<p>This thesis describes a new <i>in vitro</i> slide chamber model that makes it possible to conduct studies of molecular and cellular interactions between whole blood and biomaterials. The model proved to be a suitable tool for detection of cell and platelet binding to a biomaterial surface. It was possible to monitor activation of the blood cascade systems and cells in the fluid phase and detect surface-bound molecules.</p><p>One finding was that thrombin generation is primarily triggered by FXII on a biomaterial surface since corn trypsin inhibitor, inhibited thrombin generation in blood.</p><p>Another finding was that thrombin generation was dependent on variety types of blood cells, since thrombin generation was almost negligible in platelet-rich plasma. When various preparations of blood cells were used to reconstitute platelet-rich and platelet-poor plasma, erythrocytes were shown to be the most efficient cell type in triggering thrombin generation. Inhibition of platelet aggregation with aspirin and Ro44-9883 was associated with a decrease in thrombin generation, confirming that platelet activation is necessary for normal coagulation activation. These findings suggest that the central events consist of an initial low-grade generation of thrombin that involves erythrocytes and possibly leukocytes which leads to activation of platelets; and a second platelet-dependent amplification loop that produces most of the thrombin.</p><p>Titanium exposed to whole blood produced high amounts of thrombin. Stainless steel and PVC, generated lower amounts. This indicates that titanium might be less suitable as a biomaterial in devices that are in direct contact with blood for prolonged time. Considering the superior osteointegrating properties of titanium and titanium's response to blood, a correlation between high thrombogenicity and good osteointegration seems to exist.</p><p>Compstatin, that binds to complement component C3, effectively inhibited the generation of C3a and sC5b-9 and the binding of C3/C3 fragments to the surface. Our results suggest that a biomaterial is able to activate complement through both the classical and alternative pathways and that the classical pathway alone is able to maintain a substantial bioincompatibility reaction. The results show that complement activation is a prerequisite for activation and binding of PMNs to the surface in the <i>in vitro</i> model.</p>

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

This thesis describes a new in vitro slide chamber model that makes it possible to conduct studies of molecular and cellular interactions between whole blood and biomaterials. The model proved to be a suitable tool for detection of cell and platelet binding to a biomaterial surface. It was possible to monitor activation of the blood cascade systems and cells in the fluid phase and detect surface-bound molecules. One finding was that thrombin generation is primarily triggered by FXII on a biomaterial surface since corn trypsin inhibitor, inhibited thrombin generation in blood. Another finding was that thrombin generation was dependent on variety types of blood cells, since thrombin generation was almost negligible in platelet-rich plasma. When various preparations of blood cells were used to reconstitute platelet-rich and platelet-poor plasma, erythrocytes were shown to be the most efficient cell type in triggering thrombin generation. Inhibition of platelet aggregation with aspirin and Ro44-9883 was associated with a decrease in thrombin generation, confirming that platelet activation is necessary for normal coagulation activation. These findings suggest that the central events consist of an initial low-grade generation of thrombin that involves erythrocytes and possibly leukocytes which leads to activation of platelets; and a second platelet-dependent amplification loop that produces most of the thrombin. Titanium exposed to whole blood produced high amounts of thrombin. Stainless steel and PVC, generated lower amounts. This indicates that titanium might be less suitable as a biomaterial in devices that are in direct contact with blood for prolonged time. Considering the superior osteointegrating properties of titanium and titanium's response to blood, a correlation between high thrombogenicity and good osteointegration seems to exist. Compstatin, that binds to complement component C3, effectively inhibited the generation of C3a and sC5b-9 and the binding of C3/C3 fragments to the surface. Our results suggest that a biomaterial is able to activate complement through both the classical and alternative pathways and that the classical pathway alone is able to maintain a substantial bioincompatibility reaction. The results show that complement activation is a prerequisite for activation and binding of PMNs to the surface in the in vitro model.

Induction of collagen-induced arthritis (CIA), an animal model for human rheumatoid arthritis, is dependent on anti-collagen type II (CII) antibodies. The effector mechanism by which autoantibodies contribute to inflammatory reactions in autoimmune diseases is not well understood. In this thesis I have studied the effector pathways used by IgG anti-CII antibodies to initiate arthritis, namely the IgG Fc receptors (FcγRs) and the complement system. We have found that FcγRIII is crucial for development of CIA, as CII-immunized mice lacking this receptor do not develop arthritis and IgG1 and IgG2b anti-CII antibodies require FcγRIII to trigger arthritis when transferred to naïve mice. The antibody-mediated arthritis was further enhanced in mice deficient in the inhibitory FcγRIIB, indicating that FcγRIIB regulates the activation of FcγRIII. Furthermore, we demonstrate that FcγRIII exist as three distinct haplotypes in mice, FcγRIII:H, FcγRIII:V and FcγRIII:T. Mice expressing the FcγRIII:H haplotype are more susceptible to CIA than mice expressing the FcγRIII:V haplotype, indicating that certain FcγRIII haplotype predisposes for CIA. We also show that the most likely FcγRIII-expressing effector cell in CIA is the macrophage, since FcγRIII-expressing macrophages exclusively can induce arthritis in FcγRIII-deficient mice challenged for CIA. The complement system was also investigated in development of CIA. We found that this effector pathway is also necessary for onset of arthritis, as CIA was inhibited by treatment with anti-complement factor 5 (C5) antibodies. C5-deficient mice could neither develop CIA unless provided with C5-containing sera. Taken together, the work presented in this thesis indicates that FcγRs and the complement system are crucial for the induction of experimental arthritis. These findings are important for understanding the mechanisms behind rheumatoid arthritis and blocking of these effector pathways may in the future be used as treatment of rheumatoid arthritis.

Haptoglobin (Hp) is a serum protein known for its ability to form a tight complex with hemoglobin (Hb) and thereby inhibiting the oxidative activity of Hb. Mammalian Hp is synthesized as a precursor (proHp) that undergoes proteolytic cleavage by a previously unidentified enzyme in the endoplasmic reticulum (ER). In this study, a proHp-cleaving enzyme was isolated from human serum and identified as complement C1r-like protein (C1rLP). Co-expression of C1rLP with proHp in mammalian cells resulted in cleavage of the latter protein in the ER. Mutation of either the active site serine residue in C1rLP or the arginine residue in the cleavage site of Hp abolished the cleavage of proHp by C1rLP. RNAi studies in mammalian cells identified the proHp-cleaving enzyme as C1rLP. Hp has been found in all mammals studied to date but its presence in non-mammalian species has not been unambiguously shown. By searching currently available genomic DNA and cDNA sequence databases, a gene orthologous to mammalian Hp was found in bony fish. Hp-like protein expressed from this gene was demonstrated to be a major Hb protein in fish serum. Surprisingly, no Hp-like gene was found in the genomes of either frog or chicken. In chicken, a protein previously described as Hp was identified as PIT54, a member of a scavenger receptor cysteine-rich family of proteins. Interestingly, ostrich serum seemed to contain two Hb-binding proteins; one similar to PIT54 and one to mammalian Hp. We are not aware of any other case where the function of one gene has been taken over by another, completely unrelated gene Fish Hp (fHp) is composed of a serine proteinase-related domain preceded by an extension consisting of several aminoa acids and a signal peptide. The extension contains a consensus motif for cleavage by subtilisin-like proprotein convertases (SPCs). fHp was found to be cleaved by SPCs in the Golgi complex. Collectively, this thesis presents evidence that Hp has undergone significant changes during evolution with respect to its molecular organization and to the mechanism of its proteolytic cleavage.

FibMat2.0 is a fibrinogen multilayer developed by AddBIO. Other proteins such as immunoglobulin G (IgG) and human serum albumin (HSA) can also be used to build multilayers with the same technique. The aim of this study of FibMat2.0 was to investigate if the manufacturing of the protein multilayer would induce an immune or haemostatic response in the body. The multilayers of IgG and HSA were also studied. Methods such as null ellipsometry, imaging of coagulation and the cone-and-plate setup were used to study immune reactions, activation of the coagulation cascade, and stability of the multilayers. Small amounts of plasma proteins were adsorbed to fibrinogen multilayers, but complement proteins adsorbed only to the IgG matrix and high molecular weight kininogen (HMWK) adsorbed only to the HSA monolayer. The imaging of coagulation method indicated that the titanium surface and the HSA monolayer activate surface induced coagulation rapidly, whereas fibrinogen and IgG multilayers demonstrated longer coagulation times. Platelets and a few white blood cells were bound to titanium surfaces and fibrinogen multilayers, but not to IgG multilayers or HSA monolayers. A conclusion in this study is that the surface of an implant can be coated with FibMat2.0 without any risks, but more studies are needed to better understand the interactions between the surfaces prepared in the present study and the immune and the haemostatic systems of the human body.

B cells are normally regulated to prevent activation against self-proteins through tolerance mechanisms.  However, occasionally there is a break in tolerance and B cells can become self-reactive, which might lead to the development of autoimmune disease. The activation of self-reactive B cells is regulated by receptors on the B cell surface, such as Fc gamma receptor IIb (FcγRIIb), complement receptor type 1 (CR1), and CR type 2 (CR2). In this thesis I have studied the role of FcγRIIb, CR1 and CR2 on B cells in autoimmune arthritis. By using a model for rheumatoid arthritis, I discovered that the initial self-reactive B cell response in arthritis was associated with the splenic marginal zone B cell population. Marginal zone B cells express high levels of CR1/CR2 and FcγRIIb, suggesting that they normally require high regulation. Further, female mice deficient in CR1/CR2 displayed increased susceptibility to arthritis compared to CR1/CR2-sufficient female mice. When investigating whether sex hormones affected arthritis susceptibility, we found that ovariectomy, of the otherwise fairly resistant CR1/CR2-sufficient mice, reduced the expression of CR1 on B cells and rendered the mice more susceptible to arthritis. In humans, a significantly reduced CR1 and FcγRIIb expression was found on B cells in aging women, but not in men. This may contribute to the increased risk for women to develop autoimmune disease as reduced receptor expression may lead to the activation of self-reactive B cells. In agreement, lower CR1, CR2 and FcγRIIb expression was seen in patients with rheumatoid arthritis.   Finally, a soluble form of FcγRIIb was used to investigate FcγRIIb’s ability to bind self-reactive IgG in an attempt to treat autoimmune arthritis. Treatment of mice with established arthritis was associated with less self-reactive IgG antibodies and consequently less disease, suggesting that soluble FcγRIIb may be used as a novel treatment in arthritis.

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

Se estudió la incidencia de déficits del sistema complemento en pacientes con enfermedad meningocócica, detectándose 4 pacientes con déficit completo en la actividad hemolítica mediada por el complemento (ensayo de CH50). Tres de ellos tenían un déficit completo del factor C8, dos de los cuales pertenecían a la misma familia, y el cuarto paciente presentaba un déficit parcial de C3, secundario a la presencia de un factor activador de la vía alterna del complemento, conocido con factor nefrítico, o C3-nef. En los casos con déficit de C8 se llevó a cabo un estudio familiar, detectándose otro individuo más con déficit de C( y nueve sujetos con déficit parcial (inviduos heterocigotos). Así mismo se analizaron los niveles de complejos inmunes circulantes, mediante dos tecnicas diferentes: la fijación a C1q y la unión a células Raji. Los resultados no muestran asociación entre los niveles de CIC y la severidad del cuadro clínico. / We analyzed the incidence of complement deficiencies in patients with acute meningocococcal disease. We detected four patients with recurrent meningococcal infections having a complete lack of serum haemolitical activity (CH50 assay). Three of then had a complete absence of CD8 in serum and the four patient had an aberrant activation of the alternative complement pathway due to the presence of C3-nef. Familial studies were done in the cases of C8 deficiencies, in which a fourth member was identified, together with nine individuals with a partial (heterocygotic) C8 deficiency. Finally we evaluated the levels of circulating immune complexes (CIC) by binding to C1q or to Raji cells. Our results showed a lack of association between the severity of the disease and the levels of CIC.

Every language have some modal expression what express the possibility and necessity, for example there are modal verbs and modal adverbs in English, and modal verbs, modal adverbs and possible complement for Mandarin Chinese. However in Mandarin Chinese, some modal expressions have similar semantic functions, as ¡§¯à(neng)¡¨, ¡§·|(hui)¡¨, ¡§¥i¥H(keyi)¡¨and possible complement, it could cause confusions to the learners. Furthermore, all of these modal expressions could translated as ¡§할 수 있다¡]hal su itda¡^¡¨ in Korean, so Korean learner would feel that is very difficult. Therefore, this study will contrast the modal expressions of Mandarin Chinese and Korean, and contrast novel¡m¶É«°¤§ÅÊ(Love of the beautiful women)¡nin Mandarin Chinese and Korean, try to explain the problems what the Korean learners could have when they acquire the modal expressions in Mandarin Chinese. Palmer¡]1986¡^classify the modality as epistemic modality, deontic modality and dynamic modality. And he classify the deontic modality as permission, obligation and promise, classify the dynamic modality as volition and ability. Hofmann(1993) classify the dynamic modality as ability and capability, and Porter(2009) classify as ability and opportunity. Present paper would accept the classification of Palmer mainly and refer to the classification of Hofmann and Portner, classify the modality as epistemic modality, deontic modality and dynamic modality, the deontic modality include obligation and permission, and the dynamic modality include volition, ability, capability and opportunity. The result of the study, the counterparts of modal expressions in Mandarin Chinese and Korean is very complicated. The ¡§¯à(neng)¡¨, ¡§·|(hui)¡¨, ¡§¥i¥H(keyi)¡¨and possible complement are equivalent to ¡§¡]으¡^ㄹ 수 있다¡]eul su itda¡^¡¨ altogether, but only negation of ¡§¯à(neng)¡¨, ¡§¥i¥H(keyi)¡¨and possible complement are equivalent to the negation of ¡§¡]으¡^ㄹ 수 있다¡]eul su itda¡^¡¨ , it will cause the difficulty of Korean learners.