by Hua Dongfan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1991. / Includes bibliographical references. / Acknowledgments --- p.i / Abstract --- p.i i / Table of Contents --- p.iv / List of tables --- p.vii / Chapter Chapter 1. --- Introduction --- p.1 / Chapter 1.1. --- The English Dative --- p.1 / Chapter 1.2. --- The Learnability Problem --- p.3 / Chapter 1.3. --- Aims of the Present Study --- p.8 / Chapter Chapter 2. --- Approaches to the Acquisition of the English Dative --- p.13 / Chapter 2.1. --- Approaches to the Acquisition of the English Dative --- p.13 / Chapter 2.1.1. --- An Item-by-item Approach --- p.14 / Chapter 2.1.2. --- A Semantic and Morphophonological Approach --- p.15 / Chapter 2.1.3. --- A Formal Approach --- p.19 / Chapter 2.2. --- Empirical Studies on the Acquisition of the English Dative --- p.22 / Chapter 2.2.1. --- L1 Acquisition of the English Dative --- p.22 / Chapter (a) --- Mazurkewich and White (1984) --- p.22 / Chapter (b) --- White ( 1987 ) --- p.23 / Chapter (c) --- Gropen et al . (1989) --- p.25 / Chapter 2.2.2. --- L2 Acquisition of the English Dative --- p.27 / Chapter (a) --- Mazurkewich ( 1984 ) --- p.27 / Chapter (b) --- Le Compagnon (1984) --- p.28 / Chapter (c) --- Hawkins ( 1987 ) --- p.31 / Chapter Chapter 3. --- Research Design and Procedure --- p.36 / Chapter 3.1. --- Test Design --- p.37 / Chapter 3.1.1. --- Test 1 --- p.38 / Chapter 3.1.2. --- Hypotheses --- p.44 / Chapter 3.2. --- Test 2 --- p.46 / Chapter 3.3. --- Test 3 and Test 4 --- p.47 / Chapter 3.4. --- Cloze Test --- p.50 / Chapter 3.5. --- Subjects --- p.50 / Chapter 3.6. --- Test Procedure --- p.52 / Chapter Chapter 4 --- . Results --- p.54 / Chapter 4.1. --- Scoring Method --- p.54 / Chapter 4.2. --- Results of Test 1 --- p.55 / Chapter 4.2.1. --- Subjects' Judgments of Prepositional Datives --- p.55 / Chapter 4.2.2. --- Subjects' Judgments of Double-object Datives --- p.56 / Chapter 4.2.2.1. --- Performance of Native Speakers --- p.56 / Chapter 4.2.2.2. --- Performance of Secondary Students --- p.57 / Chapter 4.2.2.3. --- Performance of University Students --- p.59 / Chapter 4.2.2.4. --- The Effect of L1 --- p.62 / Chapter 4.3. --- Results of Test 2 --- p.63 / Chapter 4.4. --- Results of Test 3 and Test 4 --- p.64 / Chapter 4.5. --- Summary --- p.67 / Chapter Chapter 5. --- Discussion --- p.71 / Chapter 5.1. --- The Semantic Constraint on the English Dative --- p.71 / Chapter 5.2. --- Overgeneralization and the Learnability Problem --- p.76 / Chapter 5.3. --- L1 Influence on Dative Acquisition by Chinese ESL Learners --- p.81 / Chapter Chapter 6. --- Conclusions --- p.85 / References --- p.89 / Chapter Appendix I. --- Verb Check List --- p.93 / Chapter Appendix IIa. --- Instruction for Test 1 --- p.94 / Chapter Appendix IIb. --- Test Sentences for Test 1 --- p.96 / Chapter Appendix IIIa. --- Instruction for Test 2 --- p.103 / Chapter Appendix IIIb. --- Test Sentences for Test 2 --- p.104 / Chapter Appendix IVa. --- Instruction for Test 3 --- p.107 / Chapter Appendix IVb. --- Test Sentences for Test 3 --- p.108 / Chapter Appendix Va. --- Instruction for Test 4 --- p.110 / Chapter Appendix Vb. --- Test Sentences for Test 4 --- p.111 / Chapter Appendix VI. --- Cloze Test --- p.114

L'objectif est de déterminer si le sens attribué à la préposition provient d’un sémantisme initial, stable en langue mais susceptible de diversement se décliner en discours, ou s’il est constitué à partir de son environnement, par conséquent à partir des combinaisons opérées dans le discours lui-même. La réflexion est, dans la présente étude, centrée sur la relation de la préposition avec le verbe qui la sélectionne. Néanmoins, d'autres éléments environnants doivent être pris en considération dans la mesure où ils interviennent dans l’interprétation du sens. L'hypothèse est que la préposition possède une identité fondamentale qui fait qu'elle est choisie par le verbe en l'une des acceptions de ce dernier, afin de véhiculer un certain sens, et que, réciproquement, l'ensemble ainsi formé détermine la distribution du complément syntaxique de la préposition. Cette hypothèse repose sur l'adoption du principe, dans le point de vue retenu sur le fonctionnement de la langue, selon lequel la forme et le sens varient concomitamment. Autrement dit, une similitude de forme (par exemple la présence de la préposition à) est supposée intrinsèquement liée à une similitude de sens (il y a donc à rechercher un point commun sémantique à tous les verbes qui se construisent avec la préposition à) et, réciproquement, une différence de forme (par exemple la présence de la préposition à opposée à son absence) est supposée intrinsèquement liée à une différence de sens. L'adoption de ce principe heuristique impose, par le lien étroit que notre hypothèse institue entre le verbe et la préposition d’un côté, et entre le verbe et le syntagme prépositionnel de l’autre, d’envisager ces différents types de relations aussi bien dans des configurations formelles (syntaxiques) que dans des configurations sémantiques. Sur le plan formel, nous étions amenée à distinguer les différents types de groupes prépositionnels en fonction de leur lien plus ou moins proche avec le verbe. Notre hypothèse est que l’on peut rendre compte de la relation du verbe avec le syntagme prépositionnel qu’il régit au moyen d’un classement scalaire L’approche de la préposition à introduisant un groupe prépositionnel impose de séparer le complément de verbes des autres types de groupes prépositionnels(GP) dont les dénominations sont variés et contestables puisque les critères de définition fixés par la grammaire dite traditionnelle comme la suppression et le déplacement caractérisant habituellement le complément circonstanciel et la pronominalisation typique du complément sont insuffisamment distinctifs. Comme on trouve des compléments supprimables par exemple,elle chante (une chanson), dans l’emploi absolu du verbe, on trouve aussi des circonstants pronominalisables comme (il mange dans la cuisine, il y mange). Pour cela, le chapitre II présente deux contributions renouvelant ce que nous avons précédemment appelé « les théories classiques » : l'article de Lavieu (2006), s'affronte précisément aux difficultés que nous avons soulevées, et propose une reconfiguration terminologique et taxinomique distinguant entre « constituants » (où se rangent les « compléments » et les « ajouts ») et « incidents »Pour résoudre le problème de la distinction entre les différents types syntaxiques régis par à, nous avons opté pour un classement scalaire (idée inspirée de la théorie de l’actance de Lazard) qui détermine le degré de relation des verbes avec les différents actants régis par cette préposition. Ce classement a été fait au regard des mêmes tests syntaxiques sur un corpus illustrant la complémentation en à d’abord dans une structure simple et ensuite dans une structure double.Ainsi, nous avons tenté de caractériser formellement les GP en à régis par le verbe dans les différentes structures dans lesquelles ils figurent. Notre classement scalaire a montré qu’il y a une graduation dans le rapport du verbe avec le complément allant du degré le plus fort au degré le plus faible... / The objective of this thesis is to determine whether the meaning attributed to the preposition is primarily semantic or not. The aim is then to prove that though it is grammatically fixed, it can vary since it is shaped by its context, thereby assembled by multiple discourse combinations. In the present research, the focus is then centred on the study of the relationship between the preposition and the verb it determines. Accordingly, several other related elements will be studied as they intervene in the act of interpreting meaning itself. The thesis of this Ph.D dissertation suggests that the preposition has a given core identity which is determined by the verb itself and which by implication determines the meaning and the nature of the syntactic complement as well.This thesis contends then that form and meaning do vary considerably but still simultaneously. In other words, what determines form determines by implication meaning as well. The present thesis seeks then to study the different aspects and mechanics of the relationship between the verb and its preposition.In the present thesis, the study of the preposition requires all verb complements to remain separate from the different types of prepositional groups. The second chapter attempts then to re-examine the contentions of classical theories, particularly with reference to Laview’s article (2006). In order to effectively study the multiple differences and distinctions between the different syntactic classes, a scalar approach is adopted. The findings demonstrate in fact that the relationship between the verb and its complement is constantly varying. Actually, the fifth chapter of the second part includes the corpus needed to attest the research questions.The semantic research part of the present thesis, seeks in fact to further elaborate the principle that relates meaning to form. In order to demonstrate that, the approach has been based on the characteristics of the syntactic complements which are actually the main target of the analysis in the first part of the thesis.Conducting then a comparative analysis of different types of verbs which are all related by their respective preposition complement “à”, the following findings are deduced:- The nature of the verb is determined by the type of the preposition- The preposition introduces its complement meanwhile identifying the nature of the verb itself- Regardless of the nature or the type of the verb, what the preposition adds to meaning remains the sameThe main objective of this research work is then to offer new and pertinent assumptions concerning the role and function of the preposition “à”, meanwhile still taking into consideration former critical assumption advocated by Grévisse, Gougenheim, Guillaume, Pottier and Cadiot°. Since the main focus of this research work is to study the semantic identity of the preposition “à”, the approach has been based on an attempt to validate or refute all former semantic assumptions presented by former writers, exploring then the different aspects of the relationship between the verb and the preposition based of the corpus of LVF. The analysis of the different examples studied required also that a nominalisation test and a telicity test should be conducted. The following are the main findings:- There is no definite syntactic difference between the verb and the nominalised structure- The empirical study of LVF which seeks to distinguish the different dative cases reveals that the idea of interchangeability is not at the very basis of the meaning of the verb itself- The lexical dative does not automatically entail deletion of the GP since the idea of interchangeability is primarily expressed by the meaning of the verb itself- The preposition “à” designates both the origin and the recipient as well. It is a in fact linear retrospective movement which allows the process of interchangeability

Critical illness is an aetiologically and clinically heterogeneous syndrome that is characterised by organ failure and immune dysfunction. Mortality in critically ill patients is driven by inflammation-associated organ damage and a profound vulnerability to nosocomial infection. Both factors are influenced by the complement protein C5a, released by unbridled activation of the complement system during critical illness. C5a suppresses antimicrobial functions of key immune cells, in particular the neutrophil, and this suppression has been shown to be associated with poorer outcomes amongst critically ill adults. The intracellular signalling pathways which mediate C5a-induced neutrophil dysfunction are incompletely understood, and scalable tools with which to assess immune cell dysfunction in patients are lacking. This thesis aimed to develop tools with which to assess neutrophil function and delineate intracellular signalling pathways driving C5a-induced impairment. Neutrophils were isolated from healthy volunteer blood and functions (priming, phagocytosis and reactive oxygen species production) were assessed using light microscopy, confocal microscopy and flow cytometry. A new assay was developed using an Attune Nxt™ acoustic focusing cytometer (Life Technologies) which allowed the rapid assessment of multiple neutrophil functions in small samples of unlysed, minimally-manipulated human whole blood. Complete proteomes and phosphoproteomes of phagocytosing neutrophils were obtained from four healthy donors pre-treated with C5a or vehicle control. Several key insights were gained from this work and are summarised here. Firstly, C5a was found to induce a prolonged (greater than seven hours) impairment of neutrophil phagocytosis. This defect was found to be preventable by previous or concurrent phagocytosis, indicating common signalling mechanisms. Secondly, a novel assay was developed which allows the rapid assessment of multiple neutrophil functions in less than 2 mL of whole blood, and this assay can feasibly be applied in clinical settings. Thirdly, cell-surface expression of the C5a receptor was found to be markedly decreased during phagocytosis, and this decrease was not mediated by protease activity. Finally, unbiased proteomics quantified 4859 proteins and 2712 phosphoproteins respectively. This quantification is the deepest profile of the human neutrophil proteome published to date, and has revealed novel insights into the mechanisms of C5a-induced neutrophil dysfunction and phagocytosis.

Lipossomas (LUV) são estruturas compostas por uma bicamada lipídica que se organizam de forma semelhante a vesículas, contendo um compartimento aquoso em seu interior. Têm sido avaliados como potenciais carreadores de fármacos. No entanto, após sua administração, in vivo, opsoninas do soro adsorvem-se em sua superfície contribuindo para que o sistema fagocitário mononuclear (SFM) reconheça essas partículas, favorecendo sua remoção da circulação. O sistema complemento (SC) parece ter papel importante neste processo, principalmente por gerar fragmentos ativos do componente C3 (C3b/iC3b) que se depositam nas vesículas lipossomais e são reconhecidos por receptores do complemento presentes, por exemplo, nos polimorfonucleares. Antioxidantes, como a quercetina, têm demonstrado importantes e benéficos efeitos sobre a saúde humana, porém sua baixa solubilidade em água e biodisponibilidade limitam seu uso. Assim, o desenvolvimento apropriado de carreadores de flavonoides seria de grande importância para sua aplicabilidade in vivo. O objetivo do presente trabalho é avaliar a ativação das proteínas do SC por lipossomas compostos de fosfatidilcolina de soja e colesterol (PC:CHOL) ou colesteril-etil-éter (PC:CHOL-OET), contendo ou não quercetina. O consumo das vias clássica (VC) e alternativa (VA) provocado pelas diferentes vesículas foi analisado por ensaio hemolítico e a quantificação de iC3b e anticorpos naturais (IgG e IgM) na superfície dessas partículas foi realizada através de kits de ELISA. A ativação de C3 por vesículas contendo ou não quercetina foi avaliada por imunoeletroforese bidimensional (IEF). Os resultados mostram que lipossomas vazios, compostos por grande quantidade de colesterol, consomem mais os componentes do complemento para ambas as vias, VC e VA. Ainda, a substituição de colesterol por colesteril-etil éter reduziu o consumo das duas vias, mas a ativação do SC ainda é dependente da quantidade deste composto. A incorporação de quercetina não alterou o consumo de ambas as vias. O depósito de iC3b, IgG ou IgM nas vesículas compostas de PC:CHOL-OET na proporção de massa 3:1 foi o menor comparado aos demais. A IEF mostrou que vesículas PC:CHOL vazias induzem maior clivagem de C3 em relação às vesículas PC:CHOL-OET. Ainda, a incorporação de quercetina reduz a conversão de C3 em seus fragmentos. Essas observações sugerem que a preparação lipossomal PC:CHOL-OET em proporção de massa 3:1 parece ser a mais adequada para dar continuidade aos estudos que deverão culminar na tentativa de utilizá-la como carreadora de quercetina para administração in vivo / Liposomes (LUV) are structures composed by lipid bilayer that are organized similarly to vesicles, containing an aqueous compartment inside. They have been evaluated as potential drug carriers, however, after in vivo administration, serum opsonins are adsorb on the surface, contributing to their clearance from the circulation by mononuclear phagocytes system (MPS). The complement system (CS) seems to play an important role in this process, mainly to generate active fragments of the C3 component (C3b/iC3b) that are deposited in the liposomal vesicles and are recognized by complement receptors present, for example, in polymorphonuclear cells. Antioxidants such as quercetin have demonstrated significant and beneficial effects on human health, but its low water solubility and bioavailability limit their use. Thus, the proper development of flavonoids carriers would be of great importance to its applicability in vivo. The objective of this study is to evaluate the activation of SC proteins by liposomes composed of soy phosphatidylcholine and cholesterol (PC: CHOL) or cholesteryl ethyl ether (PC: CHOL-OET), with or without quercetin. The consumption of the classical (CP) and alternative pathway (AP) caused by the different vesicles was analyzed by hemolytic assay and quantification of iC3b and natural antibodies (IgG and IgM) on the surface of these particles was performed using ELISA kits. The activation of C3 by vesicles with or without quercetin was assessed by two-dimensional immunoelectrophoresis (IEF). The results show that empty liposomes, composed of large amounts of cholesterol, consume more CS components in both pathways, CP and AP. Moreover the replacement of cholesterol by cholesteryl ethyl ether reduced the consumption of both pathways, but the activation of the SC is still dependent on the amount of the compound. The incorporation of quercetin did not alter the consumption of both pathways. The deposition of iC3b, IgG or IgM in vesicles composed of PC: CHOL-OET at mass ratio of 3:1 was the lowest compared to the others. The IEF showed that empty vesicles of PC:CHOL induce less cleavage of C3 in relation to vesicles of PC: CHOL-OET. In addition, the incorporation of quercetin reduces the conversion of C3 into its fragments. These observation suggest that the liposomes PC:CHOL at mass ratio 3:1 seems to be the most appropriate to continue the studies that could culminate in an attempt to use it as a carrier to administrate quercetin in vivo

A leptospirose é causada por bactérias que pertencem ao gênero Leptospira. Em um estudo realizado por nosso grupo, observou-se que as proteases secretadas por leptospiras patogênicas foram capazes de clivar a molécula C3 do Complemento e seus fragmentos C3b e iC3b, além de Fator B, C4b e C2. Neste trabalho expandimos a análise da atividade proteolítica sobre os componentes do Complexo de Ataque à Membrana (MAC): C6, C7, C8 e C9. Nós observamos que essas proteases clivam todos os componentes do MAC inclusive o complexo solúvel formado e que essas clivagens ocorrem de modo tempo-dependente. Além disso, as clivagens dessas moléculas ocorrem de modo seletivo, pois mesmo utilizando quantidades reduzidas de sobrenadantes ainda foi possível observar produtos de clivagem. A atividade proteolítica foi inibida pela 1,10fenantrolina, indicando a participação de metaloproteases. O reconhecimento de quais moléculas do MAC são clivadas por proteases de leptospiras patogênicas pode contribuir para o desenvolvimento de estratégias terapêuticas na infecção por estes patógenos. / Leptospirosis is a zoonosis caused by spirochetes from the genus Leptospira. In a previous study, our group observed that the proteases secreted by Pathogenic Leptospira were capable of cleaving C3 of Complement, as well as the fragments C3b and iC3b, Factor B (Alternative Pathway), C4 and C2 (Classical and Lectin Pathways). In this work, we analyze the activity of the leptospiral proteases on the components of Membrane Attack Complex (MAC). We observed that the protease cleaves all MAC components including soluble complex formed and that these cleavages occur in a time-dependent manner and in a selective way, since even when reduced quantities of supernatants were used, the cleavage products were still observed. The proteolytic activity was inhibited by 1,10phenanthroline, indicating the participation of metalloproteinases. The recognition that MAC molecules are cleaved by proteases of pathogenic leptospires can contribute to the development of therapeutic strategies for the infection by these pathogens.

A leptospirose é uma zoonose causada por leptospiras patogênicas que possuem estratégias para driblar a ação do sistema complemento ao interagir com diversos reguladores negativos, como a vitronectina. Neste projeto, avaliou-se a interação de vitronectina com três proteínas de membrana de Leptospira: LigA, LigB e LcpA. Verificou-se que essas interações ocorrem nas porções C-terminais das proteínas e nos domínios de ligação com heparina da vitronectina. Essas proteínas também se ligam a C9 e são capazes de impedir a formação do poro in vitro e a formação do MAC em um ensaio clássico de hemólise. Essas proteínas de Leptospira interagem com diversos reguladores negativos do sistema complemento. Ensaios de competição demonstram que estes reguladores interagem simultaneamente com as proteínas através de sítios distintos. A caracterização funcional de proteínas e a descoberta de novos mecanismos utilizados por esse patógeno para sobreviver no hospedeiro podem auxiliar nossa compreensão no que diz respeito a aspectos relacionados à clínica e à prevenção da leptospirose. / Leptospirosis is a zoonotic disease caused by pathogenic Leptospira that have strategies to escape the action of the complement system interacting with several negative regulators, such as vitronectin. In this project, we evaluated the interaction of vitronectin with three Leptospira membrane proteins: LigA, LigB and LcpA. It has been found that such interactions occur at the C-terminal portions of these proteins and the heparin binding domain of vitronectin. These proteins also bind to C9 and are capable of preventing the formation of the pore in vitro and the formation of MAC in a classical test of hemolysis. These proteins interact with various Leptospira negative regulators of the complement system. Competition assays demonstrated that both interact with these regulatory proteins through distinct sites. The functional characterization of these proteins and the discovery of new mechanisms used by this pathogen to survive in the host may help our understanding with regard to clinical aspects and prevention of leptospirosis.

Ng, Tsz Kin. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 151-185). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Introdução: Empiema pleural em geral decorre de complicação de pneumonias e, se não identificado e tratado precocemente, pode ocasionar aumento morbidade ou mesmo mortalidade. A identificação de marcadores no líquido pleural de efusões parapneumônicas que mostrem a presença ou a evolução precoce para empiema tem significância clínica. Neste cenário, dosagens das concentrações de frações ativadas do complemento no líquido pleural podem ajudar no diagnóstico precoce do empiema. Objetivos: Comparar as concentrações de frações ativadas do complemento (C3a, C5a e C5b9) em efusões pleurais induzidas em ratos por inoculação intrapleural de bactérias ou por irritante químico estéril (terebentina). Métodos: Trinta e nove ratos Wistar machos, peso médio de 414g (290 a 546g), realizaram anestesia geral com isofluorano inalatório por máscara, e toracocentese no 4º espaço intercostal com abocath conectado a oscilômetro de pressão para confirmar posição intrapleural. Os animais foram divididos em 3 grupos: SA (n=17) - inoculação de Staphylococcus aureus; SP (n=12) - inoculação de Streptococcus pneumoniae; C (n=10) – inoculação de terebintina (efusão pleural estéril, controle). Doze horas após a inoculação intrapleural foi coletado liquido pleural por toracocentese, sob controle ecográfico, e realizadas dosagens de C3a, C5a e C5b9 pelo método ELISA. Resultados: A dosagem de C3a foi de 1066,82 μg/ml (937,29 – 1196,35 μg/ml) no grupo SA, 1188,28 μg/ml (1095,65 – 1280,92 μg/ml) no SP, e de 679,13 μg/ml (601,29 – 756,98 μg/ml) no C (p<0,001). A dosagem de C5a foi de 55.727 ng/ml (41,22 – 70,23 ng/ml) no grupo SA, 520.107 ng/ml (278,92-761,3 ng/ml) no SP, e de 5.268 ng/ml (1,68 – 8,85 ng/ml) no C (p<0,001). A dosagem de C5b9 foi de 15,02 ng/ml (13,1 – 16,94 ng/ml) no SA, de 16,63 ng/ml (14,37 – 18,9 ng/ml) no SP, e de 14,05 ng/ml (9,8 – 18,29 ng/ml) no C (p=0,692). A avaliação das curvas ROC demonstrou área sob a curva de 0,987 (IC95% 0,953-1) para o C3a; 1 para C5a (1-1) e 0,757 (0,523-0,990). Conclusões: As frações ativadas dos complementos C3a e C5a foram significativamente maiores nos empiemas induzidos experimentalmente por inoculação intrapleural de Staphylococcus aureus e Streptococcus pneumoniae do que com aquelas observadas após inoculação intrapleural de terebentina. A dosagem elevada destas frações ativadas do complemento foi útil para o diagnóstico do empiema pleural induzido em ratos. / Background Pleural empyema is a well-known complication of pneumonia. If treatment is delayed, empyema may increase morbidity and mortality in affected patients. Therefore, the identification of empyema biomarkers in parapneumonic pleural effusion is desirable. Previous research has suggested complement activation products as candidate empyema markers. Objective To compare the levels of complement activation products C3a, C5a, and C5b9 in pleural effusion induced by Staphylococcus aureus (SA), Streptococcus pneumoniae (SP), or turpentine (control). Method Thirty-nine male Wistar rats (mean weight 414g; 290-546g) were allocated as follows: 17 animals in the SA group, 12 in the SP group, and 10 in the control group. Bacteria or turpentine were injected into the pleural space. After 12h, intrapleural fluid was collected using ultrasound-guided thoracentesis. Levels of complement activation products were determined using ELISA kits. Results Two SA and 1 SP animals died before 12h. Mean levels were as follows: C3a: 1066.82 μg/mL (937.29-1196.35 μg/mL) in SA, 1188.28 μg/mL (1095.65-1280.92 μg/mL) in SP, and 679.13 μg/mL (601.29-756.98 μg/mL) in controls (p<0.001); C5a: 55.727 ng/mL (41.22-70.23 ng/mL) in SA, 520.107 ng/mL (278.92-761.3 ng/mL) in SP, and 5.268 ng/mL (1.68-8.85 ng/mL) in controls (p<0.001); C5b9: 15.02 ng/mL (13.1-16.94 ng/mL) in SA, 16.63 ng/mL (14.37-18.9 ng/mL) in SP, and 14.05 ng/mL (9.8-18.29 ng/mL) in controls (p=0.692). ROC analysis revealed an area under the curve of 0.987 (95%CI: 0.953-1) for C3a; 1 (1-1) for C5a; and 0.757 for C5b9 (0.523-0.990). Conclusions In the present rat model, complement activation fragments C3a and C5a accurately detected infected pleural effusion.

Embora camundongos sejam resistentes à infecção por Leptospira interrogans, eles têm sido pouco utilizados para se entender os mecanismos imunes efetores contra esta bactéria. Adler & Faine mostraram em 1976 que esta resistência é dependente da resposta imune, uma vez que camundongos imunossuprimidos tornavam-se suscetíveis à L. interrogans. Outros autores mostraram que camundongos portadores de imunodeficiência grave combinada também morriam após inoculação de L. interrogans. Sabendo da importância do Sistema Complemento em infecções bacterianas, investigamos se animais C5 deficientes (C5-) são mais suscetíveis à infecção por L. interrogans que animais C5 normais (C5+). Observamos que camundongos C5- possuem menores porcentagens de linfócitos T CD8+ na circulação periférica e maiores níveis de IL-12p40 no rim e de TNF e IL-6 no pulmão que os animais C5+. Animais C57Bl/6 (B6) C5+ possuem maior porcentagem de linfócitos T CD4+ que B6 C5-, além de lesões hepáticas mais intensas, mostrando um efeito dependente de C5 e do fundo genético dos camundongos. / Although mice are resistant to Leptospira interrogans infection, they are not usual models to study the imune response against this bacteria. Adler and Faine demonstrated in 1976 the importance of the imune response, since immunosuppressed mice were suceptible to L. interrogans. Other authors showed that mice that had severe combined immunodeficiency also died when inoculated with L. interrogans. Due to the activity of the Complement System in infections, we analyzed whether C5 deficient (C5-) mice are more susceptible than C5 sufficient (C5+) mice to L. interrogans infection. C5- mice have lower percentages of T CD8+ lymphocytes in peripheral circulation and upper levels of IL-12p40 in the kidney and TNF and IL-6 in the lungs than C5+ mice. C57Bl/6 (B6) C5+ mice has higher percentages of T CD4+ lymphocytes than B6 C5- mice, in addition to stricter liver injures, exhibiting an effect dependent of C5 and of the genetic background.

A leptospirose é uma zoonose causada por leptospiras patogênicas. Para estabelecer a infecção, estas bactérias desenvolveram estratégias de escape ao sistema complemento. Neste trabalho demonstramos que o sobrenadante de cultura de leptospiras patogênicas é capaz de inibir as três vias do complemento. Observamos que esse sobrenadante possui atividade proteolítica sobre C3, C3b e iC3b, além do FB (via alternativa), C2 e C4b (via clássica e das lectinas). As proteínas C3, C4, C2 e FB também foram clivadas quando soro humano normal (SHN) foi utilizado como fonte de complemento. Demonstramos que as proteases atuam em conjunto com os reguladores do hospedeiro Fator I e Fator H na clivagem de C3b. As clivagens foram inibidas pela 1,10-fenantrolina, sugerindo a participação de metaloproteases. Metaloproteases de leptospira da família das termolisinas foram produzidas como proteínas recombinantes e clivaram C3 no SHN. Concluímos que proteases de leptospiras patogênicas podem desativar moléculas do complemento e são potencias alvos para novas terapias em leptospirose. / Leptospirosis is a zoonotic disease caused by pathogenic Leptospira. To establish the infection, these bacteria have developed strategies to escape the complement system. In this work, we demonstrate that culture supernatant from pathogenic Leptospira is capable of inhibiting the three complement pathways. We observe that this supernatant possess proteolytic activity under C3, C3b and iC3b, FB (alternative pathway), C2 and C4b (classical and lectin pathways). The proteins C3, C4, C2 and FB were also cleaved when normal human serum (NHS) was used as a source of complement. We demonstrate that these proteases act together with the host regulators Factor I and Factor H in C3b cleavage. The cleavages were inhibited by 1,10-phenanthroline, suggesting the involvement of metalloproteinases. Leptospira metalloproteinases from the thermolysin family were produced as recombinant proteins and cleaved C3 in NHS. We concluded that proteases from pathogenic Leptospira can inactivate complement molecules and are potential targets for new therapies in leptospirosis.