically established diagnosis of leishmaniasis were used. The muscles selected were: triceps brachial, extensor carpi radialis, biceps femoris and gastrocnemius. The electromyograph abnormalities included decreased (68% of dogs) and increased (24%) insertion activity; abnormal spontaneous activity (52%) in the form of positive sharp waves, fibrilation potentials and bizarre high frequency discharges and abnormal recruitment of motor unit action potential (72%) during voluntary activity. Histological examination showed various degrees of degeneration and myonecrosis (80%), perimysial and endomysial lymphocytes infiltrations (48%), variation in myofibers sizes (60%), and replacement of muscle fibres by fibrous tissue (56%) and adipose tissue (40%). These changes were considered to be consistent with a chronic phase of polymyositis in 52% of the dogs, in acute phase for 8% and variable phase for 16% of the dogs. / Doutor

Aims: To elucidate the aetiopathological mechanisms underlying the IIMs, through a combination of genotyping, serotyping and clinical phenotyping in a large cohort of Caucasian idiopathic inflammatory myopathy (IIM) patients. Methods: A cross-sectional study of prevalent IIM cases, ascertained through the Adult Onset Myositis Immunogenetic Collaboration, was performed. Cases were confirmed as possessing myositis according to Bohan and Peter (Bohan and Peter 1975a; Bohan and Peter 1975b). IIM clinical subtypes studied included polymyositis (PM), dermatomyositis (DM) and myositis associated with other connective tissue disease (myositis/CTD-overlap). Genotyping of major histocompatibility complex genes, including HLA-B, -DR, -DQ, tumour necrosis factor alpha (TNF-α), was performed using commercial kits. Serotyping of a comprehensive range of myositis specific/associated antibodies (MSA/MAAs) was undertaken. Results: Clinical subsets are described within the serological groupings, suggesting that the classification of the IIMs appears to be better served by the serotype than by the clinical subgrouping of disease. The IIMs possess HLA class I and II haplotype associations and genetic differences observed between PM and DM are accounted for by serological differences. The TNF-308A association is not independent of HLA class I, due to the strong LD within the MHC, but does form part of a haplotype with these factors. An absence of routinely tested for MSA/MAAs makes cancer associated myositis (CAM) more likely, especially in the DM subgroup. An antibody against a 155 and 140kDa doublet is associated with the development of CAM. Outcome measures in the IIMs show construct validity. HLA-DRB1*07 appears to predict a milder clinical phenotype with less disability. No convincing gene-environmental interaction was found capable of altering disease susceptibility or clinical phenotype. Conclusions: Myositis disease subtypes therefore appear to be defined by specific haplotypes acting as risk factors for the development of various MSAs and MAAs.

Objetivo: Avaliar a função gonadal de homens com miopatias inflamatórias idiopáticas (MII). Métodos: Vinte e cinco pacientes com MII foram avaliados e comparados com 25 homens saudáveis. Os pacientes foram subdivididos em dois sub-grupos de acordo com as alterações dos espermatozóides: grupo A (n=10, duas ou mais das seguintes alterações: terato/oligo/astenozoospermia ou azoospermia) e grupo B (n=15, teratozoospermia). Foram realizados: exame urológico, ultra-sonografia testicular, análise dos espermatozóides (critérios da OMS e Kruger), pesquisa dos anticorpos anti-espermatozóides e dosagens hormonais Nos subgrupos foram também avaliados: Disease Activity Score (DAS), Visual Analogue Scale (VAS), Manual Muscle Testing (MMT), myositis disease activity assessment visual analogue scales (MYOACT), myositis intention to treat activity index (MITAX), Myositis damage index [MDI], enzimas musculares e tratamento. Resultados: Pacientes apresentaram alterações nos espermatozóides comparados com controles com freqüência maior de nível elevado de FSH (20% versus 0%, p=0,05). O sub-grupo A apresentou freqüência e mediana maior do nível de CK (p=0,001 e p=0,001) assim como DAS (p=0,01), VAS (p=0,051), MMT (p=0,003). As medianas dos volumes testiculares foram menores no grupo A (direito, p=0,015 e esquerdo, p=0,025). As medianas dos parâmetros espermáticos foram reduzidas no grupo A [contagem total (p=0,0001); motilidade (p=0,0001); morfologia pela OMS (p=0,0001) e por Kruger (p=0,0001). A mediana de FSH foi elevada (p=0,035) e de androstenediona foi reduzida (p=0,02) no sub-grupo A. Afrequência de ciclofosfamida foi similar nos grupos (30% versus 6%, p=0,26). Conclusões: Atividade da doença foi o principal fator contribuinte para disfunção gonadal. Hipogonadismo hipergonadotrófico pode explicar as alterações anatômicas e funcionais observadas / Objective: To perform a global gonad evaluation in male idiopathic inflammatory myopathies (IIM) patients. Methods: Twentyfive consecutive IIM were compared to 25 age-matched healthy subjects. Patients were subdivided in two groups according to the severity of sperm abnormalities: group A (at least two of the following terato/oligo/asthenozoospermia or azoospermia) and group B (teratozoospermia). Patients and controls underwent a systematic assessment consisting of: urologic examination, testicular ultrasound, semen analysis and hormones. Patients´ serum CK levels, visual analogue scale (VAS), disease activity score (DAS), manual muscle testing (MMT), myositis disease activity assessment visual analogue scales (MYOACT), myositis intention to treat activity index (MITAX), and Myositis damage index (MDI) were evaluated. Results: Several sperm variables were significantly altered compared to controls (p<0.05). The subgroup analysis according to the severity of sperm alterations revealed that the frequency of elevated CK and its median level was significantly higher in group A (p=0.001 and p=0.001), as also was DAS, VAS and MMT (p=0.01; p=0.051 and p=0.03). The median of testicular volumes were lower in group A (right p=0.015 and left p=0.025). All median sperm parameters were lower in group A (total sperm count, p=0.0001; total motile sperm count, p=0.0001; and sperm morphology by Kruger p=0.0001 and WHO p=0.0001). Higher median FSH (p=0.035) and lower median androstenedione levels (p=0.02) were observed in group A. The frequency of cyclophosphamide was similar in both groups (30% vs. 6%, p=0.26). Conclusions: Active disease was the major contributing factor for severe gonad dysfunction. The hypergonadotrophic hypogonadism may explain the anatomical and dysfunctional alterations observed