Shiga toxin-producing enterohemorrhagic Escherichia coli (EHEC) is an emerging food- and water-borne pathogen, causing approximately 73,000 annual infections in the United States and an estimated 1.5 million infections globally. E. coli O157:H7, the most frequently associated EHEC strain, is primarily transmitted through consumption of contaminated ground beef and produce and leads to hemorrhagic colitis in humans. In 5% to 15% of infected patients, circulating Shiga toxins (Stx1, Stx2) cause hemolytic uremic syndrome (HUS), characterized by the presence of thrombocytopenia, hemolytic anemia, and thrombotic microangiopathy, contributing to acute kidney injury (AKI). Current treatment is supportive and antibiotic therapy is contraindicative as it increases toxin production. Therapeutics for EHEC-induced HUS need to be identified to minimize kidney injury and uncontrolled coagulopathy. Well-characterized animal models of HUS and EHEC infection are available and provide avenues for potential therapeutic discovery. Baboons (Papio) challenged with endotoxin-free Shiga toxins develop full spectrum HUS, and mice infected with Stx2-producing Citrobacter rodentium (Cr Stx2+), a genetically modified enteric mouse pathogen, develop severe Stx2-mediated kidney injury. Initial studies have shown that soluble thrombomodulin (sTM), an anti-coagulant, is a promising therapeutic in preventing severe kidney injury in pediatric patients. In these studies, we determined whether complement was activated in baboons challenged with Shiga toxins, and evaluated whether intraperitoneal injection of sTM would reduce disease severity from mice infected with Cr Stx2+. D-dimer and cell injury markers (HMGB1, histones) confirmed the presence of coagulopathy and cell injury in Stx challenged baboons. Studies revealed that complement activation is not required for the development of thrombotic microangiopathy and HUS induced by EHEC Shiga toxins in these pre-clinical models. Soluble thrombomodulin treatment in Cr Stx2+ infected mice significantly decreased colonization but did not alter mortality. However, gene expression of kidney injury markers (NGAL, KIM-1) decreased significantly compared to no treatment indicating sTM-associated cytoprotectivity. The C. rodentium mouse model does not develop the coagulopathy seen in HUS patients and sTM treatment may be more effective in the baboon toxemia model. Soluble thrombomodulin is a promising therapeutic for EHEC-induced HUS and should be further evaluated in Stx challenged baboons.

A leptospirose é uma zoonose de distribuição mundial, com maior incidência nas regiões tropicais. As bactérias que causam a doença pertencem ao gênero Leptospira, família Leptospiracea e ordem Spirochaetales. A leptospirose é mantida na natureza pela colonização persistente dos túbulos renais proximais dos animais portadores. Uma estratégia adotada por estas espiroquetas para sobreviver à ação do sistema imune inato do hospedeiro é a capacidade que possuem de interagir com os reguladores do sistema complemento Fator H (FH) e proteína de ligação a C4b (C4BP). O sistema complemento é um componente vital da imunidade inata, uma vez que desempenha um papel crucial na defesa do hospedeiro, particularmente contra bactérias Gram-negativas. Dados recentes gerados por nosso grupo mostraram que C4BP interage com a proteína de superfície LcpA de Leptospira. Com cerca de 20 kDa, essa proteína é capaz de se ligar a C4BP purificado ou solúvel no soro de maneira dose-dependente. Uma vez ligado à proteína, C4BP permanece funcional agindo como cofator de Fator I na clivagem de C4b. O presente estudo teve como principal objetivo avaliar a interação da proteína LcpA com FH humano, principal regulador solúvel da via alternativa do sistema complemento. A proteína LcpA e suas porções N-Terminal, Intermediária e CTerminal recombinantes foram purificadas por cromatografia de afinidade a metal a partir da fração insolúvel. A interação dessas proteínas com FH foi avaliada por dois métodos distintos: ELISA e Western blot com overlay. Os resultados indicaram que a porção C-Terminal da proteína LcpA é responsável pela interação com FH. Curiosamente, C4BP também se liga a esse domínio da proteína. Uma vez que esses dois reguladores solúveis do sistema complemento interagem com o mesmo segmento da LcpA, realizaram-se, a seguir, ensaios de competição com o objetivo de avaliar se ambos compartilhariam os mesmos sítios de interação. Os dados mostraram que FH e C4BP devem se ligar a sequências distintas desta proteína. Com o objetivo de se avaliar a funcionalidade de FH ligado à LcpA, realizou-se um ensaio para investigar sua atividade de co-fator de Fator I na clivagem de C3b. Produtos de degradação de 46 kDa e 43 kDa da cadeia α' de C3b foram detectados, indicando que FH permanece funcional. Em se tratando de uma proteína com funções relacionadas ao processo de evasão ao sistema imune inato, decidiu-se realizar ensaios de desafio em modelo de hamster com a finalidade de se avaliar seu potencial imunoprotetor. Os três ensaios realizados indicaram que a proteína não é capaz de conferir proteção. Os ensaios de ELISA visando à avaliação dos títulos de anticorpos mostraram que LcpA não é imunogênica, fato que explica os resultados dos ensaios de desafio observados. Portanto, embora interaja com moléculas do hospedeiro e pareça contribuir para o processo de evasão ao sistema imune inato, essa proteína de membrana não se mostrou promissora como candidato vacinal contra leptospirose. / Leptospirosis is a zoonosis of global distribution, with higher incidence in tropical areas. The bacteria that cause the disease belong to the genus Leptospira, family Leptospiracea and order Spirochaetales. Leptospirosis is maintained in nature by persistent colonization of proximal renal tubules of carrier animals. One strategy adopted by these spirochetes to escape from host´s innate immune system is the ability to interact with the complement regulators Factor H (FH) and C4b Binding Protein (C4BP). The complement system is a vital component of the innate immune system, being crucial for host´s defense, particularly against Gram-negative bacteria. According to our recent published data, C4BP interacts with the leptospiral surface protein LcpA. This 20 kDa outer membrane protein binds both purified and serum C4BP in a dose-dependent manner. Once bound, C4BP remains functional acting as a cofactor for Factor I in the cleavage of C4b. In the present study we evaluated the interaction of LcpA with human FH, the main soluble regulator of the alternative pathway of complement. The intact protein as well as its N-terminal, intermediate and C-terminal portions were purified by metal-affinity chromatography from the insoluble pellet. The interaction of these proteins with FH was evaluated by two distinct methods: ELISA and Western blot overlay. Our results indicate that the C-terminal domain of LcpA mediates interaction with FH, and also with C4BP. Since both complement regulators interact with the same fragment of LcpA, we next performed competition assays to assess if they would share binding sites. According to our data, FH and C4BP have distinct binding sites on LcpA. Cofactor activity of FH bound to immobilized LcpA was confirmed by detecting the C3b α' chain cleavage fragments of 46 and 43 kDa upon incubation with Factor I, thus indicating that it remains functionally active. Given the LcpA´s role in host´s innate immune evasion, we also evaluated its vaccine potential in a hamster model. Data from three challenge assays indicated that the protein can not afford protection. Low ELISA antibody titers of hamsters immunized with LcpA were observed, which strongly suggests that this protein is not immunogenic. In conclusion, LcpA interacts with host´s molecules and seems to contribute to the bacterial immune evasion. Nevertheless, this outer membrane protein is not a promising vaccine candidate against leptospirosis.

A ocorrência de trombose está freqüentemente associada com a presença de um ou mais fatores de riscos, os quais podem ser genéticos e/ou adquiridos, tais como as mudanças hormonais que ocorrem durante a gravidez, a terapia de reposição hormonal e o uso de contraceptivos hormonais combinados (CHC). A inflamação, por sua vez, é uma importante resposta do organismo às agressões e envolve vários mecanismos biológicos relacionados entre si e altamente regulados, tais como: coagulação, fibrinólise, ativação do sistema complemento (SC), antioxidação e regulação hormonal. Fisiologicamente, os sistemas complemento e da coagulação compartilham componentes. A ativação do fator XII da coagulação é controlada pela mesma proteína reguladora da ativação do sistema complemento, o inibidor de C1. A deficiência do inibidor de C1 leva a uma patologia conhecida como angioedema hereditário. No entanto, uma manifestação clínica similar ao angioedema tem sido descrita em mulheres que usam CHC ou recebem terapia de reposição hormonal com estrogênio (E). Esta influência do estrogênio na coagulação e no SC também é evidenciada pela ação regulatória do E sobre a expressão do fator XII e dos seus níveis plasmáticos. Considerando o efeito pleiotrópico do E, e as interações do SC e da hemostasia, o objetivo desse estudo foi avaliar o efeito de diferentes CHC sobre: a) a atividade hemolítica (AH) do SC e ativação das vias clássica/lectina e alternativa; b) a atividade opsonizante do SC em mediar o burst oxidativo dos neutrófilos; e c) a função dos receptores para complemento (CR) em mediar o burst oxidativo dos neutrófilos. Nós estudamos 5 CHC diferentes e observamos que a) drospirenona + 30g E mostrou uma tendência a aumentar o burst oxidativo mediado por CR; b) gestodeno + 20g E mostrou redução da capacidade opsonizante do SC; c) levonorgestrel + 30g E e gestodeno + 20g E promoveram uma redução no número de neutrófilos positivos para a expressão de CR1; d) drospirenona + 30g E e drospirenona + 20g E promoveram um aumento da AH da via clássica (VC) do SC; e) levonorgestrel + 30g E promoveu uma redução da AH da VC do SC; f) drospirenona + 30g E, gestodeno + 20g E e levonorgestrel + 30g E promoveram uma diminuição do nível sérico de C4d, produto da ativação das vias clássica/lectina do SC; g) levonorgestrel + 30g E apresentou um aumento da concentração sérica de inibidor de C1; h) nenhum dos CHC mostrou diferenças na ativação da via alternativa do SC. Os resultados mostram a importância de considerar os diferentes grupos de CHC nas comparações com o Grupo Controle, uma vez que algumas diferenças foram significativas apenas para CHC em particular. Estas observações podem contribuir para o entendimento dos mecanismos envolvidos na fisiopatologia dos processos inflamatórios associados ao uso de estrogênios. / The occurrence of thrombosis is often associated with the presence of one or more risk factors, which may be genetic and/or acquired, such as hormonal changes that occur during pregnancy, hormone replacement therapy and the use of combined hormonal contraceptives (CHC). The inflammation in turn, is an important body\'s response to the aggression and involves several biological mechanisms related and highly regulated, such as coagulation, fibrinolysis, activation of the complement system (CS), oxidation and hormonal regulation. Physiologically, the complement and coagulation systems share components. Activation of coagulation factor XII is controlled by the same regulatory protein activation of the complement inhibitor C1. The deficiency of C1 inhibitor leads to a condition known as hereditary angioedema. However, a clinical manifestation similar to angioedema has been reported in women using CHC or receiving hormone replacement therapy with estrogen (E). The influence of E on coagulation and the CS is also evidenced by the regulatory action of E on the expression of factor XII and its plasma levels. Considering the pleiotropic effects of E, and the interactions of CS and hemostasis, the goal of this study was to evaluate the effect of different CHC on: a) hemolytic activity (HA) CS and activation of classical/lectin and alternative pathways, b) the opsonizing activity of the CS in mediating the oxidative burst of neutrophils, and c) the function of receptors for complement (CR) in mediating the oxidative burst (OB) of neutrophils. We studied 5 different CHC and data showed: a) drospirenone + 30g E increase of the OB neutrophils mediated by CR; b) gestodene + 20g E had a reduced opsonizing ability; c) levonorgestrel + 30g E and gestodene + 20g E promoted a reduction of neutrophils positive for the expression of CR1, d) drospirenone + 30g E and drospirenone + 20g E promoted an increase in HA for classical pathway (CP); e) levonorgestrel + 30g E reduced the HA for CP; f) drospirenone + 30g E and gestodene + 20g E and levonorgestrel + 30g E reduced the serum level of C4d; g) levonorgestrel + 30g E showed an increase of the serum level of C1 inhibitor; h) none of CHC showed differences in activation of the alternative pathway in CS. The results show the importance of considering the different groups of CHC in comparison with the control group, since some differences were significant only for CHC in particular. These observations may contribute to the understanding of the mechanisms involved in the pathophysiology of inflammatory processes associated with estrogen use.

The human immune-regulatory protein, complement receptor type 1 (CR1, CD35), occurs on erythrocytes where it serves as the immune adherence receptor. It interacts with C3b, C4b, C1q and mannan-binding lectin (MBL). It additionally binds the Plasmodium falciparum protein, Rh4, in the non-sialic acid-dependent erythrocye-invasion pathway, and is also important for rosetting, via an interaction with P. falciparum erythrocyte membrane protein 1 (PfEMP1). A C3b/C4b, and PfEMP1 binding site lies in CCP modules 15-17 (out of 30 in CR1), while polymorphisms that afford advantage to some populations in dealing with severe malaria occur in CCPs 24-25, begging the question central to this thesis – do these polymorphism modulate function, and if so how? We hypothesized that the CR1 architecture apposes CCPs 15-17 and CCPs 24-25 using the exceptionally long linker between CCPs 21 and 22 as a hinge, thus polymorphic variants in CCPs 24-25 modulate functionality in CCPs 15-17. To test this, a panel of recombinant CR1 protein fragments (CCPs 21, 21-22, 20-23, 15-17, 17, 10-11, 17-25, 15-25 and 24-25) were produced in Pichia pastoris along with polymorphic forms of the relevant constructs. After purification, biophysical and biological methods were used to assess whether the linker does indeed act as a hinge, and the comparative abilities of the CCPs 15-25 variants (along with soluble CR1 (sCR1), CCPs 1-3 and the panel of CR1 fragments) to interact with a range of ligands were measured. We found no evidence from NMR for face-to-face contacts between CCPs 21 and 22 that would be consistent with the long linker permitting a 180-degree bend between them. Indeed, based on scattering and analytical ultracentrifugation data, CCPs 20-23 form an extended rather than a bent-back structure. All of the four Knops blood-group variants of the CCPs 15-25 proteins produced similar results according to dynamic light scattering and AUC indicating no structural difference or change in self-association state between variants. In addition, based on the data collected from surface plasmon resonance (SPR), ELISA and fluid-phase cofactor (for factor I) assays, there were no evidence of any difference between the polymorphic forms with respect to their interactions with C3b, C4b, C1q and MBL. Only weak interaction was observed for sCR1, and all CCPs 15-25 variants, with the relevant part of PfEMP1, and there was no measurable difference amongst the variants in disrupting rosettes. The sCR1-Rh4.9 interaction was confirmed by SPR; affinities measured between the binding domain of Rh4 and the panel of CR1 fragments identified CCPs 1-3 (site 1) as the main interaction site. It seemed unlikely therefore that CCPs 24 and 25 could modulate Rh4 binding; indeed none of the four CR1 15-25 variants bound Rh4.9 appreciably. Thus we concluded that allotypic variations in CCPs 24-25 have no measurable effect on the architecture as well as binding of CR1 to its host or parasite ligands The inferred selective pressure acting on these variants likely arise from some other (i.e. besides malaria) geographically localised infectious diseases.

Glioblastoma Multiforme (GBM) is an extremely aggressive grade IV brain tumour that is highly infiltrative and can spread to other parts of the brain quickly. It is the most common primary brain tumour where patients have a median survival of 14.6 months. Symptoms vary depending upon the location of the tumour and include seizures, progressive headaches and focal neurological deficit. The poor prognosis is characterised by deregulation of many key signalling pathways involving survival, growth, apoptosis and evasion of immune surveillance. In this study, we investigated whether complement factor H related protein 5 (CFHR5) from primary GBM cells direct from patients exhibited functional activity similar to factor H. The presence of CFHR5 was validated by western blot and ELISA technique from B30, B31 and B33 primary GBM cells. The functional capacity of CFHR5 was examined through the alternative pathway, co-factor, and decay acceleration assay. We demonstrated that CFHR5 was able to inhibit the alternative pathway through the same mechanism as factor H. Emerging evidence had shown that the innate immune protein surfactant protein D (SP-D) and recombinant human SP-D (rhSP-D) were able to induce apoptosis in eosinophilic leukaemic cells. We studied the ability of rhSP-D to induce apoptosis in U87 GBM cells through apoptotic and viability assays. rhSP-D was unable to mediate cell death and instead increased cell viability. This led us to investigate the expression of SP-D in U87 and B30 GBM cells through western blot, ELISA and immuno-fluorescence detection. We demonstrated novel information about the production of SP-D by GBM cells. To extend our study, we investigated the interaction of THP-1 macrophage with rhSP-D bound U87 cells. We carried out live cell imaging, RT-qPCR, and cell viability assays, to study the changes in cytokine expression and viability of cells. THP-1 did not engulf U87 cells; however, it did reduce the number of cells and decrease the expression of pro-tumourigenic cytokines. This study highlights the ability of primary GBM cells to evade innate immune detection by the secretion of functionally active CFHR5. It also demonstrated the ability of U87 to evade destruction by rhSP-D and THP-1 highlighting the extremely aggressive behaviour of the tumour and lack of new treatment to improve prognosis in over a decade.

A inflamação envolve vários mecanismos biológicos relacionados entre si e altamente regulados: coagulação, fibrinólise, sistema complemento (SC), antioxidação e regulação hormonal. Fisiologicamente, o SC e a coagulação compartilham componentes regulatórios e várias interações entre o SC e a hemostasia têm sido propostas, mas não elucidadas. O interesse científico sobre as interações do SC com a hemostasia é crescente, devido ao risco de tromboembolismo venoso (TEV) associado ao uso de contraceptivos orais combinados (COC). Nosso grupo tem investigado os efeitos de COC sobre o SC, hemostasia e moléculas de adesão. Os resultados confirmam vantagens do etinilestradiol (EE)/levonorgestrel (LNG), um COC de 2ª geração sobre os de 3ª geração, mantendo-o como referência para comparação com novos COC, os quais buscam componentes mais naturais. Os componentes hormonais nos COC podem influenciar as interações entre o endotélio e o SC, as quais medeiam adesão de neutrófilos ao endotélio, podendo favorecer o TEV. Desde o advento dos COC busca-se constantemente minimizar seus efeitos indesejáveis com redução das doses, diferentes combinações de progestógenos/estrógenos e vias de administração. O 17? estradiol (E2) é o estrógeno natural e encontra-se combinado com o progestógeno acetato de nomegestrol (NOMAC), mais seletivo para o receptor de progesterona, minimizando efeitos indesejáveis sobre o metabolismo. Considerando-se os benefícios do EE/LNG e a aprovação do E2/NOMAC, o objetivo deste estudo foi avaliar o efeito destes COC sobre: 1) o SC por ativação das Via Clássica (VC) e Via das Lectinas (VL), concentração de C4d e concentração sérica e atividade do inibidor de C1 (C1inh) e 2) os níveis séricos de moléculas de adesão intercelular (ICAM)-1 e vascular (VCAM)-1 e E-selectina. A atividade hemolítica (AH) sérica da VC foi avaliada por ensaio cinético nos grupos de usuárias ou não dos COC e em pool de soro humano normal (SHN) exposto aos componentes dos COC. Os demais parâmetros (VL, C4d, C1inh e moléculas de adesão) foram avaliados por ensaios imunoenzimáticos. Os resultados foram: 1) não houve diferenças na AH sérica da VC; 2) exceto para EE+LNG, a AH da VC in vitro foi menor quando o pool de SHN foi exposto aos componentes individuais das formulações dos COC (EE, LNG, E2 e NOMAC) e à combinação E2+NOMAC comparada ao controle (pool SHN sem componentes das formulações dos COC), menor para E2+NOMAC comparada ao EE+LNG e menor para o NOMAC comparado ao LNG; 3) não houve diferenças na ativação da VL; 4) não houve diferenças na concentração sérica do fragmento C4d; 5) a concentração sérica do C1inh foi maior para o grupo EE/LNG, enquanto a atividade do mesmo foi maior para o grupo E2/NOMAC quando comparadas ao grupo controle; 6) a concentração sérica de E-selectina foi maior para o grupo E2/NOMAC comparada ao grupo controle e 7) não houve diferenças para ICAM-1 e VCAM-1. E2/NOMAC teve efeito sobre maior número de parâmetros comparados ao grupo controle, significativos para a maior atividade do C1inh e maior concentração de E-selectina. Este é o primeiro resultado, ao nosso conhecimento, sobre a relação do NOMAC e E-selectina. Observaram-se tendências para redução da AH da VC em consonância com a redução do C4d. Esta observação deve ser destacada uma vez que E2+NOMAC e seus componentes individualizados promoveram redução significativa na AH in vitro do SHN. Juntos estes dados sugerem ao E2/NOMAC um efeito sobre a regulação negativa da atividade do SC, enquanto o aumento de E-selectina sugere um efeito pró-inflamatório. Quanto ao EE/LNG, a maioria dos parâmetros manteve-se sem diferenças em relação ao grupo controle. O impacto destas observações depende de aprofundamento na investigação dos efeitos dos COC sobre os sistemas biológicos e de estudos clínicos controlados, os quais juntos podem refletir benefícios em muitos aspectos para a saúde humana, em particular, a contracepção e terapias de reposição hormonal / Inflammation involves several biologically related and highly regulated biological mechanisms: coagulation, fibrinolysis, complement system (CS), antioxidation and hormonal regulation. Physiologically, CS and coagulation share regulatory components and various interactions between CS and hemostasis have been proposed but not elucidated. The scientific interest in CS interactions with hemostasis is increasing because of the risk of venous thromboembolism (VTE) associated with combined oral contraceptive (COC) use. Our group has investigated the effects of COC on CS, hemostasis and adhesion molecules. Results confirm the advantages of ethinylestradiol (EE)/levonorgestrel (LNG), a 2nd generation COC over the 3rd generation, maintaining it as a reference for comparison with new COC, which seek more natural components. Hormonal components in COC can influence the interactions between the endothelium and the CS, which mediates neutrophil adhesion to the endothelium and may favor VTE. Since the advent of COC, it has been consistently sought to minimize its undesirable effects by reducing dosages, different combinations of progestogens/estrogens and routes of administration. E2 or 17? estradiol is the natural estrogen and is combined with a new progestin, the nomegestrol acetate (NOMAC), which is more selective for the progesterone receptor, minimizing undesirable effects on metabolism. Considering the benefits of EE/LNG and the approval of E2/NOMAC, the objective of this study was to evaluate the effect of these COC on: 1) CS by measurement of activation of Classical Pathway (CP) and Lectin Pathway (LP), serum concentration of C4d fragment, serum concentration and activity of C1 inhibitor (C1inh) and 2) serum levels of soluble adhesion molecules: intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1 and E-selectin. The serum hemolytic activity (HA) of CP was assessed by kinetic assay in the COC and non-COC users and in normal human serum (NHS) groups exposed to COC components. Other parameters (LP, C4d, C1inh and soluble adhesion molecules) were evaluated by immunoenzymatic assays. The results were: 1) there was no difference in serum HA of CP; 2) except for EE + LNG, the HA of CP in vitro was lower when the pool of NHS was exposed to the individual components of the COC formulations (EE, LNG, E2 and NOMAC) and the E2+NOMAC combination compared to the control group (pool of NHS without hormonal components), lower for E2+NOMAC compared to EE+LNG and lower for NOMAC compared to LNG; 3) there was no difference in the activation of the LP; 4) there was no difference in the serum concentration of the C4d fragment; 5) the serum concentration of the C1inh was higher for EE/LNG group, whereas its activity was higher for E2/NOMAC group when compared to the control group; 6) the serum concentration of E-selectin was higher for E2/NOMAC group compared to the control group and 7) there was no difference for ICAM-1 and VCAM-1. E2/NOMAC had an effect on a higher number of parameters compared to control group, significant for the higher activity of C1inh and higher concentration of E-selectin. This is the first result, to our knowledge, about the association of NOMAC wiht E-selectin. Tendencies to reduce the HA of the CP were observed in consonance with the reduction of C4d. This observation should be highlighted since E2+NOMAC and its individualized components promoted a significant reduction in the HA in vitro of NHS. Together these data suggest to E2/NOMAC an effect on the down-regulation of CS activity, while the increase of soluble E-selectin suggests a pro-inflammatory effect. Regarding EE/LNG, most of the parameters have no difference compared to control group. The impact of these observations depends on further research into the effects of COC on biological systems and on controlled clinical studies, which together may reflect benefits in many respects to human health, in particular contraception and hormone replacement therapies

Monoclonal antibodies (mAbs) have become an important part of therapy for a number of cancers. The first mAb to be approved for clinical use is rituximab, which is currently used for the treatment of various B cell malignancies. Despite its clinical value, the mechanisms in which rituximab induces tumor regression are unclear. Growing evidence suggests that multiple mechanisms involving complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) are involved. However, the direct interactions between CDC and ADCC have yet to be investigated. My studies examine the relationship between complement fixation and the activation of NK cells by utilizing in vitro assays, a syngeneic murine lymphoma model, and clinical samples from patients. Using these systems, I demonstrate that the initiation of the complement cascade inhibits NK cell activation and ADCC induced by rituximab in vitro. I also show that depletion of complement enhances the activation of NK cells and improves the efficacy of mAb therapy in a murine model. Lastly, I demonstrate that NK cell activation correlates with decreased complement activity in patients after rituximab treatment. The studies described in this dissertation have furthered the understanding of the mechanisms involved in antibody therapy. These results have described a novel inhibitory role for complement activity in the anti-tumor responses of mAbs. Furthermore, these findings suggest that strategies to circumvent the inhibitory effect of complement may improve how current mAbs are used and the how mAbs are designed in the future.

The Major Histocompatibility Complex (MHC) is a gene-dense region located on the short arm of chromosome 6 (6p21.31). This region contains the highly polymorphic HLA genes as well as many other genes with immunological and non-immunological function. The susceptibility genes of many human disorders have been mapped to genes within the MHC. However, the genes themselves and indeed the locations of the genes, for many of the disorders, remain a mystery. This is a result of the high degree of linkage disequilibrium (LD) that exists between loci within the MHC. The high LD is explained by the genomic structure of the MHC. The MHC contains several blocks of DNA within which recombination is extremely rare, whereas the boundaries of the blocks are defined as "hotspots" of recombination. Most disease association studies have used the highly polymorphic HLA class I and class II genes which are separated by an, as yet, undefined number of blocks and several hundred kilobases of DNA. The MHC gamma block resides in the central region of the MHC between the blocks that contain the HLA class I and class It genes. As such, typing for polymorphisms in the gamma block is critical for MHC disease gene mapping studies. The gamma block contains approximately 20 known genes including the complement C4 genes. The gamma block can contain between I and 3 tandemly arranged C4 genes. The C4 protein exists as either the C4A or C413 isotype and is polymorphic with up to 40 allotypes being reported. However, the majority of Caucasian haplotypes can be explained by the common C4A3 / C4B1 or C4AQ0 / C4B1 complotypes with the remaining haplotypes explained by just a few other complotypes. For this reason, and because C4 allotyping is a technically difficult procedure, C4 allotyping is rarely used in MHC disease association studies. / The molecular heterogeneity of human C4 genes has not been extensively studied. However, the C4A3 and C4131 genes have been completely sequenced and are >99% identical at the DNA level across 41 exons and 15 kb of DNA. This high degree of homology and the presence of up to 3 C4 genes on any MHC haplotype makes PCR separation of the C4 genes difficult for subsequent genetic studies. The aim of this study was to extensively characterise the molecular heterogeneity of the human C4 genes and thereby: 1. determine the extent of human C4 gene polymorphism 2. confirm previous studies which have defined isotype specific sequences 3. characterise the C4 protein polymorphisms at the DNA level 4. determine if common C4 allotypes can be subtyped on a molecular basis 5. identify C4 gene polymorphisms that can be used as targets for DNA based typing methods 6. apply DNA based C4 typing methods in MHC disease association studies 7. provide insights into MHC haplotype evolution. In contrast to separating the C4 genes, a novel approach whereby the C4 genes were amplified and sequenced simultaneously was applied in this study. The DNA from 24 homozygous workshop cell lines, representing different ancestral haplotypes (AHs), was studied. Comparison of the C4 genes from different AHs revealed that the C4d region of the C4 a-chain is most polymorphic, but that polymorphic amino acid residues are also present in other regions of C4. The highest degree of polymorphisms was seen in the introns. In addition, the presence of the isotype specific sequences in exon 26 was confirmed and primers were designed to specifically amplify, and thereby separate, the C4A and C4B genes. / Comparison of the C4 gene sequences representing the same C4 allotype revealed that most C4 allotypes are heterogeneous and may be split into several subtypes. The polymorphisms observed at the sequence level did not correlate with C4 allotypes defined by electrophoretic mobility. However, it could be shown that the differences in electrophoretic mobility of the C4 allotypes are due to cumulative charge differences. Seven polymorphic amino acids were found to account for the different migration rates of the C4 allotypes analysed in this study. In addition, a number of haplospecific single nucleotide polymorphisms (SNPs) were identified within the C4 genes. Haplospecific SNPs are informative markers enabling the genetic mapping of recombinant AHs, an approach which can be used to identify disease susceptibility genes. Haplospecific SNPs located in the C4 gene region are important markers as they represent a separate block of the MIIC (i.e. the gamma block). The frequency of one such SNP marker has been shown for a diabetes patient group and a control population. Although further studies are required to elucidate the role of the gamma block genes in susceptibility to diabetes, this study demonstrates a possible approach for the mapping of MHC disease susceptibility genes, which can also be applied in studies of other MHC associated diseases. To conclude, the present study adds to our knowledge of the C4 gene polymorphism, provides insights into MHC and C4 gene evolution and enables future studies to examine the significance of the C4 genes and other gamma block genes in susceptibility to MHC associated diseases.

Atherosclerosis is a slow-developing disease of large and medium sized arteries, and is the premier cardiovascular disease that underlies myocardial and cerebral infarction, aneurysm, stroke and gangrene of the extremities. At least 17 million people die of atherosclerotic complications each year worldwide, with another 15 million surviving unstable events. Despite therapeutic advances such as drug-eluting stents and statins, which reduce cardiovascular events by around 25%, there is an urgent need for additional strategies to complement these treatments and further reduce morbidity and mortality. Inflammation plays a fundamental role in mediating all stages of atherogenesis. The innate immune response has long been implicated in atherogenesis, and activation of the complement system has been associated with all stages of disease. In particular, C5b-9 (membrane attack complex) has been detected in human plaques and may be pathogenic. Since C5b-9 is produced in plaques then the complement activation product 5a (C5a) must also be generated. However, very little is known about the role of C5a in atherogenesis. Indeed, elevated levels of serum C5a have been detected in patients with advanced atherosclerosis and recently the classical C5a receptor, CD88, has been detected on most of the cells found in human atherosclerotic plaques. To date, no studies examining specific C5a receptor antagonism in an animal model of atherosclerosis have been performed. This thesis explored the potential therapeutic benefits of inhibiting C5a, using the C5a receptor antagonist, PMX53, in the ApoE knockout (ApoE-/-) mouse model of atherosclerosis. In Chapter 2, expression of both receptors to C5a, CD88 and C5L2, in aortae of ApoE -/- mice was explored. CD88 and C5L2 mRNA expression was detected in the aorta of ApoE -/- mice at 3, 5, 9,12, 17 and 25 weeks of age. CD88 expression in ApoE -/- mice increased with time, and with macrophage accumulation within the plaque, as indicated by an increase in expression of the macrophage marker, F4/80. Expresssion of CD88 was significantly increased at 17 and 26 weeks of age, compared with age-matched wild-type controls. C5L2 was also expressed albeit at much lower levels compared with wild-type controls. Having established the presence of C5a receptors in ApoE -/- mice, in Chapter 3, the effects PMX53-treatment on ApoE -/- mice on a normal chow diet was examined. PMX53 treatment (3 mg/kg; tri-weekly s.c., plus ~1mg/kg/day p.o. for 20 weeks) resulted in a significant reduction in neointimal area and therefore the intima:media ratio in the brachiocephalic artery compared to untreated controls (P < 0.05; n = 6-8). PMX53 treatment also reduced collagen content and outward remodelling of the brachiocephalic artery. In Chapter 4, studies exploring the effects of PMX53-treatment in the more inflammatory environment created by a high fat (or Western-type diet) were explored. Male ApoE -/- mice were treated with PMX53 from 5 – 25 weeks of age (3 mg/kg; tri-weekly s.c., plus v ~1mg/kg/day p.o.). Mice were placed on a high fat diet from 10 weeks of age. While PMX53- treated did not affect neo-intimal area, it did result in a significant increase in cell density (P<0.01; n=12) and a significant reduction in buried caps (P < 0.05; n = 12) in the brachiocephalic artery compared with untreated animals. Interestingly, PMX53-treated mice also had significantly reduced total cholesterol compared with untreated controls (P < 0.05; n = 12). These results provide the first evidence for a role for C5a in plaque destabilisation and cholesterol metabolism. Finally, Chapter 5 described the expression of CD88 and C5L2 in cultured primary rat vascular SMC was explored. Expression of CD88 and C5L2 was detected by Western blot; immunocytochemical analysis demonstrated intracellular expression of both C5L2 and CD88. Conversely, radioligand binding experiments suggested the presence of ~25000 cell surface receptors with a high affinity to C5a (KD = 0.3 nM). After establishing the presence of receptors to C5a, experiments were conducted to determine whether C5a has any functional effects on these cells. C5a induced a moderate increase in TNF-α release after 4 hours of treatment (P < 0.05, n = 3), but did not affect SMC proliferation (n = 3). In summary, this study is the first to demonstrate the benefits of specifically inhibiting C5a in a mouse model of atherosclerosis. These findings suggest that C5a plays a role in atherogenesis in ApoE -/- mice and that the C5a receptor antagonist PMX53 may have therapeutic potential in human atherosclerotic disease.

<p>Today there are a vast number of medical devices in temporary or permanent contact with human tissues. Blood-biomaterial contact is known to trigger the complement system and results in generation of fluid phase anaphylatoxins C3a and C5a, and surface-bound C3b and iC3b. All these products together are able to attract and activate leukocytes and trigger release of inflammatory mediators leading to a systemic inflammation indirectly causing hemostatic problems and even organ failure. The aim of this study was to identify how complement is triggered on a biomaterial surface and to find ways to regulate this activation.</p><p>The finding that complement activation on biomaterials can be divided into initiation and amplification will facilitate regulation of complement activation biomaterial surfaces. This concept is also compatible with the two techniques to regulate complement activation on a surface.</p>