Biophysical characterisation of protein – ligand interactions can provide vital information to dissect complex biochemical binding mechanisms. C3d has been shown to interact with a number of different protein ligands, namely CD21 SCR1-2, S. aureus Efb-C, S. aureus Ehp, S. aureus Sbi and complement regulatory protein factor H. Although much is known about the relationship of C3d and CD21 in the induction of humoral immunity, the structural aspects of this interaction remained controversial until very recently. The aim of this thesis was to gain a detailed understanding of the C3d/CD21 SCR1-2 interaction using different biophysical methods and to identify potential low molecular weight inhibitors of the interaction. A crystal structure of the C3d/CD21 complex solved by Szakonyi et al. in 2001 indicated the C3d binding site on CD21 was in the SCR2 domain. It did not agree with mutagenesis studies and recent NMR titration experiments show that only residues from the SCR1 domain of CD21 appear to mediate binding under physiologically relevant ionic strength. In the current work, NMR was employed to monitor ligand binding to C3d and to provide residue specific information that reflects a physiologically relevant binding mode to complement the crystallographic model solved by van den Elsen and Isenman in 2011. Microcalorimetric analysis on the site-directed mutagenesis studies also supported a model of hydrophobically- and electrostatically-driven protein-protein interaction for C3d and CD21 SCR1-2. Complement C3d forms a non-specific thioester linkage with antigen, which then binds to CD21 SCR1-2 and coligates with membrane immunoglobulin of the B cell receptor. While the interactions triggers B cell activation and hence the production of antibody under normal circumstances, it has been demonstrated that the interactions also lead to undue B cell activation and auto-antibody production. There is a well established collagen-induced arthritis (CIA) mouse model to support the significance of C3d and CD21 in disease susceptibility. To this end, a high-throughput SPR-based screening platform was set up to screen a fragment library against C3d, so as to identify low molecular weight compounds that could serve as a starting point for drug development programme. Unfortunately, the work did not yield C3d-binding inhibitors and future work could include screening large fragment libraries that are designed to target protein-protein interfaces.

by Wang Lidi. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 125-127). / Acknowledgement --- p.iii / Abstract --- p.iv / Introduction --- p.1 / Chapter Chapter I --- " In Search of a Definition for ""Word""" --- p.6 / Chapter 1.1 --- Productivity --- p.6 / Chapter 1.2 --- Headedness --- p.9 / Chapter 1.3 --- Constraints on Word Structure --- p.26 / Chapter 1.4 --- A-N Constructions in Chinese: --- p.30 / Chapter 1.5 --- Anaphoric Island --- p.38 / Chapter Chapter II --- Morphological Properties of V-C Compounds --- p.46 / Chapter 2.1 --- VOCs ´ؤ A Misnomer --- p.46 / Chapter 2.2 --- Morphological Properties of V-C Compounds --- p.51 / Chapter 2.3 --- The Typology of V-C Constructions --- p.62 / Chapter Chapter III --- V-C Compounds as Noun Incorporation --- p.70 / Chapter 3.1 --- Syntactic and Lexical Derivations in Grammar --- p.72 / Chapter 3.2 --- Two Types of Causative Constructions --- p.74 / Chapter 3.3 --- V-C Compounds as Noun Incorporation --- p.78 / Chapter Chapter IV --- V-C Compound and Event Quantifier --- p.86 / Chapter 4.1 --- The Gerundive Derivation --- p.89 / Chapter 4.2 --- Position of the Event Quantifier --- p.92 / Chapter 4.3 --- V-C Compound and Event Quantifier --- p.97 / Chapter 4.4 --- The Double-object Construction --- p.107 / Chapter 4.5 --- Verb Reduplication --- p.115 / Conclusion --- p.119 / Notes --- p.122 / References --- p.125

Malaria has been a major driving force in the evolution of the human genome. In sub-Saharan African populations, two neighbouring polymorphisms in the Complement Receptor 1 (CR1) gene, named Swain-Langley (Sl2) and McCoy (McCb), occur at high frequencies, consistent with selection by malaria. This thesis investigates the association between these two polymorphisms and severe malaria. Previous studies into this area have produced conflicting findings. Using a large case-control study of severe malaria in Kenyan children and statistical models adjusted for confounders, I found that the Sl2 polymorphism was associated with markedly reduced odds of cerebral malaria and death, while the McCb polymorphism was associated with increased odds of cerebral malaria. I also identified an interaction between Sl2 and α+thalassaemia, with the protective association of Sl2 greatest in children with normal α-globin. Following these epidemiological findings, I explored potential biological hypotheses which might explain them. The first approach examined whether the Sl2 and McCb polymorphisms affected how CR1 forms clusters on erythrocyte membranes, a process which is key in the binding and transfer of immune complexes from erythrocytes to macrophages. Using erythrocytes from Kenyan children, I performed immunofluorescence assays (IFAs) with confocal microscopy to quantify CR1 cluster number and volume. I found no association between the Sl2 and McCb polymorphisms and either the number or volume of CR1 clusters formed. The second approach investigated whether the cerebral malaria-specific associations seen with Sl2 and McCb might be due to expression of CR1 by human brain endothelial cells (HBEC). The immortalised cell line HBEC-5i was investigated for expression of CR1 using IFA, flow cytometry, western blotting, functional C3b degradation assays, mass spectrometry, immunoprecipitation and siRNA knockdown experiments. A pool of α-CR1 monoclonal antibodies recognised an intracellular antigen in permeabilised HBEC-5i cells which was a similar molecular weight to CR1 on western blotting. However, when the α-CR1 monoclonal antibodies were tested individually, only E11 recognised an HBEC-5i antigen. Further investigative approaches did not support the presence of CR1 on HBEC-5i cells, instead suggesting that E11 was not specific for CR1 and was instead recognising a protein in the Golgi apparatus. The final approach was to examine whether the Sl2 and McCb polymorphisms might influence the binding of the complement components mannose binding lectin, C1q and L-ficolin to the LHR-D region of CR1. I aimed to generate recombinant proteins of the LHR-D region which included the polymorphisms. Site-directed mutagenesis of the region was successful and subcloning and expression of the mutant amplicons will be performed at a later date. In summary, I have identified opposing associations between the Sl2 and McCb polymorphisms and cerebral malaria, which do not appear to be due to differences in CR1 clustering or expression of CR1 by human brain endothelial cells. My investigation into whether the polymorphisms might influence complement component binding is ongoing.

Uma criança brasileira (LAS) vítima de infecções recidivantes e vasculite, cujos pais são consangüíneos em segundo grau apresentou 0,15 µg/mL de C3 plasmático e atividades hemolíticas nulas pelas vias clássica e alternativa, já outras proteínas do complemento e Igs estavam normais (exceto IgG4, que foi indetectável). Diferentemente de sua mãe os fibroblastos da criança não foram capazes de sintetizar as cadeias a e b de C3, como observado por SDS-PAGE. O probando possui dois alelos C3S, assim como seu irmão mais novo e saudável, enquanto a mãe é FS. A migração de leucócitos, em resposta ao soro do probando ativado com LPS foi menor que a obtida com soro normal e estatisticamente semelhante àquela gerada por SHN inativado a 56oC (SHNi). A ingestão e a morte de C. albicans, opsonizadas por soro do probando, por fagócitos normais foram semelhantes às dos fungos opsonizados por SHNi. Nós concluimos que, em conseqüência da incapacidade de sintetizar C3, o probando não é capaz de exercer as funções imunológicas dependentes do complemento, resultando em uma maior susceptibilidade a infecções. / A brasilian child (LAS) victim of recurrent infections whose parents have second degree consanguinity presented 0.15 µg/mL of serum C3 and no hemolytic activities either after activation of the classical or alternative pathways. His mother presented C3 alpha and beta chains of normal sizes, while LAS's fibroblasts did not secrete any C3 as observed by SDS-PAGE. The proband possesses two C3S alleles, like his younger and healthy brother whereas his mother is FS. Leukocyte migration across nitrocellulose membrane in response to the proband's LPS-activated serum was less intense than that obtained in response to normal serum. Phagocytosis and killing of C. albicans opsonized with the proband's serum was comparable to fungi opsonized with inactivated serum, incdicating that chemotactic and opsonic activities of the proband's serum are greatly diminished. We colclude that as a consequence of C3 deficiency the proband's complement system is uncapable of performing it's normal effector functions resulting in greater susceptibility to infections.

Francisella tularensis is a facultative intracellular bacterium and the causative agent of tularemia, a severe and potentially fatal disease in humans. This pathogen is extremely infectious by the aerosol route and inhalation of as few as 10 organisms can cause severe pneumonic disease. Consequently, F. tularensis was developed as a bioweapon by several nations and is considered a category A select agent by the Centers for Disease Control and Prevention. The ability of F. tularensis to cause overwhelming infections at low infectious doses suggests this organism has adapted efficient mechanisms to evade containment by the host innate immune system. The goal of this thesis was to better understand the mechanisms by which Francisella modulates innate host defenses, with particular focus on interactions between this pathogen and two important effectors of innate immunity: neutrophils and the complement system. We demonstrate that F. tularensis profoundly modulates neutrophil lifespan during infection, delaying spontaneous apoptosis by inhibiting both the intrinsic and extrinsic apoptotic pathways to maintain an intracellular niche for persistence and proliferation. Furthermore, we show that F. tularensis can override activation of the apoptotic program induced by extracellular apoptotic signals that may drive neutrophil apoptosis at the site of infection. Initial characterization of the molecular mechanisms behind apoptosis inhibition by this pathogen suggests that F. tularensis employs multiple, redundant mechanisms to promote global anti-apoptosis in the cell. Transcriptome analyses of infected PMNs using oligonucleotide microarrays show that 365 unique apoptosis and cell survival genes are differentially regulated between 3-24 hr, several of which directly modulate intrinsic and extrinsic pathway signaling. Moreover, we demonstrate that levels of the potent caspase inhibitor, X-linked inhibitor of apoptosis protein (XIAP), are maintained over the course of infection, which may represent an important mechanism of caspase inhibition by this pathogen. We also confirm reports that F. tularensis can activate complement during incubation in nonimmune serum, and demonstrate for the first time that natural IgM antibodies bind to the bacterial surface and mediate complement opsonization to promote phagocytosis by both human neutrophils and macrophages. Finally, we identify the first neutrophil receptors, CR1 and CR3, involved in the uptake of complement-opsonized F. tularensis. In sum, our data presented here significantly advance our understanding of the host-pathogen relationship between F. tularensis and components of innate immunity, and suggest that this pathogen modulates both neutrophil and complement function to evade innate immune defenses and cause disease.

Demyelination in the CNS is known to involve several immune effector mechanisms, including complement proteins. For this dissertation project the central hypothesis that C3 and downstream effector complement proteins exacerbate demyelination through activation of glial cells was tested. To investigate the role of C3 and downstream complement proteins in demyelination and remyelination pathology in vivo we utilized the cuprizone model. We used C3 knockout mice (C3-/-), which are lacking the central C3 protein and subsequently all downstream complement effector proteins, and transgenic mice expressing C3a or C5a under the control of the glial GFAP promoter. Interestingly, we found no changes in demyelination or remyelination pathology between C3-/- and control mice. However, C3a and C5a transgenic mice had exacerbated demyelination and slightly delayed remyelination in the corpus callosum compared to WT mice. Transgenic mice had increased cellularity in the corpus callosum due to increased activation and/or migration of microglia. There was also evidence of T cells in the corpus callosum during demyelination in C5a transgenic mice, suggesting C5a may modulate BBB permeability. During early remyelination oligodendrocytes migrated to the corpus callosum in higher numbers in C3a and C5a transgenic mice, thus enabling these mice to remyelinate as effectively as WT mice by the end of the ten week study. To determine the effects of anaphylatoxins on individual glial subsets, we created murine recombinant C3a and C5a proteins. We found that the MAPK pathway proteins JNK1 and ERK1/2 were activated in glia upon stimulation with recombinant anaphylatoxin proteins. When microglia and mixed glial cultures were stimulated with C3a and/or C5a, we observed an increase in the production of proinflammatory cytokines and chemokines. In contrast, anaphylatoxin-treated primary astrocytes had suppressed cytokine and chemokine production compared to untreated astrocytes. In vitro, primary microglia and astrocytes did not significantly migrate in response to stimulation with C3a or C5a proteins, suggesting migration may not be a primary anaphylatoxin-mediated function in the CNS. Overall, our findings show that anaphylatoxin production in the brain plays a negative proinflammatory role during demyelination and that anaphylatoxin proteins can activate individual subsets of glia, initiating the production of inflammatory mediators.

Atypical hemolytic uremic syndrome (aHUS) is characterized by acute renal failure, thrombocytopenia, and microangiopathic hemolytic anemia. aHUS is far less common and more severe than typical HUS, which is caused by E. coli infection and manifests as diarrheal illness. The pathogenesis of the disease is linked to dysregulation of the alternative pathway of the complement cascade. Mutations in the complement regulators factor H (CFH), membrane cofactor protein (MCP), factor B (CFB), and factor I (CFI) have been implicated in aHUS. These loss or gain of function mutations lead to uncontrolled complement activity and immune-mediated host cell damage. Establishing a genetic etiology is important as it helps to direct treatment during the acute phase of disease and when transplantation is considered. It has been shown in previous studies that the age of onset as well the severity of the disease is correlated with the type of mutation a patient is found to carry. In forty percent of aHUS patients a mutation in CFH, MCP, CFB, CFI, C3 or THBD is not detected. These data strongly suggest that other genetic factors are involved in the pathogenesis of aHUS and that comprehensive mutation detection in aHUS patients is essential to provide diagnostic and prognostic information, and improve their clinical care. My thesis work has aimed to identify the other genetic contributors to this disease. To achieve this goal we began by screening the largest American cohort of aHUS patients for mutations in CFH, MCP, CFB, CFI, C3, THBD as well as CFHR5. This study identified over thirty novel mutations and suggests a more comprehensive genetic screening method that would better serve patients. To complement these studies multiplex ligation-dependent probe amplification was used to detect genetic rearrangements within the factor H related genes. A number of unique fusion proteins were seen in aHUS patients, all of which are predicted to affect the function of CFH. To discover mutations in novel genes that are causally related to aHUS, we have optimized a platform called CASCADE (Capture and Sequencing of Complement-Associated Disease Exons), which is based on targeted-genome capture and next-generation sequencing. This study revealed an unexpected role for ADAMTS13 and other genes in the coagulation pathway as modifiers of aHUS. Using functional assays we show two of the ADAMTS13 variants alter the behavior of this protein. This work has changed how we view this disease by identifying several novel candidate genes, for which we hope future analysis will lead to a better understanding of their role in aHUS. Using this knowledge we can provide better and more personalized treatments for patients.

The complement system is a principal component of innate immunity. Recent studies have underscored the importance of C5a and other complement components in inflammatory and neuropathic pain, although the underlying mechanisms are largely unknown. In particular, it is unclear how the complement system communicates with nociceptors and which ion channels and receptors are involved. Here we demonstrate that inflammatory thermal and mechanical hyperalgesia induced by complete Freund’s adjuvant were accompanied by C5a upregulation and were markedly reduced by C5a receptor (C5aR1) knockout (KO) or treatment with the C5aR1 antagonist PMX53. Direct administration of C5a into the mouse hindpaw produced strong thermal and mechanical hyperalgesia, an effect that was absent in TRPV1 KO mice, and was blocked by the TRPV1 antagonist AMG9810. Immunohistochemistry of mouse plantar skin showed prominent expression of C5aR1 in macrophages. Additionally, C5a evoked strong Ca2+ mobilization in macrophages. Macrophage depletion in transgenic macrophage Fas-induced apoptosis (MAFIA) mice abolished C5a-dependent thermal and mechanical hyperalgesia. Examination of inflammatory mediators following C5a injection revealed a rapid upregulation of nerve growth factor (NGF), a mediator known to sensitize TRPV1. Pre-injection of an NGF-neutralizing antibody or Trk inhibitor GNF-5837 prevented C5a-induced thermal hyperalgesia. Notably, NGF-induced thermal hyperalgesia was unaffected by macrophage depletion. Collectively, these results suggest that C5a induces thermal and mechanical hyperalgesia by triggering macrophage-dependent signaling that involves mobilization of NGF and NGF-dependent sensitization of TRPV1. Our findings highlight the importance of macrophage-to-neuron signaling in pain processing and identify C5a, NGF and TRPV1 as key players in this cross-cellular communication.

published_or_final_version / Linguistics / Master / Master of Philosophy

No description available.