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.

C3 glomerulopathy (C3G) encompasses a group of severe renal diseases characterized by “dominant C3” deposition in the renal glomerulus. Patients typically present as nephritic nephrotics, with hematuria, hypertension, heavy proteinuria and edema. Within ten years of diagnosis, 50% of affected patients progress to end-stage renal disease and require dialysis or renal transplantation. No treatment is available to halt disease progression and thus both disease recurrence and allograft loss are common after transplantation. Genetic studies of C3G have firmly implicated dysregulation of the alternative pathway (AP) of complement in disease pathogenesis. In addition to genetic factors, acquired factors like autoantibodies can also exaggerate AP activity in the circulation to cause C3G. Although AP dysregulation in the circulation (i.e. fluid-phase dysregulation) has been well studied in these patients, AP activity in the glomerular microenvironment is not well understood. In this body of work, we used MaxGel, an ex-vivo surrogate for the glomerular extracellular matrix, to study AP activity and regulation. We showed that C3 convertase can be assembled on MaxGel and elucidated the dynamics of its formation and decay in the presence of complement regulators. We confirm that on MaxGel factor H (fH) inhibits C3 convertase formation and accelerates its decay, while properdin has a stabilizing effect. We also show that the complement factor H-related proteins (FHRs) are vital to the regulation of AP activity. Consistent with our MaxGel data, CFHR gene-fusion events have been reported as genetic drivers of disease in a few familial cases of C3G. One such familial case in which we identified and characterized the rearrangement event results from a novel CFHR5-CFHR2 fusion gene. The fusion gene is translated into a circulating FHR-5/-2 protein that consists of the first two SCRs of FHR-5 followed by all four SCRs of FHR-2. The structural repetition of SCR1-2 followed by another SCR1-2 motif facilitates the formation of complex FHR-1, FHR-2 and FHR-5 multimers, which have enhanced affinity for C3b and by out-competing fH, lead to impaired C3 convertase regulation in the glomerular microenvironment. Finally, we tested gene therapy as a tool to rescue the disease phenotype and restore fluid-phase AP complement control in a mouse model of C3G (Cfh-/-/huCR1-Tg mice). Using the piggyBac transposon system, we introduced a construct derived from complement regulator 1 (CR1) into Cfh-/-/huCR1-Tg mice. Delivery of sCR1-AC via hydrodynamic tail vein injection provided constitutive circulatory expression of sCR1-AC, and in animals followed for 6 months, we found that long-term expression of this complement regulator rescued the renal phenotype. These results suggest that sCR1 may be a potential therapy for patients with this disease.

<p>Both Multiple Sclerosis (MS) and Guillan-Barré syndrome (GBS) are neurological inflammatory demyelinating autoimmune diseases, with a probable antibody contribution. Complement proteins in both MS and GBS does play a role in inflammation and demyelination at pathogenesis, according to earlier scientific evidence. The aim of this examination project work was to investigate systemic and intrathecal complement activation in MS and GBS, to gain further knowledge that might be useful for development of future therapeutics targeting immune responses during those diseases. An additional aim was to develop a new ELISA method for detection of complement iC3.</p><p>By using sandwich ELISA, complement proteins C1q, C4, C3, fH and C3a were measured in plasma and cerebrospinal fluid (CSF) from persons within 4 different diagnostic groups; MS, other neurological diseases (OND), GBS and controls (C). An ELISA method to detect iC3 (hydrolysed C3) was also developed, including usage of SDS-PAGE. Results based on raw data and statistical analysis show significantly elevated levels of C3a (C3a/C3) in MS and decreased C3 in plasma. In CSF low levels of C4 and C3a/C3 in MS were detected, though correlation of C3a and C1q was positive. GBS reveal high levels of all complement proteins analysed in CSF except for C3, and a positive correlation of C3a and C1q as well as C3a and fH was found.</p><p>These results indicate that MS patients have systemic complement activation; however the activation pathway is not determined. Complement activation in MS may also occur intrathecally, with correlation analysis indicating a possible activation via the classical pathway. MS patients suffering from a more acute relapsing-remitting (RR) MS have a more prominent systemic complement activation compared to MS patients responding to beta-interferon treatment. Systemic increased C3a/C3 ratio may be a possible biomarker to distinguish more acute RR MS in an earlier step of MS pathogenesis and should be further investigated. GBS patients have an intrathecal complement activation that seems to occur via the classical pathway.</p>

Akademisk avhandling--Lund. / Extra t.p., with thesis statement, inserted. Errata slip inserted. Bibliography: p. [117]-125.

The complement fragments C3a and C5a were purified from zymosan-activated human serum by column chromatographic procedures after the bulk of the proteins had been removed by acidic polyethylene glycol precipitation. In the isolated in situ perfused rat liver C3a increased glucose and lactate output and reduced flow. Its effects were enhanced in the presence of the carboxypeptidase inhibitor DL-mercaptomethyl-3-guanidinoethylthio-propanoic acid (MERGETPA) and abolished by preincubation of the anaphylatoxin with carboxypeptidase B or with Fab fragments of an anti-C3a monoclonal antibody. The C3a effects were partially inhibited by the thromboxane antagonist BM13505. C5a had no effect. It is concluded that locally but not systemically produced C3a may play an important role in the regulation of local metabolism and hemodynamics during inflammatory processes in the liver.

Biologically inspired materials are being developed with the aim of improving the integration of medical implants and minimizing non-desirable host reactions. A promising strategy is the design of topographically patterned surfaces that resemble those found in the extracellular environment. Nanoporous alumina has been recognized as a potential biomaterial and as an important template for the fabrication of nanostructures. In this thesis in vitro studies were done to elucidate the role of alumina nanoporosity on the inflammatory response. Specifically, by comparing alumina membranes with two pore sizes (20 and 200 nm in diameter). Complement and platelet activation were evaluated as well as monocyte/macrophage behaviour. Whole blood was incubated with the alumina membranes and thereafter the biomaterial surfaces were evaluated in terms of protein and platelet adhesion as well as procoagulant properties. The fluid phase was analyzed for complement activation products and platelet activation markers. Besides, human mononuclear cells were cultured on the alumina membranes and cell adhesion, viability, morphology and release of pro-inflammatory cytokines were evaluated. The results indicated that nanoporous alumina with 200 nm pores promotes higher complement activation than alumina with 20 nm pores. In addition, platelet response to nanoporous alumina was found to be highly dependent on the material porosity, as reflected by differences in adhesion, PMP generation and procoagulant characteristics. A clear difference in monocyte/macrophage adhesion and activation was found between the two pore size alumina membranes. Few but highly activated cells adhered to the 200 nm membrane in contrast to many but less activated monocytes/macrophages on the 20 nm surface. The outcome of this work emphasizes that nanotopography plays an important role in the host response to biomaterials. Better understanding of molecular interactions on nano-level will undoubtedly play a significant role in biomaterial implant development and will contribute to design strategies for controlling specific biological events.

Today there is an increasing clinical demand and expectation of patients for biomaterials, which underscores the importance of discovering the correlations between biomaterials and biological systems, especially blood. When an artificial material makes contact with blood, the first event is a rapid adsorption of plasma protein on the material surface, on top of which the innate immune system is triggered, with potentially detrimental consequences. The work presented in this thesis, reported in four papers, was designed to investigate complications associated with (a) biomaterial-induced immune systems, including activation mechanisms and crosstalk between cascades on the biomaterial surface, and with (b) clinical investigations. In Paper I and Paper II, a series of studies led to the development of a direct prediction of the subsequent biological events based on the pattern of initially bound proteins. A reciprocal relationship was demonstrated between activation of the contact system and the complement system when they were induced on artificial material surfaces. Based on these studies, a robust and simple method for biocompatibility testing was proposed and validated, yielding high specificity and sensitivity when compared to today’s gold standard. Paper III investigated biomaterial-induced activation of complement and leukocytes in dialysis treatment-related conditions. The results suggested that citrate is more biocompatible than the conventionally used acetate. This reduction in activation could be further enhanced with higher citrate concentrations, suggesting that dialysis fluid containing citrate is a promising alternative to acetate dialysis fluid. Paper IV investigated complement initiation mechanisms with clinical implications. An experimental system was set up to revisit the initiation of the complement alternative pathway, and correlations were found between chaotropic or nucleophilic agents and iC3 generation under physiologically relevant conditions. A clinical study of hepatic encephalopathy patients indicated a direct correlation between elevated plasma ammonia and iC3 formation, as well as with complement activation in vivo.  Taken together, these studies have provided a model for a robust biomaterial test and have investigated biomaterial-induced complications in the fluid phase in clinically related conditions; furthermore, the basic mechanisms of complement activation have been dissected in relation to disease symptoms. Keywords: Complement system, contact system, blood, biomaterials, biocompatibility, in vitro screening, iC3, dialysis

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.

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.

O sistema complemento participa da patogenia da Doença Hepática Alcoólica (DHA), onde C3 contribui para o acúmulo de triglicerídeos (tg) e C5 para injúria e inflamação hepática. Investigamos o papel de C5 em modelo de DHA aplicando as linhagens B6 e A/J, e geramos a linhagem congênica B6.A-Hc0 (B6 C5def). B6 e A/J foram tratados com dieta contendo ou não etanol, ou maltodextrina, por 6, 8 e 10 semanas. Em ambas as linhagens a dieta com etanol induziu hepatomegalia, acúmulo de tg e redução de IL6 e IL12 hepáticos ao longo das semanas. Os A/J tratados com etanol exibiram aumento de leucócitos circulantes, IL10 e NO hepáticos e menor acúmulo de tg hepáticos em relação aos B6. Os B6 tratados com etanol exibiram redução de IL1b, IL10 e NO hepáticos. Tratando a linhagem congênica por 10 semanas com a dieta com etanol observamos aumento de IL17 e IL10 e redução de IL1b e TGFb hepáticos nos B6.A-Hc0 em relação aos B6. Independentemente da dieta, houve aumento sérico de AST, FA, albumina, colesterol, tg e redução hepática de IL6, IL12 e IFNg nos B6.A-Hc0. Portanto, C5 promoveu um ambiente hepático pró-inflamatório e pareceu influenciar os valores séricos das enzimas de função e síntese hepática, citocinas e do perfil lipídico no modelo de DHA. / The complement system may be involved in the pathogenesis of Alcoholic Fatty Liver Disease (ALD). Murine models of ALD showed that C3 contributes to the accumulation of triglycerides (tg) in liver and the C5 seems to be involved with hepatic inflammation. We here investigated the contribution of C5 in ALD using C57Bl/6 (B6) and A/J (C5 deficient), and B6 C5 deficient congenic mice (B6.A-Hc0). B6 and A/J were treated with modified diet containing ethanol or maltodextrin for 6-10 weeks. In both strains, the ethanol diet induced hepatomegaly, increased liver tg and decreased IL6 and IL12 levels. However, total blood leukocytes counting, IL10 and NO liver production increased only in A/J. In addition, only in B6 the IL1b levels increased while IL10 and NO decreased in liver. A/J mice suffered more inflammatory damage and accumulated less liver tg than B6. In B6.A-Hc0 IL17 and IL10 increased while of IL1b and TGFb decreased when compared to B6. Independently of the diet, levels of AST, FA, albumin, cholesterol, tg increased in B6.A-Hc0 serum and IL6, IL12 and IFNg reduced in liver. In conclusion, C5 promoted a pro-inflammatory environment in the liver and influenced the serum levels of hepatic enzymes, cytokines and lipid profile.