L’activation du système complément, qui se produit à la suite d’une infection ou d’un trauma, génère de puissants signaux moléculaires capables d’initier la réaction inflammatoire. Parmi ces signaux, le fragment actif C5a permet de recruter sur le site lésé les cellules qui expriment son récepteur (le C5aR/CD88). Bien que le C5aR/CD88 soit initialement connu pour être exprimé par les cellules inflammatoires, il est établi que de nombreuses cellules non immunitaires expriment ce récepteur indiquant son implication dans d’autres processus. Nos résultats ont permis de démontrer que l’activation du système du complément au niveau de la pulpe dentaire est réalisée non seulement à partir des protéines plasmatiques mais aussi des protéines synthétisées par les fibroblastes pulpaires. Ainsi, l’activation locale du système du complément, produit à la suite d’une infection, d’un trauma ou de l’application de biomatériaux, génère du C5a qui induit la migration des cellules progénitrices. Ce travail démontre pour la toute première fois l’implication du fragment actif C5a dans le recrutement de progénitrices pulpaires, étape clef au processus de régénération pulpo-Dentinaire. Ces travaux pourraient donc constituer une piste sérieuse dans l’établissement de nouvelles thérapies permettant de cibler les cellules progénitrices au cours du processus de régénération. / After tissue injury or infection, Complement activation provides powerful signals initiating the inflammatory reaction. These events are mediated by biologically active fragments such as C5a which attracts cells expressing its receptor (C5aR/CD88) to the injury site. Besides inflammatory cells as the main C5aR-Expressing cells, various tissue cells have been reported to express this receptor suggesting its involvement in other processes. In order to investigate the possible relationship between complement activation and pulp regeneration, we investigated Complement activation in the dental pulp and progenitor cell migration from their perivascular niches to the pulp injury site to initiate the regeneration process.Our results indicate that complement activation in the dental pulp is the result of both plasma and fibroblast secreted complement proteins. Thus upon local complement activation, which can occur after pathological injury or biomaterials application, C5a induces pulp progenitors’ migration which is critical in initiating the regenerative processes. To our knowledge, this is the first work to demonstrate the involvement of C5a biologically active fragment in the recruitment human pulp progenitor cells. This may provide a useful future therapeutic tool in targeting the progenitor cells in a dentin/pulp regeneration process.

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.

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.

Investigation of activated remodelling in the healing of experimental stress fractures and the influence of anti-inflammatory treatments Lisa Jane Kidd Abstract Targeted and focal remodelling are important processes in bone homeostasis and pathology. However, the factors that initiate and direct remodelling to repair microcracks, or respond to excess loading are still poorly understood. The rat ulna-loading (RUL) model has been widely used to examine modelling and remodelling responses to axial cyclic loading. However the model has not yet been fully characterised. Stress fractures are common amongst athletes, dancers and military recruits, but there is almost no information available on the mechanism of healing of these fractures. Although cyclooxygenase-2 (COX-2) is a key mediator of bone resorption and bone formation, very little information is available on the effect of non-steroidal antiinflammatories (NSAIDs) on stress fracture healing. Remodelling may play a role in the pathogenesis of stress fractures, and there is growing interest in the potential use of bisphosphonates to prevent them. Nonetheless, the effect of bisphosphonates on stress fracture healing is not known. PMX53 is a C5a receptor antagonist developed as a novel anti-inflammatory agent. It is effective against inflammatory arthritis, but has not been tested in any fracture models. The aims of this study were to undertake a detailed examination of the histology, histomorphometry and gene expression of the healing and remodelling process initiated by RUL, and to use this model to determine the effects of selective and non-selective NSAIDs, a bisphosphonate and PMX53 on stress fracture healing. To characterise the RUL model, fatigue fractures were created by loading ulnae until displacement was observed to increase by between 4% and 50%. Ulnae were bulk-stained in basic fuchsin and processed for undecalcified histology. For all remaining experiments, loading was stopped when the displacement had increased by 10%. For detailed histology and histomorphometry, ulnae were decalcified, paraffin embedded and stained with toluidine blue, saffranin-O or for tartrate resistant acid phosphatase (TRAP). Ulnae were examined at 1, 2, 4, 6, 8, and 10 weeks after loading. The effects of DFU (a selective COX-2 inhibitor, 2 mg/kg po), ibuprofen (a non-selective NSAID, 30 mg/kg po) and PMX53 (10 mg/kg po) were examined at 2, 4 and 6 weeks after loading. Effects of risedronate (a bisphosphonate) were examined at a high (1.0 mg/kg po) and low dose (0.1 mg/kg po) at 2, 6 and 10 weeks after loading. RUL did not create isolated intracortical microcracks, but curvilinear fatigue fractures that occurred at a standard position in the medial cortex of the distal ulna diaphysis. These stress fractures induced rapid periosteal woven bone formation and direct intracortical remodelling along the fracture line that originated at the periosteum and progressed towards the medullary cavity. Basic multicellular units (BMUs) could be followed through serial sections extending along the fracture line towards the centre of the bone. Quantitative, real-time PCR was performed at 4 hours, 24 hours, 4 days, 7 days and 14 days after fatigue fracture. Following each period, bones were dissected and mRNA was extracted using standard protocols. Gene expression was compared between loaded and unloaded ulnae and to an unloaded control group. Four hours after loading, there was a marked, 220-fold increase (P<0.0001) in expression of Interleukin-6 (IL-6). There were also prominent peak increases in mRNA expression for Osteoprotegerin (OPG), cyclooxygenase-2 (COX-2), and vascular endothelial growth factor (VEGF) (all P<0.0001). At 24 hours there was a peak increase in mRNA expression for IL-11 (73-fold increase, P<0.0001). At 4 days there was a significant increase in mRNA expression for Bcl-2, COX-1, bone morphogenic protein (BMP)-2, insulin-like growth factor (IGF)-1, osteopontin (OPN), and stromal cell derived factor SDF-1. At 7 days there was a significant increase in mRNA expression of Receptor activator of nuclear factor kappa β ligand (RANKL) and OPN. The dramatic, early up-regulation of IL-6 and IL-11 suggests they play a central role in initiating signalling events for stress fracture healing. Treatment with PMX53 did not affect any measures of woven bone formation or stress fracture remodelling. There were no treatment effects of Ibuprofen or DFU on the area of woven bone. DFU treatment resulted in a significant reduction in the area of porosity (resorption) and BMU area along the fracture line at 2 weeks after fracture. Ibuprofen treatment resulted in a significant reduction in length and area of BMUs and new bone formation along the fracture line at 6 weeks (p < 0.05). This is the first report to demonstrate a negative effect on stress fracture healing of both a selective COX-2 inhibitor and a non-selective NSAID. These data confirm the importance of cyclooxygenase in bone resorption and formation during remodelling. Bisphosphonates are potent inhibitors of osteoclastic bone resorption Two, 6 and 10 weeks after loading, measures of resorption and new bone formation were significantly reduced along the fracture line by high dose risedronate treatment, but not by the low dose. Only the porosity along the fracture line 2 weeks after loading was significantly reduced by the low dose risedronate. The low dose more closely resembles the clinical dose used to treat patients. Woven bone formation and consolidation were not affected by the low or high doses of risedronate. In conclusion, fatigue fractures in the rat ulna are highly reproducible, induce exuberant periosteal woven bone formation, and heal by direct remodelling along the fracture line. Remodelling is associated with gene expression for molecules typically associated with bone resorption and formation, angiogenesis and cell signalling. Remodelling of the stress fracture line was adversely affected by treatment with selective and non-selective COX inhibitors, by high dose treatment with risedronate, but not by PMX53, a C5a antagonist.

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.

Slutsteget i immunförsvarets komplementaktivering innefattar en klyvning av komplementprotein C5, till C5a och C5b, vilket initierar bildandet av terminalt komplementkomplex (TCC) som i form av membran-attack-komplex (MAC) bildar cytotoxiska porer i bland annat gramnegativa bakterier. Bildandet av MAC kan blockeras av endogena regulatorer och TCC frisätts då som ett lösligt komplex, sC5b-9, i plasma. I examensarbetet studerades en variant av TCC, som i tidigare studier visats bildas oberoende av C3- och C5-konvertas när serum surgjorts till pH &lt; 7,0 in vitro. Syftet med studien var att studera om detta atypiska TCC (aTCC) bildades hos grisar, som i en mekonium aspirationssyndrom (MAS)-modell, erhållit ett sänkt systemiskt pH in vivo. I syftet ingick också att etablera en ELISA-baserad metod för att analysera aTCC. I en sandwich ELISA användes monoklonal anti-C5a/C5a (desArg) (klon T13/9) som fångande antikropp och monoklonal anti-C9 (klon aE11) som detekterande antikropp för att analysera aTCC i plasmaprover från 18 MAS-grisar, samt i ett kontrollmaterial bestående av grisserum som surgjorts till pH 6,8 och 6,4 in vitro. Mängden aTCC i kontrollproverna ökade när pH sänktes men innehållet av aTCC i plasmaproverna minskade över MAS-studiens förlopp. När den relativa förändringen i aTCC relaterades till MAS-grisarnas slutliga pH kunde ett signifikant samband ses (p = 0,02) som visade att en större förändring i aTCC sammanföll med ett lägre slutligt pH. Nivåerna av aTCC var generellt sett högre i plasmaproverna jämfört med kontrollproverna vilket skulle kunnat bero på skillnader i plasma vs serum avseende aTCC eller att proverna kom från grisar med olika ålder och vikt. Avsaknad av grisspecifik standard och negativ kontroll samt lågt signal/brusförhållande bidrar till felkällor för analysen och denna kräver fortsatt optimering. / The late steps of complement activation involves a cleavage of complement protein C5, to C5a and C5b, which initiates the formation of terminal complement complex (TCC). The final complex is referred to as the membrane-attack-complex (MAC) which forms cytotoxic pores in, inter alia, gram-negative bacteria. The formation of MAC can be inhibited by endogenous regulators and the TCC is then released as a soluble complex, sC5b-9, in plasma. In the degree project, another type of TCC was studied, which in previous studies had shown to form independently of C3 and C5 convertases when serum was acidified to pH &lt;7.0 in vitro. The purpose of the study was to investigate whether this atypical TCC (aTCC) was formed in piglets, which in a model of meconium aspiration syndrome (MAS), received a reduced systemic pH in vivo. The purpose was also to establish an ELISA for analyzing aTCC. Sandwich ELISA, with monoclonal anti-C5a / C5a (desArg) (clone T13/9) as a capture antibody and monoclonal anti-C9 (clone aE11) as a detection antibody, was used to analyse aTCC in plasma samples from 18 MAS piglets, and in control samples consisting of pig serum acidified to pH 6.8 and 6.4 in vitro. The amount of aTCC in the control samples increased when the pH was lowered, but the content of aTCC in the plasma samples decreased over the course of the MAS study. When the relative change in aTCC was related to the final pH of the MAS pigs, a significant relationship could be seen (p = 0.02) which showed that a major change in the aTCC coincided with a lower final pH. aTCC were generally higher in plasma samples compared with control samples, which could be due to differences in plasma vs serum for aTCC or that the samples came from pigs of different age and weight. Lack of pig-specific standard and negative control as well as low signal to noise ratio contribute to sources of error for the analysis and this requires continued optimization.

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.

No description available.

The human specific gram-negative bacterium Neisseria meningitidis (Nme, meningococci) is a common colonizer of the upper respiratory tract. Upon becoming invasive, Nme can cause meningitis and life-threatening sepsis. The most important immune defense mechanism in invasive meningococcal disease (IMD) is the complement mediated killing of bacteria. The complement cascade is activated through different pathogen associated patterns and finally leads to the lysis of the bacteria by the membrane attack complex. In addition to the direct bacterial killing, the complement system is also an important player in different inflammatory processes. A hallmark of IMD is an overreaction of the immune system and the release of the potent anaphylatoxins C3a and C5a by the complement system is an important factor hereby. There are three anaphylatoxin receptors (ATRs), the C3aR, the C5aR1 and the C5aR2, capable of detecting these anaphylatoxins. It has already been shown that blocking the ATR C5aR1 strongly benefitted the outcome of IMD in a murine sepsis model. However, the roles of ATRs C3aR and C5aR2 in IMD are still unclear. This work aims to analyze the role of these ATRs in meningococcal sepsis and to identify possible underlying mechanisms. Furthermore, a possible involvement of the complement system, the ATRs and the type II CRISPR/Cas system on nasopharyngeal colonization is analyzed. In vivo depletion experiments showed that without neutrophils or monocytes/macrophages the complement system alone was not able to clear a low dose Nme infection, which highlights the importance of cellular components in IMD. Analyzing the role of the ATRs in knock-out mice with high dose Nme infections, revealed that the lack of C5aR2, like the lack of C5aR1, was beneficial for the outcome of meningococcal induced sepsis. In contrast, the lack of C3aR in knock-out mice was detrimental. The positive outcome associated with the C5aRs could be reproduced by using an antagonist against both C5aRs or an antagonist specifically against C5aR1 in WT mice. These findings are giving hope to future therapeutic applications. Next, a possible contribution of neutrophils to this positive outcome was analyzed. Absence of C5aR1 led to a decrease of degranulation by neutrophils in a murine whole blood model, while the other ATRs showed no effect. Neutrophil analysis in human whole blood, on the other hand, revealed a reduced oxidative burst and IL-8 secretion upon inhibition of all three ATRs. A functional difference between the C5aRs and the C3aR in neutrophils was observed in phagocytosis, which was reduced upon C3aR inhibition, but was unaltered with C5aR1 or C5aR2 inhibition. Possible underlying mechanisms in the phosphorylation of ERK1/2 were analyzed in bone marrow derived macrophages isolated from ATR knock-out mice. The later phosphorylation of ERK1/2 in macrophages without C5aR1 or C5aR2 expression might explain, why blocking the C5aRs is beneficial for the outcome of IMD in mice. In contrast to these findings, the colonization of the nasopharynx in huCEACAM 1 expressing mice by Nme did not seem to depend on the Complement system factors C3 and C5 nor the ATRs. Additionally, no difference in the colonization could be observed in this model using Nme mutants lacking different parts of the type 2 CRISPR/Cas system. Conclusively, this work highlights the importance of the complement system, the ATRs and the cellular components in IMD. Contrariwise, these factors did not play a role in the analyzed nasopharyngeal infection model. The beneficial effects of C5aR1 and C5aR2 lack/inhibition in IMD might have medicinal applications, which could support the standard therapies of IMD in the future. / Das human spezifische pathogene Gram-negative Bakterium Neisseria meningitidis (Nme, Meningokokken) ist ein Kommensale angesiedelt im Nasopharynx. Bei invasiver Erkrankung können Nme Meningitis oder eine lebensbedrohliche Sepsis verursachen. Die wichtigste Verteidigung des Immunsystems in invasiver Meningokokken-Erkrankung (IMD) ist die Abtötung von Bakterien durch das Komplementsystem. Die Komplementkaskade wird durch verschiedene pathogenassoziierte Muster in Gang gesetzt und resultiert in dem Aufbau des Membranangriffskomplex, welcher die Bakterien schließlich lysiert. Darüber hinaus spielt das Komplementsystem auch eine wichtige Rolle in verschiedenen inflammatorischen Prozessen im Körper. Ein charakteristisches Merkmal von IMD ist eine übermäßige Reaktion des Immunsystems und dabei ist die Freisetzung der Anaphylatoxine C3a und C5a, durch das Komplementsystem, ein wichtiger Faktor. Es gibt drei Anaphylatoxin Rezeptoren (ATR), den C3aR, den C5aR1 und den C5aR2, welche die jeweiligen Anaphylatoxine erkennen. In murinen Modellen wurde bereits gezeigt, dass die Inhibition des C5aR1 einen positiven Einfluss auf den Verlauf von IMD hat. Im Kontrast dazu sind die Rollen der ATRs C3aR und C5aR2 in IMD weiter unklar. Diese Arbeit hat als Ziel, die Rolle der ATRs in Meningokokken induzierter Sepsis zu untersuchen und mögliche zugrundeliegende Mechanismen zu finden. Des Weiteren soll ein möglicher Einfluss des Komplementsystems, der ATRs und des Typ II CRISPR/Cas Systems auf die Kolonisation durch Nme im Nasopharynx untersuchen werden. In vivo Depletions-Versuche zeigten, dass ohne Neutrophile oder Monozyten/Makrophagen das Komplementsystem allein nicht in der Lage war eine Nme-Infektion mit einer niedrigen Infektionsdosis zu beseitigen. Dies zeigt die Wichtigkeit von Immunzellen neben dem Komplementsystem in IMD. Experimente mit hohen Nme-Dosen in ATR knock-out Mäusen zeigten, dass die fehlende Expression von C5aR2, wie die von C5aR1, sich positiv auf den Ausgang von IMD auswirkte. Im Gegensatz dazu, verschlimmerte das Fehlen des C3aR Rezeptors den Ausgang der IMD. Die positive Wirkung in den C5aR knock-out Mäusen, konnte auch mit der Gabe von einem gegen beide C5aRs oder einem spezifisch gegen C5aR1 gerichteten Antagonisten in WT Mäusen beobachtet werden. Diese Ergebnisse geben Hoffnung auf eine mögliche zukünftige therapeutische Applikation. Als nächstes wurde eine mögliche Beteiligung von Neutrophilen an dem positiven Ausgang von IMD in Abhängigkeit von den ATRs untersucht. Eine fehlende C5aR1 Expression führte zu einer verminderten Degranulation durch Neutrophile in dem verwendeten murinen Vollblutmodel, wohingegen die fehlende Expression der anderen ATRs keinen Effekt zeigte. Im Gegensatz dazu, zeigten Versuche mit humanem Vollblut einen verminderten Oxidativen Burst sowie eine verminderte Ausschüttung von IL-8 bei der Blockade von allen drei ATRs. Ein Unterschied zwischen den C5aRs und dem C3aR zeigte sich hingegen in der Phagozytose, welche mit C3aR Inhibierung reduziert war, aber unverändert nach der Inhibierung von C5aR1 oder C5aR2 blieb. Mögliche zugrundeliegende Mechanismen in der Phosphorylation von ERK1/2 wurden anschließend in Knochenmark-gereiften Makrophagen von ATR knock-out Mäusen untersucht. Ohne C5aR1 oder C5aR2 Expression wurde eine verzögerte Phosphorylierung von ERK1/2 in den Makrophagen beobachtet, was erklären könnte warum die Blockade von C5aRs den Ausgang von Meningokokken induzierter Sepsis in Mäusen positiv beeinflusst. Im Gegensatz zu diesen Ergebnissen wurde die Kolonisation des Nasopharynx durch Nme in huCEACAM-1 exprimierenden Mäusen, weder durch die Komplementfaktoren C3 und C5 noch durch die ATRs beeinflusst. Zusätzlich konnte auch kein Unterschied in der Besiedelung des Nasopharynx durch Nme-Mutanten, die verschiedene Mutationen des Typ 2 CRISPR/Cas Systems besaßen, beobachtet werden. Diese Arbeit zeigt die Wichtigkeit des Komplementsystems, der ATRs und der Immunzellen in IMD. Zusätzlich zeigt diese Arbeit, dass das Komplementsystem und die ATRs jedoch keine Auswirkungen auf die Kolonisation des Nasopharynx in Mäusen haben. Die äußerst positive Auswirkung auf IMD, wenn C5aR1 und C5aR2 nicht gebildet oder blockiert werden, könnte medizinisch von Bedeutung sein und eventuell in der Zukunft die Standarttherapie bei IMD unterstützen.

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