Immune mechanisms in atherosclerosis; Focus on the role of the complement system

Atherosclerosis is an inflammatory disease characterized by accumulation of lipids in the inner layer of the arterial wall. During atherogenesis, various structures that are recognized as non-self by the immune system, such as modified lipoproteins, are deposited in the arterial wall. Accordingly, atherosclerotic lesions and blood of humans and animals with atherosclerotic lesions show signs of activation of both innate and adaptive immune responses. Although immune attack is initially a self-protective reaction, which is meant to destroy or remove harmful agents, a chronic inflammatory state in the arterial wall accelerates atherosclerosis. Indeed, various modulations of the immune system of atherosclerosis-prone animals have provided us with convincing evidence that immunological mechanisms play an important role in the pathogenesis of atherosclerosis. This thesis focuses on the role of complement system, a player of the innate immunity, in atherosclerosis. Complement activation via any of the three different pathways (classical, alternative, lectin) proceeds as a self-amplifying cascade, which leads to the generation of opsonins, anaphylatoxins C3a and C5a, and terminal membrane-attack complex (MAC, C5b-9), all of which regulate the inflammatory response and act in concert to destroy their target structures. To prevent uncontrolled complement activation or its attack against normal host cells, complement needs to be under strict control by regulatory proteins. The complement system has been shown to be activated in atherosclerotic lesions, modified lipoproteins and immune complexes containing oxLDL, for instance, being its activators. First, we investigated the presence and role of complement regulators in human atherosclerotic lesions. We found that inhibitors of the classical and alternative pathways, C4b-binding protein and factor H, respectively, were present in atherosclerotic lesions, where they localized in the superficial proteoglycan-rich layer. In addition, both inhibitors were found to bind to arterial proteoglycans in vitro. Immunohistochemical stainings revealed that, in the superficial layer of the intima, complement activation had been limited to the C3 level, whereas in the deeper intimal layers, complement activation had proceeded to the terminal C5b-9 level. We were also able to show that arterial proteoglycans inhibit complement activation in vitro. These findings suggested to us that the proteoglycan-rich layer of the arterial intima contains matrix-bound complement inhibitors and forms a protective zone, in which complement activation is restricted to the C3 level. Thus, complement activation is regulated in atherosclerotic lesions, and the extracellular matrix is involved in this process. Next, we studied whether the receptors for the two complement derived effectors, anaphylatoxins C3a and C5a, are expressed in human coronary atherosclerotic lesions. Our results of immunohistochemistry and RT-PCR analysis showed that, in contrast to normal intima, C3aR and C5aR were highly expressed in atherosclerotic lesions. In atherosclerotic plaques, the principal cells expressing both C3aR and C5aR were macrophages. Moreover, T cells expressed C5aR, and a small fraction of them also expressed C3aR, mast cells expressed C5aR, whereas endothelial cells and subendothelial smooth muscle cells expressed both C3aR and C5aR. These results suggested that intimal cells can respond to and become activated by complement-derived anaphylatoxins. Finally, we wanted to learn, whether oxLDL-IgG immune complexes, activators of the classical complement pathway, could have direct cellular effects in atherogenesis. Thus, we tested whether oxLDL-IgG immune complexes affect the survival of human monocytes, the precursors of macrophages, which are the most abundant inflammatory cell type in atherosclerotic lesions. We found that OxLDL-IgG immune complexes, in addition to transforming monocytes into foam cells, promoted their survival by decreasing their spontaneous apoptosis. This effect was mediated by cross-linking Fc receptors with ensuing activation of Akt-dependent survival signaling. Our finding revealed a novel mechanism by which oxLDL-IgG immune complexes can directly affect the accumulation of monocyte-macrophages in human atherosclerotic lesions and thus play a role in atherogenesis.

Innate immune system and adiponectin in diabetic nephropathy in type 1 diabetes

Introduction: The pathogenesis of diabetic nephropathy remains a matter of debate, although strong evidence suggests that it results from the interaction between susceptibility genes and the diabetic milieu. The true pathogenetic mechanism remains unknown, but a common denominator of micro- and macrovascular complications may exist. Some have suggested that low-grade inflammation and activation of the innate immune system might play a synergistic role in the pathogenesis of diabetic nephropathy. Aims of the study: The present studies were undertaken to investigate whether low-grade inflammation, mannan-binding lectin (MBL) and α-defensin play a role, together with adiponectin, in patients with type 1 diabetes and diabetic nephropathy. Subjects and methods: This study is part of the ongoing Finnish Diabetic Nephropathy Study (FinnDiane). The first four cross-sectional substudies of this thesis comprised 194 patients with type 1 diabetes divided into three groups (normo-, micro-, and macroalbuminuria) according to their albumin excretion rate (AER). The fifth substudy aimed to determine whether baseline serum adiponectin plays a role in the development and progression of diabetic nephropathy. This follow-up study included 1330 patients with type 1 diabetes and a mean follow-up period of five years. The patients were divided into three groups depending on their AER at baseline. As a measure of low-grade inflammation, highly sensitive CRP (hsCRP) and α-defensin were measured with radio-immunoassay, and interleukin-6 (IL-6) with high- sensitivity enzyme immuno-assay. Mannan-binding lectin and adiponectin were determined with time-resolved immunofluorometric assays. The progression of albuminuria from one stage to the other served as a measure of the progression of diabetic nephropathy. Results: Low-grade inflammatory markers, MBL, adiponectin, and α-defensin were all associated with diabetic nephropathy, whereas MBL, adiponectin, and α-defensin per se were unassociated with low-grade inflammatory markers. AER was the only clinical variable independently associated with hsCRP. AER, HDL-cholesterol and the duration of diabetes were independently associated with IL-6. HbA1c was the only variable independently associated with MBL. The estimated glomerular filtration rate (eGFR), AER, and waist-to-hip ratio were independently associated with adiponectin. Systolic blood pressure, HDL-cholesterol, total cholesterol, age, and eGFR were all independently associated with α-defensin. In patients with macroalbuminuria, progression to end-stage renal disease (ESRD) was associated with higher baseline adiponectin concentrations. Discussion and conclusions: Low-grade inflammation, MBL, adiponectin, and defensin were all associated with diabetic nephropathy in these cross-sectional studies. In contrast however, MBL, adiponectin, and defensin were not associated with low-grade inflammatory markers per se. Nor was defensin associated with MBL, which may suggest that these different players function in a coordinated fashion during the deleterious process of diabetic nephropathy. The question of what causes low-grade inflammation in patients with type 1 diabetes and diabetic nephropathy, however, remains unanswered. We could observe in our study that glycemic control, an atherosclerotic lipid profile, and waist-to-hip ratio (WHR) were associated with low-grade inflammation in the univariate analysis, although in the multivariate analysis, only AER, HDL-cholesterol, and the duration of diabetes, as a measure of glycemic load, proved to be independently associated with inflammation. Notably, all these factors are modifiable with changes in lifestyle and/or with a targeted medication. In the follow-up study, elevated serum adiponectin levels at baseline predicted the progression from macroalbuminuria to ESRD independently of renal function at baseline. This observation does not preclude adiponectin as a favorable factor during the process of diabetic nephropathy, since the rise in serum adiponectin concentrations may remain a mechanism by which the body compensates for the demands created by the diabetic milieu.

The Protection of Traditional Knowledge Associated with Genetic Resources: The Role of Databases and Registers

"The Protection of Traditional Knowledge Associated with Genetic Resources: The Role of Databases and Registers" ABSTRACT Yovana Reyes Tagle The misappropriation of TK has sparked a search for national and international laws to govern the use of indigenous peoples knowledge and protection against its commercial exploitation. There is a widespread perception that biopiracy or illegal access to genetic resources and associated traditional knowledge (TK) continues despite national and regional efforts to address this concern. The purpose of this research is to address the question of how documentation of TK through databases and registers could protect TK, in light of indigenous peoples increasing demands to control their knowledge and benefit from its use. Throughout the international debate over the protection of TK, various options have been brought up and discussed. At its core, the discussion over the legal protection of TK comes down to these issues: 1) The doctrinal question: What is protection of TK? 2) The methodological question: How can protection of TK be achieved? 3) The legal question: What should be protected? And 4) The policy questions: Who has rights and how should they be implemented? What kind of rights should indigenous peoples have over their TK? What are the central concerns the TK databases want to solve? The acceptance of TK databases and registers may bring with it both opportunities and dangers. How can the rights of indigenous peoples over their documented knowledge be assured? Documentation of TK was envisaged as a means to protect TK, but there are concerns about how documented TK can be protected from misappropriation. The methodology used in this research seeks to contribute to the understanding of the protection of TK. The steps taken in this research attempt to describe and to explain a) what has been done to protect TK through databases and registers, b) how this protection is taking place, and c) why the establishment of TK databases can or cannot be useful for the protection of TK. The selected case studies (Peru and Venezuela) seek to illustrate the complexity and multidisciplinary nature of the establishment of TK databases, which entail not only legal but also political, socio-economic and cultural issues. The study offers some conclusions and recommendations that have emerged after reviewing the national experiences, international instruments, work of international organizations, and indigenous peoples perspectives. This thesis concludes that if TK is to be protected from disclosure and unauthorized use, confidential databases are required. Finally, the TK database strategy needs to be strengthened by the legal protection of the TK itself.

When sharing becomes a liability: An intellectual capital approach to describing the dichotomy of knowledge protection versus sharing in intra- and interorganizational relationships

The tension created when companies are collaborating with competitors – sometimes termed co-opetition - has been subject of research within the network approach. As companies are collaborating with competitors, they need to simultaneously share and protect knowledge. The opportunistic behavior and learning intent of the partner may be underestimated, and collaboration may involve significant risks of loss of competitive edge. Contrastingly, the central tenet within the Intellectual Capital approach is that knowledge grows as it flows. The person sharing does not lose the knowledge and therefore knowledge has doubled from a company’s point of view. Value is created through the interplay of knowledge flows between and within three forms of intellectual capital: human, structural and relational capital. These are the points of departure for the research conducted in this thesis. The thesis investigates the tension between collaboration and competition through an Intellectual Capital lens, by identifying the actions taken to share and protect knowledge in interorganizational collaborative relationships. More specifically, it explores the tension in knowledge flows aimed at protecting and sharing knowledge, and their effect on the value creation of a company. It is assumed, that as two companies work closely together, the collaborative relationship becomes intertwined between the two partners and the intellectual capital flows of both companies are affected. The research finds that companies commonly protect knowledge also in close and long-term collaborative relationships. The knowledge flows identified are both collaborative and protective, with the result that they sometimes are counteracting and neutralize each other. The thesis contributes to the intellectual capital approach by expanding the understanding of knowledge protection in interorganizational relationships in three ways. First, departing from the research on co-opetition it shifts the focus from the internal view of the company as a repository of intellectual capital onto the collaborative relationships between competing companies. Second, instead of the traditional collaborative and sharing point of departure, it takes a competitive and protective perspective. Third, it identifies the intellectual capital flows as assets or liabilities depending on their effect on the value creation of the company. The actions taken to protect knowledge in an interorganizational relationship may decrease the value created in the company, which would make them liabilities.

The struggle for protection of environmental refugees: who's responsibility? Discourses from the United Nations and Non-governmental organizations

This thesis identifies, examines and problematizes some of the discourses that have so far come to light on the issue of protection for environmental refugees. By analyzing the discourses produced by the United Nations Office of the High Commissioner for Refugees (UNHCR) and two non-governmental organizations - the Environmental Justice Foundation (EJF) and Equity and Justice Working Group Bangladesh (EquityBD), I examine the struggling discourses that have emerged about how protection for environmental refugees has been interpreted. To do this, I rely on Ernesto Laclau and Chantal Mouffe's theory and method of discourse analysis. The results show that responsibilization is the main point of struggle in the discussions on the protection of environmental refugees. As a floating signifier, it was utilized by the discourses produced by the UNCHR and the selected NGOs in contingent ways and with different political objectives. The UNHCR discourse responsibilized both the environmental refugees for their own protection and the individual states. The EJF and EquityBD, by contrast, allocated responsibility for the protection of environmental refugees to the international community. These contingent understandings of responsibilization necessitated different justifications. While the EJF discourse relied on humanitarianism for the assistance of environmental refugees, the EquityBD discourse constructed a rights based, more permanent solution. The humanitarian based discourse of the EJF was found to be inextricably linked with the neoliberal discourse produced by the UNHCR. Both these discourses encouraged environmental refugees to stay in their homelands, undermining the politics of protection. Another way in which protection was undermined was by UNHCR's discourse on securitization. In this context, climate change induced displacement became threat to developed countries, the global economy and transnational classes. The struggling discourses about who/what has been allocated responsibility for the protection of environmental refugees also meant that identities of the displaced be constructed in specific ways. While the UNHCR discourse constructed as voluntary migrants and predators, the EJF and EquityBD discourses portrayed them as victims. However, even though the EJF discourse constructed them as victims, their reliance on humanitarianism could also be interpreted as a way of giving the environmental refugee a predator like identity. These discourses on responsibilization and identity formation clashed with each other in the aim of achieving a hegemonic position in discussions and debates about the protection of environmental refugees.

Innate Immune Recognition of RNA-virus infection in human macrophages

Innate immunity and host defence are rapidly evoked by structurally invariant molecular motifs common to microbial world, called pathogen associated molecular patterns (PAMPs). In addition to PAMPs, endogenous molecules released in response to inflammation and tissue damage, danger associated molecular patterns (DAMPs), are required for eliciting the response. The most important PAMPs of viruses are viral nucleic acids, their genome or its replication intermediates, whereas the identity and characteristics of virus infection-induced DAMPs are poorly defined. PAMPs and DAMPs engage a limited set of germ-line encoded pattern recognition receptors (PRRs) in immune and non-immune cells. Membrane-bound Toll-like receptors (TLRs), cytoplasmic retinoic acid inducible gene-I (RIG-I)-like receptors (RLRs) and nucleotide-binding oligomerization domain-like receptor (NLRs) are important PRRs involved in the recognition of the molecular signatures of viral infection, such as double-stranded ribonucleic acids (dsRNAs). Engagement of PRRs results in local and systemic innate immune responses which, when activated against viruses, evoke secretion of antiviral and pro-inflammatory cytokines, and programmed cell death i.e., apoptosis of the virus-infected cell. Macrophages are the central effector cells of innate immunity. They produce significant amounts of antiviral cytokines, called interferons (IFNs), and pro-inflammatory cytokines, such as interleukin (IL)-1β and IL-18. IL-1β and IL-18 are synthesized as inactive precursors, pro-IL-1β and pro-IL-18, that are processed by caspase-1 in a cytoplasmic multiprotein complex, called the inflammasome. After processing, these cytokines are biologically active and will be secreted. The signals and secretory routes that activate inflammasomes and the secretion of IL-1β and IL-18 during virus infections are poorly characterized. The main goal of this thesis was to characterize influenza A virus-induced innate immune responses and host-virus interactions in human primary macrophages during an infection. Methodologically, various techniques of cellular and molecular biology, as well as proteomic tools combined with bioinformatics, were utilized. Overall, the thesis provides interesting insights into inflammatory and antiviral innate immune responses, and has characterized host-virus interactions during influenza A virus-infection in human primary macrophages.

The struggle for protection of environmental refugees: who's responsibility? Discourses from the United Nations and Non-governmental organizations

This thesis identifies, examines and problematizes some of the discourses that have so far come to light on the issue of protection for environmental refugees. By analyzing the discourses produced by the United Nations Office of the High Commissioner for Refugees (UNHCR) and two non-governmental organizations - the Environmental Justice Foundation (EJF) and Equity and Justice Working Group Bangladesh (EquityBD), I examine the struggling discourses that have emerged about how protection for environmental refugees has been interpreted. To do this, I rely on Ernesto Laclau and Chantal Mouffe's theory and method of discourse analysis. The results show that responsibilization is the main point of struggle in the discussions on the protection of environmental refugees. As a floating signifier, it was utilized by the discourses produced by the UNCHR and the selected NGOs in contingent ways and with different political objectives. The UNHCR discourse responsibilized both the environmental refugees for their own protection and the individual states. The EJF and EquityBD, by contrast, allocated responsibility for the protection of environmental refugees to the international community. These contingent understandings of responsibilization necessitated different justifications. While the EJF discourse relied on humanitarianism for the assistance of environmental refugees, the EquityBD discourse constructed a rights based, more permanent solution. The humanitarian based discourse of the EJF was found to be inextricably linked with the neoliberal discourse produced by the UNHCR. Both these discourses encouraged environmental refugees to stay in their homelands, undermining the politics of protection. Another way in which protection was undermined was by UNHCR's discourse on securitization. In this context, climate change induced displacement became threat to developed countries, the global economy and transnational classes. The struggling discourses about who/what has been allocated responsibility for the protection of environmental refugees also meant that identities of the displaced be constructed in specific ways. While the UNHCR discourse constructed as voluntary migrants and predators, the EJF and EquityBD discourses portrayed them as victims. However, even though the EJF discourse constructed them as victims, their reliance on humanitarianism could also be interpreted as a way of giving the environmental refugee a predator like identity. These discourses on responsibilization and identity formation clashed with each other in the aim of achieving a hegemonic position in discussions and debates about the protection of environmental refugees.

Water quality assessment in water protection planning

Yhteenveto: Veden laadun arviointi vesiensuojelun suunnittelussa