Το συμπλήρωμα είναι βασικός παράγοντας της φυσικής ανοσίας (innate immunity) και αποτελεί γέφυρα για την ενεργοποίηση της ειδικής ανοσίας (adaptive immunity). Αποτελείται από ένα σύστημα πρωτεϊνών, που συναντώνται ως ανενεργά προένζυμα και ενεργοποιούνται μέσω πρωτεόλυσης πυροδοτώντας έναν καταρράκτη αντιδράσεων. Οι οδοί ενεργοποίησης του συμπληρώματος καταλήγουν στον σχηματισμό ενός λυτικού συμπλόκου (Membrane Attack Complex – MAC) που καταστρέφει τους παθογόνους μικροοργανισμούς. Οι πρωτεΐνες, συστατικά του συμπλόκου, ανήκουν στην οικογένεια MACPF (MAC – Perforin). Σημαντικό ρόλο στη λειτουργία τους παίζει η πρωτεϊνική περιοχή (module) FIMAC (Factor I Membrane Attack Complex). Η περιοχή αυτή υπάρχει στα συστατικά C6 (Complement component 6) και C7 (Complement component 7) του συμπλόκου MAC και φαίνεται να είναι η περιοχή πρόσδεσής τους με την περιοχή C345C του C5b. Επιπλέον, η περιοχή FIMAC υπάρχει και στον παράγοντα Ι του Συμπληρώματος (Complement Factor I, CFI), ο οποίος συμμετέχει στην αποσταθεροποίηση της κομβερτάσης C3/C5, μέσω του καρβοξυτελικού άκρου της FIMAC που φαίνεται να συνδέεται αλλοστερικά με την πρωτεϊνική περιοχή SP (Serine Protease), που αποτελεί την ενεργή του περιοχή. Το μοντέλο, για την ανάλυση της περιοχής FIMAC βασίστηκε στην περιοχή της πρωτεΐνης φολιστατίνη (follistatin, FS) FD (follistatin Domain), η οποία αναλύθηκε πρόσφατα κρυσταλλογραφικά και με την οποία εμφανίζει ομοιότητα στην αλληλουχία της. Η δομή FD αποτελεί ένα υβρίδιο μιας αμινοτελικής περιοχής EGF (Epidermal Growth Factor) και μίας καρβοξυτελικής περιοχής του ωοβλεννοειδούς (ovomucoid) που ομοιάζουν με τις πρωτεϊνικές περιοχές KAZAL και συναντώνται σε πολλούς αναστολείς σερινικών πρωτεασών. Η περιοχή FD περιέχεται, επίσης, στην πρωτεΐνη αγκρίνη (agrin, AGRN) που αποτελεί συστατικό της βασικής μεμβράνης και παίζει σημαντικό ρόλο στην νευρομυϊκή σύναψη. Η μελέτη των πρωτεϊνικών περιοχών FIMAC και KAZAL αναφορικά με την λειτουργία, την δομή και την εξέλιξή τους αποτέλεσε το αντικείμενο της παρούσας εργασίας. Για την εκπόνηση αυτής της μελέτης χρησιμοποιήθηκαν δεδομένα από βάσεις βιολογικών δεδομένων και εργαλεία βιοπληροφορικής ανάλυσης. Πρωτογενές υλικό της μελέτης αποτέλεσαν οι νουκλεοτιδικές και αμινοξικές αλληλουχίες των γονιδίων C6, C7, CFI, AGRN και FS σε όλους τους οργανισμούς που βρέθηκαν (σπονδυλωτά και ασπόνδυλα), και πιο συγκεκριμένα οι αλληλουχίες που αντιστοιχούν στις πρωτεϊνικές περιοχές FIMAC και KAZAL. Οι πρωτοταγείς δομές των FIMAC ( ̴ 78αα) και KAZAL ( ̴ 55αα) διαφέρουν ως προς το μήκος τους, με μερικές εξαιρέσεις που αφορούν περιοχές KAZAL των πρωτεϊνών AGRN και FS (>80αα). Οι δευτεροταγείς δομές των FIMAC και KAZAL παρουσιάζουν μεγάλη ομοιότητα, φέροντας δομές α – έλικας και β – πτυχωτής επιφάνειας στην αλληλουχία τους. Τέλος, τα μοντέλα προσομοίωσης τριτοταγούς δομής και των δύο περιοχών FIMAC και KAZAL οπτικοποιούν τη διαμόρφωση των δομών της α – έλικας και των β – πτυχωτών επιφανειών στον χώρο. Αξιοσημείωτη είναι η παρουσία σημαντικού αριθμού κυστεϊνικών καταλοίπων στις αλληλουχίες των περιοχών FIMAC (8 – 10 Cys), με μεγαλύτερη συγκέντρωση στο αμινοτελικό άκρο, και KAZAL (4 – 6 Cys) με ομοιόμορφη κατανομή. Εξελικτικά, η εμφάνιση γονιδίων που συμμετέχουν και στις τρεις οδούς ενεργοποίησης του συμπληρώματος και καταλήγουν στη διαμόρφωση του συμπλόκου MAC συνοδεύεται με την εμφάνιση των χονδριχθύων. Πιο συγκεκριμένα, όσον αφορά τα γονίδια του συμπληρώματος που περιλαμβάνουν τις περιοχές FIMAC και KAZAL, ο CFI πρωτοεμφανίζεται στα άγναθα, το C6 στους χονδριχθείς και το C7 στους οστεϊχθείς. Η παρουσία των γονιδίων AGRN και FS έχει πιστοποιηθεί νωρίτερα εξελικτικά στο στάδιο των κεφαλοχορδωτών καθώς και στους πλατυέλμινθες των πρωτοστομίων. Έτσι, φαίνεται ότι η περιοχή KAZAL στις πρωτεΐνες AGRN και FS στα ασπόνδυλα αποτελεί προγονική περιοχή όλων των FIMAC και KAZAL που υπάρχουν σήμερα. Ωστόσο, νέες πρωτεϊνικές περιοχές KAZAL εμφανίστηκαν και αργότερα κατά την εξέλιξη των ειδών. Η συντηρητικότητα και των δύο περιοχών FIMAC και KAZAL στο επίπεδο της γονιδιακής τους κληρονόμησης είναι μεγάλη. Όλα τα intron phases των εξονίων που κωδικοποιούν τις περιοχές KAZAL και FIMAC σε όλα τα γονίδια όπου συναντώνται είναι 1, εκτός από τα αντίστοιχα εξόνια για την περιοχή FIMAC της γραμμικής θέσης 1 στις πρωτεΐνες C6 και C7 (intron phase 2), που φαίνεται να είναι εξελικτικά μεταγενέστερες περιοχές, και δημιουργήθηκαν με διπλασιασμό εξονίου, σε μεταγενέστερα εξελικτικά στάδια. / The complement system is a key component of the innate immune system and links the innate and adaptive immunity. It consists of more than 35 soluble and membrane proteins that initially are found as inactivated proenzymes and they can be activated by a proteolytic cascade. All three pathways that activate the complement leads to the formation of a Membrane Attack Complex (MAC) that lyses the pathogenic microorganisms. Proteins that participate in the formation of MAC, belongs to the MACPF (MAC – Perforin) family. The FIMAC (Factor I Membrane Attack Complex) module plays significant role in the function of MACPF proteins. The Complement proteins 6 (C6) and 7 (C7) that are components of the MAC, include the FIMAC module in their sequences and it seems that this module is their binding region with C345C of C5b. Moreover, the FIMAC module exists in Complement Factor I (CFI), which is a serine protease (SP) and degrades C4b and C3b molecules. The carboxyl - terminal of FIMAC module binds allosteric with the SP region in CFI and seems to be important for the function of CFI as a serine protease. The model for the analysis of FIMAC module was based in Follistatin Domain (FD) of follistatin (FS) protein, which has been analyzed by crystallography. FIMAC and FD modules seem to have homologous sequences. The FD structure is a hybrid of an amino – terminal EGF (Epidermal Growth Factor) subdomain and of a carboxyl – terminal similar to ovomucoid subdomain, which is called KAZAL and is present in many serine protease inhibitors. The FD module is also present in the Agrin (AGRN) protein. AGRN is an extracellular matrix molecule released by the nerve and is critical for the formation of the neuromuscular junction. The subject of this work was the study of FIMAC and KAZAL modules concerning their function, structure and evolution. There were used data from biological databases and bioinformatic tools for analysis. The nucleotide and amino acid sequences of C6, C7, CFI, AGRN and FS genes from the organisms that were found (vertebrates and invertebrates), and more specific, FIMAC and KAZAL sequences, were the primary material of this study. There are differences in the length of the primary structures of FIMAC ( ̴ 78aa) and KAZAL ( ̴ 55aa) modules, except for some KAZAL modules of AGRN and FS proteins (>80aa). The secondary structures of FIMAC and KAZAL modules seem to be similar as both of them contain α-helix and β-sheet conformations. Simulation models of tertiary structure of both FIMAC and KAZAL modules revealed a common conformation of α-helix and β-sheet in space. The presence of cysteine residues are very conserved and seem to be important in FIMAC (8 – 10 Cys) and KAZAL (4 – 6 Cys) modules, although the concentration of cysteine residues in FIMAC modules are denser in amino – terminal region compared with their corresponding concentration in KAZALs, where they follow an equable distribution. Evolutionary, the genes that participate in all three pathways of complement activation and result in MAC formation, first appeared on chondrichthyes. Moreover, FIMAC and KAZAL modules included in CFI sequence found firstly on agnatha, on chondrichthyes in C6 sequences and on osteichthyes in C7 sequences. The presence of AGRN and FS genes were certified earlier in evolution on cephalochordates and platyelminthes of protostomes. As a result, it seems that the KAZAL modules of AGRN and FS proteins in invertebrates are the ancestors of all FIMAC and KAZAL modules. Nevertheless, new KAZAL modules appeared later during evolution of species. At the genomic level, exons corresponding to the FIMAC and KAZAL modules are highly conserved in different taxa. Intron phases of all exons corresponding to the FIMAC and KAZAL modules in all genes are 1, except for exons of FIMAC modules in first position of C6 and C7 genes (phase 2) that seem to be evolutionary posterior and were emerged by exon duplication, later in evolution.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22696号 / 医博第4640号 / 新制||医||1045(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 羽賀 博典, 教授 妹尾 浩, 教授 木村 剛 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DGAM

In experimental membranous nephropathy, complement C5b-9-induced glomerular epithelial cell (GEC) injury leads to breakdown of glomerular peimselectivity and proteinuria. This study addresses mechanisms that limit complement-mediated injury, focusing on Ras. Complement-mediated injury was attenuated in cultured GEC expressing a constitutively active form of Ras (V12Ras), compared with Neo (control) GEC. V12Ras GEC showed constitutive activation of phosphatidylinositol 3-kinase and extracellular signal-regulated kinase pathways, but inhibition of these pathways did not reverse the protective effect of Ras. V12Ras GEC showed smaller and rounder morphology, decreased F- to G-actin ratio, decreased activity of the Rho GTPase, Rac, and decreased Src activity. In V12Ras GEC, disruption or stabilization of the F-actin cytoskeleton reversed the protective effect of V12Ras on complement-mediated injury. Thus, the protective effect of V12Ras may be dependent on remodeling of the actin cytoskeleton. Furthermore, the reduction of Src activity due to Ras activation may alter the equilibrium in activities of Rho GTPases, a family of proteins known regulate the actin cytoskeleton. Activation of Ras signaling is a novel pathway to consider in developing strategies for cytoprotection in complement-mediated injury.

No description available.

No description available.

A presença de uma inflamação crônica no músculo, a associação com outras doenças e a presença de auto-anticorpos, sugere o envolvimento de um mecanismo autoimune na patogênese da DMJ. Trinta e sete fragmentos musculares de pacientes com o diagnóstico de DMJ foram estudados com o objetivo de avaliar a expressão de MHC classes I e II, C5b-9 e fenotipagem das células inflamatórias CD4, CD8, CD20 e CD68 em tecido muscular e correlacionar com os principais parâmetros clínicos, laboratoriais, histológicos e terapêuticos desta doença. Os achados foram comparados à expressão em oito fragmentos musculares de pacientes com polimiosite (PM), cinco de dermatomiosite (DM) e quatro de distrofia. As expressões de MHC-I, MHC-II e C5b-9 foram identificadas por imunohistoquímica, através da técnica de imunoperoxidase StreptABComplex/HRP; as células CD20 e CD68, pelo sistema LSAB+ e CD4 e CD8, pela técnica EnVision-AP. A expressão de MHC-I apresentou positividade em 97,2% dos casos, enquanto que a expressão de MHC-II foi observada em apenas 21,6% dos casos. C5b-9 (83,8% de positividade), correlacionou-se com a presença de calcinose e envolvimento cardíaco. A presença de linfócitos CD4 (81,1% de positividade), CD8 (86,5% de positividade) e CD20 (62,2 % de positividade), e CD68 (97,2% de positividade) correlacionaram-se com o grau de inflamação observada na histologia muscular. A presença de CD4 e CD68, e marcação de C5b-9 também se correlacionaram com a intensidade de fraqueza muscular, e laboratorialmente, CD4 correlacionou-se com níveis elevados de CK e CD20 com DHL. Na DMJ observou-se maior expressão de C5b-9, CD4 e CD8 e menor expressão de MHC-I e II em comparação à DM e PM. A expressão destes marcadores foi sempre menor na distrofia. A expressão de MHC-I, adjuvante ao envolvimento dos linfócitos CD4 e CD8, sugere um mecanismo inicial celular citotóxico relacionado a maior gravidade do envolvimento muscular. A concomitância da maior expressão de C5b-9 foi um fator preditivo de comprometimento sistêmico e demanda de terapêutica imunossupresssora. Os resultados deste estudo apontam para o papel do MHC-I e II, C5b-9, CD4, CD8, CD20 e CD68 na patogênese da DMJ / The presence of chronic muscle inflammation, in association with other diseases and seric autoantibodies in JDM patients, suggest the involvement of an autoimmune mechanism in the pathogenesis of this inflammatory myopathy. Thirty seven muscle biopsy specimens from patients with JDM were analyzed in order to assess the expression of MHC-I and II, C5b-9, CD4, CD8, CD20 and CD68 and to correlate with the clinical, laboratorial, histological and therapeutical parameters. These findings were compared to the expression in five dermatomyositis (DM), eight polymyositis (PM) and four dystrophy cases. Immunohistochemical reactions for MHC-I and II and C5b-9 (StreptABCcomplex/HRP), CD4, CD8 (EnVision-AP) and CD20, CD68 (LSAB+) were evaluated. MHC-I expression was positive in 97.2% of the cases, whilst MHC-II was positive in only 21.6% of the cases. C5b-9 expression (positivity of 83.8%) correlated with calcinosis and cardiac involvement. The presence of lymphocytes CD4 (positivity of 81.1%), CD8 (positivity of 86.5%), CD20 (positivity of 62.2%), and CD68 (positivity of 97.2%) correlated with inflammation in muscular histology. The presence of CD4 and CD8 and expression of C5b-9 also correlated with the severity of muscle weakness, and CD4 expression correlated with serum levels of CK and CD20 with LDH. In JDM, the expressions of C5b-9, CD4 and CD8 were statistically more significant when compared to PM and DM, while expressions of MHC-I and II were lower in JDM. All expressions were lower in dystrophy. MHC-I expression, adjuvant to the presence of CD4 and CD8 lymphocytes, corroborates the involvement of the cytotoxic cellular mechanism of muscular lesion in JDM, which correlates to severity. Concomitantly, C5b-9 expression was a predictive factor of systemic involvement and of the need for imunossupressive treatment. The results of this study indicate for the function of MHC-I and II, C5b-9, CD4, CD8, CD20 e CD68 at JDM pathogenesis

A presença de uma inflamação crônica no músculo, a associação com outras doenças e a presença de auto-anticorpos, sugere o envolvimento de um mecanismo autoimune na patogênese da DMJ. Trinta e sete fragmentos musculares de pacientes com o diagnóstico de DMJ foram estudados com o objetivo de avaliar a expressão de MHC classes I e II, C5b-9 e fenotipagem das células inflamatórias CD4, CD8, CD20 e CD68 em tecido muscular e correlacionar com os principais parâmetros clínicos, laboratoriais, histológicos e terapêuticos desta doença. Os achados foram comparados à expressão em oito fragmentos musculares de pacientes com polimiosite (PM), cinco de dermatomiosite (DM) e quatro de distrofia. As expressões de MHC-I, MHC-II e C5b-9 foram identificadas por imunohistoquímica, através da técnica de imunoperoxidase StreptABComplex/HRP; as células CD20 e CD68, pelo sistema LSAB+ e CD4 e CD8, pela técnica EnVision-AP. A expressão de MHC-I apresentou positividade em 97,2% dos casos, enquanto que a expressão de MHC-II foi observada em apenas 21,6% dos casos. C5b-9 (83,8% de positividade), correlacionou-se com a presença de calcinose e envolvimento cardíaco. A presença de linfócitos CD4 (81,1% de positividade), CD8 (86,5% de positividade) e CD20 (62,2 % de positividade), e CD68 (97,2% de positividade) correlacionaram-se com o grau de inflamação observada na histologia muscular. A presença de CD4 e CD68, e marcação de C5b-9 também se correlacionaram com a intensidade de fraqueza muscular, e laboratorialmente, CD4 correlacionou-se com níveis elevados de CK e CD20 com DHL. Na DMJ observou-se maior expressão de C5b-9, CD4 e CD8 e menor expressão de MHC-I e II em comparação à DM e PM. A expressão destes marcadores foi sempre menor na distrofia. A expressão de MHC-I, adjuvante ao envolvimento dos linfócitos CD4 e CD8, sugere um mecanismo inicial celular citotóxico relacionado a maior gravidade do envolvimento muscular. A concomitância da maior expressão de C5b-9 foi um fator preditivo de comprometimento sistêmico e demanda de terapêutica imunossupresssora. Os resultados deste estudo apontam para o papel do MHC-I e II, C5b-9, CD4, CD8, CD20 e CD68 na patogênese da DMJ / The presence of chronic muscle inflammation, in association with other diseases and seric autoantibodies in JDM patients, suggest the involvement of an autoimmune mechanism in the pathogenesis of this inflammatory myopathy. Thirty seven muscle biopsy specimens from patients with JDM were analyzed in order to assess the expression of MHC-I and II, C5b-9, CD4, CD8, CD20 and CD68 and to correlate with the clinical, laboratorial, histological and therapeutical parameters. These findings were compared to the expression in five dermatomyositis (DM), eight polymyositis (PM) and four dystrophy cases. Immunohistochemical reactions for MHC-I and II and C5b-9 (StreptABCcomplex/HRP), CD4, CD8 (EnVision-AP) and CD20, CD68 (LSAB+) were evaluated. MHC-I expression was positive in 97.2% of the cases, whilst MHC-II was positive in only 21.6% of the cases. C5b-9 expression (positivity of 83.8%) correlated with calcinosis and cardiac involvement. The presence of lymphocytes CD4 (positivity of 81.1%), CD8 (positivity of 86.5%), CD20 (positivity of 62.2%), and CD68 (positivity of 97.2%) correlated with inflammation in muscular histology. The presence of CD4 and CD8 and expression of C5b-9 also correlated with the severity of muscle weakness, and CD4 expression correlated with serum levels of CK and CD20 with LDH. In JDM, the expressions of C5b-9, CD4 and CD8 were statistically more significant when compared to PM and DM, while expressions of MHC-I and II were lower in JDM. All expressions were lower in dystrophy. MHC-I expression, adjuvant to the presence of CD4 and CD8 lymphocytes, corroborates the involvement of the cytotoxic cellular mechanism of muscular lesion in JDM, which correlates to severity. Concomitantly, C5b-9 expression was a predictive factor of systemic involvement and of the need for imunossupressive treatment. The results of this study indicate for the function of MHC-I and II, C5b-9, CD4, CD8, CD20 e CD68 at JDM pathogenesis