Effect of Nucleic Acid Reactive Antibodies on Tumor Cells Grown in Vivo

Nucleic acid reactive antibodies have been reported to inhibit various nucleio acid mediated functions in cell free systems. These antibodies were also shown to inhibit the growth of transformed cells in culture due to the high rate of endocytosis in transformed cells as compared to normal cells. In this report, we have tested the possibility of nucleic acid reactive antibodies inhibiting the growth of tumor cells in vivo. The life span of mice bearing Dalton's lymphoma ascites tumor cells was increased, when they were immunized with conjugates of guanosine-BSA, GMP-BSA and tRNA-MBSA complex before transplanting the tumor cells. A similar effect was also observed when mice were injected intraperitoneally with antibodies to guanosine oi GMP along with the tumor cells. The specificity was ascertained, as immunization with non-specific antigens did not show any significant effect on tumor bearing mice. The results shows that nucleic acid. reactive antibodies inhibit the growth of tumor cells in vivo.

Hormonal modulation of riboflavin carrier protein secretion by immature rat Sertoli cells in culture

We report here that a protein species with biochemical and immunological similarity with chicken egg riboflavin carrier protein (RCP) is synthesized and secreted by immature rat Sertoli cells in culture. When quantitated by a specific heterologous radioimmunoassay, optimal concentrations of FSH (25 ng/ml) brought about 3-fold stimulation of RCP secretion. FSH, in the presence of testosterone (10−6 M) brought about 6-fold stimulation of secretion of RCP over the control cultures which were maintained in the absence of these two factors. The aromatase inhibitor (1,4,6-androstatrien-3,17-dione) curtailed 85% of the enhanced secretion of RCP, suggesting that the hormonal stimulation is mediated through in situ synthesized estrogen and this could be confirmed with exogenous estradiol-17 β which brought about 3 — fold enhancement of secretion of RCP at a concentration of 10−6 M. When tamoxifen (10 μM) was added along with FSH and testosterone, there was 75% decrease in the enhanced secretion of RCP. Addition of this anti-estrogen together with exogenous estradiol resulted in 55% decrease in elevated levels of RCP. Cholera toxin (1 μg/ml) and 8-bromo-cyclic AMP (0.5 mM) mimicked the action of FSH on the secretion of RCP thus suggesting that FSH stimulation of RCP production may be mediated through cyclic AMP. These findings suggest that estrogen mediates RCP induction in hormonally stimulated sertoli cells presumably to function as the carrier of riboflavin to the developing germ cells through blood-testis barrier in rodents.

Expression of Human Growth Hormone in Silkworm Larvae through RecombinantBombyx moriNuclear Polyhedrosis Virus

We have generated a recombinantBombyx morinuclear polyhedrosis virus, vBmhGH, harboring the full-length human growth hormone gene (2.4-kb genomic DNA, with four introns and the signal peptide sequences) under the control of the polyhedrin promoter. BmN cells in culture infected with the recombinant virus showed the presence of RNA corresponding to the authentic growth hormone mRNA as well as its incompletly processed precusor. Electrophoretic analysis and immunoprecipitation of proteins of recombinant virus-infected BmN cells revealed the presence of the growth hormone protein. Infection of silkworm larvae with vBmhGH led to the synthesis and efficient secretion of the protein into hemolymph. The recombinant human growth hormone was biologically active in a radioreceptor competition binding assay. The secreted protein was isolated and purified to homogeneity by a single step immunoaffinity chromatography, to a specific activity of 2.4 × 104U/mg. The recombinant hGH retained the immunological and biolological properties of the native peptide. We conclude that BmNPV vectors can be used successfully for expressing chromosomal genes harboring multiple introns.

Short- and long-term consequences of food resources on Ural owl Strix uralensis reproduction

Life-history theory states that although natural selection would favour a maximisation of both reproductive output and life-span, such a combination can not be achieved in any living organism. According to life-history theory the reason for the fact that not all traits can be maximised simultaneously is that different traits compete with each other for resources. These relationships between traits that constrain the simultaneous evolution of two or more traits are called trade-offs. Therefore, during different life-stages an individual needs to optimise its allocation of resources to life-history components such as growth, reproduction and survival. Resource limitation acts on these traits and therefore investment in one trait, e.g. reproduction, reduces the resources available for investment in another trait, e.g. residual reproduction or survival. In this thesis I study how food resources during different stages of the breeding event affect reproductive decisions in the Ural owl (Strix uralensis) and the consequences of these decisions on parents and offspring. The Ural owl is a suitable study species for such studies in natural populations since they are long-lived, site-tenacious, and feed on voles. The vole populations in Fennoscandia fluctuate in three- to four-year cycles, which create a variable food environment for the Ural owls to cope with. The thesis gives new insight in reproductive costs and their consequences in natural animal populations with emphasis on underlying physiological mechanisms. I found that supplementary fed Ural owl parents invest supplemented food resources during breeding in own self-maintenance instead of allocating those resources to offspring growth. This investment in own maintenance instead of improving current reproduction had carry-over effects to the following year in terms of increased reproductive output. Therefore, I found evidence that reduced reproductive costs improves future reproductive performance. Furthermore, I found evidence for the underlying mechanism behind this carry-over effect of supplementary food on fecundity. The supplementary-fed parents reduced their feeding investment in the offspring compared to controls, which enabled the fed female parents to invest the surplus resources in parasite resistance. Fed female parents had lower blood parasite loads than control females and this effect lasted until the following year when also reproductive output was increased. Hence, increased investment in parasite resistance when resources are plentiful has the potential to mediate positive carry-over effects on future reproduction. I further found that this carry-over effect was only present when potentials for future reproduction were good. The thesis also provides new knowledge on resource limitation on maternal effects. I found that increased resources prior to egg laying improve the condition and health of Ural owl females and enable them to allocate more resources to reproduction than control females. These additional resources are not allocated to increase the number of offspring, but instead to improve the quality of each offspring. Fed Ural owl females increased the size of their eggs and allocated more health improving immunological components into the eggs. Furthermore, the increased egg size had long-lasting effects on offspring growth, as offspring from larger eggs were heavier at fledging. Limiting resources can have different short- and long-term consequences on reproductive decisions that affect both offspring number and quality. In long-lived organisms, such as the Ural owl, it appears to be beneficial in terms of fitness to invest in long breeding life-span instead of additional investment in current reproduction. In Ural owls, females can influence the phenotypic quality of the offspring by transferring additional resources to the eggs that can have long-lasting effects on growth.

Blockade of PD-1/PD-L1 promotes adoptive T-Cell immunotherapy in a tolerogenic environment

Adoptive cellular immunotherapy using in vitro expanded CD8+ T cells shows promise for tumour immunotherapy but is limited by eventual loss of function of the transferred T cells through factors that likely include inactivation by tolerogenic dendritic cells (DC). The coinhibitory receptor programmed death-1 (PD-1), in addition to controlling T-cell responsiveness at effector sites in malignancies and chronic viral diseases is an important modulator of dendritic cell-induced tolerance in naive T cell populations. The most potent therapeutic capacity amongst CD8+ T cells appears to lie within Tcm or Tcm-like cells but memory T cells express elevated levels of PD-1. Based on established trafficking patterns for Tcm it is likely Tcm-like cells interact with lymphoid-tissue DC that present tumour-derived antigens and may be inherently tolerogenic to develop therapeutic effector function. As little is understood of the effect of PD-1/PD-L1 blockade on Tcm-like CD8+ T cells, particularly in relation to inactivation by DC, we explored the effects of PD-1/PD-L1 blockade in a mouse model where resting DC tolerise effector and memory CD8+ T cells. Blockade of PD-1/PDL1 promoted effector differentiation of adoptively-transferred Tcm-phenotype cells interacting with tolerising DC. In tumour-bearing mice with tolerising DC, effector activity was increased in both lymphoid tissues and the tumour-site and anti-tumour activity was promoted. Our findings suggest PD-1/PD-L1 blockade may be a useful adjunct for adoptive immunotherapy by promoting effector differentiation in the host of transferred Tcmlike cells. © 2015 Blake et al.

Functional and cellular analysis of autoimmune regulator (AIRE) protein

Autoimmune diseases are a major health problem. Usually autoimmune disorders are multifactorial and their pathogenesis involves a combination of predisposing variations in the genome and other factors such as environmental triggers. APECED (autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy) is a rare, recessively inherited, autoimmune disease caused by mutations in a single gene. Patients with APECED suffer from several organ-specific autoimmune disorders, often affecting the endocrine glands. The defective gene, AIRE, codes for a transcriptional regulator. The AIRE (autoimmune regulator) protein controls the expression of hundreds of genes, representing a substantial subset of tissue-specific antigens which are presented to developing T cells in the thymus and has proven to be a key molecule in the establishment of immunological tolerance. However, the molecular mechanisms by which AIRE mediates its functions are still largely obscure. The aim of this thesis has been to elucidate the functions of AIRE by studying the molecular interactions it is involved in by utilizing different cultured cell models. A potential molecular mechanism for exceptional, dominant, inheritance of APECED in one family, carrying a glycine 228 to tryptophan (G228W) mutation, was described in this thesis. It was shown that the AIRE polypeptide with G228W mutation has a dominant negative effect by binding the wild type AIRE and inhibiting its transactivation capacity in vitro. The data also emphasizes the importance of homomultimerization of AIRE in vivo. Furthermore, two novel protein families interacting with AIRE were identified. The importin alpha molecules regulate the nuclear import of AIRE by binding to the nuclear localization signal of AIRE, delineated as a classical monopartite signal sequence. The interaction of AIRE with PIAS E3 SUMO ligases, indicates a link to the sumoylation pathway, which plays an important role in the regulation of nuclear architecture. It was shown that AIRE is not a target for SUMO modification but enhances the localization of SUMO1 and PIAS1 proteins to nuclear bodies. Additional support for the suggestion that AIRE would preferably up-regulate genes with tissue-specific expression pattern and down-regulate housekeeping genes was obtained from transactivation studies performed with two models: human insulin and cystatin B promoters. Furthermore, AIRE and PIAS activate the insulin promoter concurrently in a transactivation assay, indicating that their interaction is biologically relevant. Identification of novel interaction partners for AIRE provides us information about the molecular pathways involved in the establishment of immunological tolerance and deepens our understanding of the role played by AIRE not only in APECED but possibly also in several other autoimmune diseases.

Endoplasmic reticulum aminopeptidases in the pathogenesis of ankylosing spondylitis

There has been significant progress in our understanding of the pathogenesis of AS. The advent of genome-wide association studies has increased the known loci associated with AS to more than 40. The endoplasmic reticulum resident aminopeptidases (ERAP) 1 and 2 were identified in this manner and are of particular interest. There appears to be a genetic as well as a functional interaction of ERAP1 and 2 with HLA-B27 based on the known functions of these molecules. Recent studies on the structure, immunological effects and the peptide-trimming properties of ERAP 1 and 2 have helped to provide insight into their pathogenic potential in AS. In this review, we explore the role of ERAP 1 and 2 in the pathogenesis of AS. © The Author 2015.

Targeting antigen to diverse APCs inactivates memory CD8+ T cells without eliciting tissue-destructive effector function

Memory T cells develop early during the preclinical stages of autoimmune diseases and have traditionally been considered resistant to tolerance induction. As such, they may represent a potent barrier to the successful immunotherapy of established autoimmune diseases. It was recently shown that memory CD8+ T cell responses are terminated when Ag is genetically targeted to steady-state dendritic cells. However, under these conditions, inactivation of memory CD8+ T cells is slow, allowing transiently expanded memory CD8+ T cells to exert tissue-destructive effector function. In this study, we compared different Ag-targeting strategies and show, using an MHC class II promoter to drive Ag expression in a diverse range of APCs, that CD8+ memory T cells can be rapidly inactivated by MHC class II+ hematopoietic APCs through a mechanism that involves a rapid and sustained downregulation of TCR, in which the effector response of CD8+ memory cells is rapidly truncated and Ag-expressing target tissue destruction is prevented. Our data provide the first demonstration that genetically targeting Ag to a broad range of MHC class II+ APC types is a highly efficient way to terminate memory CD8+ T cell responses to prevent tissue-destructive effector function and potentially established autoimmune diseases. Copyright © 2010 by The American Association of Immunologists, Inc.

Microbe-induced activation of inflammatory cytokine response in human cells

Human body is in continuous contact with microbes. Although many microbes are harmless or beneficial for humans, pathogenic microbes possess a threat to wellbeing. Antimicrobial protection is provided by the immune system, which can be functionally divided into two parts, namely innate and adaptive immunity. The key players of the innate immunity are phagocytic white blood cells such as neutrophils, monocytes, macrophages and dendritic cells (DCs), which constantly monitor the blood and peripheral tissues. These cells are armed for rapid activation upon microbial contact since they express a variety of microbe-recognizing receptors. Macrophages and DCs also act as antigen presenting cells (APCs) and play an important role in the development of adaptive immunity. The development of adaptive immunity requires intimate cooperation between APCs and T lymphocytes and results in microbe-specific immune responses. Moreover, adaptive immunity generates immunological memory, which rapidly and efficiently protects the host from reinfection. Properly functioning immune system requires efficient communication between cells. Cytokines are proteins, which mediate intercellular communication together with direct cell-cell contacts. Immune cells produce inflammatory cytokines rapidly following microbial contact. Inflammatory cytokines modulate the development of local immune response by binding to cell surface receptors, which results in the activation of intracellular signalling and modulates target cell gene expression. One class of inflammatory cytokines chemokines has a major role in regulating cellular traffic. Locally produced inflammatory chemokines guide the recruitment of effector cells to the site of inflammation during microbial infection. In this study two key questions were addressed. First, the ability of pathogenic and non-pathogenic Gram-positive bacteria to activate inflammatory cytokine and chemokine production in different human APCs was compared. In these studies macrophages and DCs were stimulated with pathogenic Steptococcus pyogenes or non-pathogenic Lactobacillus rhamnosus. The second aim of this thesis work was to analyze the role of pro-inflammatory cytokines in the regulation of microbe-induced chemokine production. In these studies bacteria-stimulated macrophages and influenza A virus-infected lung epithelial cells were used as model systems. The results of this study show that although macrophages and DCs share several common antimicrobial functions, these cells have significantly distinct responses against pathogenic and non-pathogenic Gram-positive bacteria. Macrophages were activated in a nearly similar fashion by pathogenic S. pyogenes and non-pathogenic L. rhamnosus. Both bacteria induced the production of similar core set of inflammatory chemokines consisting of several CC-class chemokines and CXCL8. These chemokines attract monocytes, neutrophils, dendritic cells and T cells. Thus, the results suggest that bacteria-activated macrophages efficiently recruit other effector cells to the site of inflammation. Moreover, macrophages seem to be activated by all bacteria irrespective of their pathogenicity. DCs, in contrast, were efficiently activated only by pathogenic S. pyogenes, which induced DC maturation and production of several inflammatory cytokines and chemokines. In contrast, L. rhamnosus-stimulated DCs matured only partially and, most importantly, these cells did not produce inflammatory cytokines or chemokines. L. rhamnosus-stimulated DCs had a phenotype of "semi-mature" DCs and this type of DCs have been suggested to enhance tolerogenic adaptive immune responses. Since DCs have an essential role in the development of adaptive immune response the results suggest that, in contrast to macrophages, DCs may be able to discriminate between pathogenic and non-pathogenic bacteria and thus mount appropriate inflammatory or tolerogenic adaptive immune response depending on the microbe in question. The results of this study also show that pro-inflammatory cytokines can contribute to microbe-induced chemokine production at multiple levels. S. pyogenes-induced type I interferon (IFN) was found to enhance the production of certain inflammatory chemokines in macrophages during bacterial stimulation. Thus, bacteria-induced chemokine production is regulated by direct (microbe-induced) and indirect (pro-inflammatory cytokine-induced) mechanisms during inflammation. In epithelial cells IFN- and tumor necrosis factor- (TNF-) were found to enhance the expression of PRRs and components of cellular signal transduction machinery. Pre-treatment of epithelial cells with these cytokines prior to virus infection resulted in markedly enhanced chemokine response compared to untreated cells. In conclusion, the results obtained from this study show that pro-inflammatory cytokines can enhance microbe-induced chemokine production during microbial infection by providing a positive feedback loop. In addition, pro-inflammatory cytokines can render normally low-responding cells to high chemokine producers via enhancement of microbial detection and signal transduction.

Genetic aspects of outcome in kidney transplantation: cytokine and thrombosis associated candidate genes and gene expression biomarkers

Kidney transplantation (Tx) is the treatment of choice for end stage renal disease. Immunosuppressive medications are given to prevent an immunological rejection of the transplant. However, immunosuppressive drugs increase e.g. the risk of infection, cancer or nephrotoxicity. A major genetic contributors to immunological acceptance of the graft are human leukocyte antigen (HLA) genes. Also other non-HLA gene polymorphisms may predict the future risk of complications before Tx, possibly enabling individualised immunotherapy. Graft function after Tx is monitored using non-specific clinical symptoms and laboratory markers. The definitive diagnosis of graft rejection however relies on a biopsy of the graft. In the acute rejection (AR) diagnostics there is a need for an alternative to biopsy that would be an easily repeatable and simple method for regular use. Frequent surveillance of acute or subclinical rejection (SCR) may improve long-term function. In this thesis, associations between cytokine and thrombosis associated candidate genes and the outcome of kidney Tx were studied. Cytotoxic and co-stimulatory T lymphocyte molecule gene expression biomarkers for the diagnosis of the AR and the SCR were also investigated. We found that polymorphisms in the cytokine genes tumor necrosis factor and interleukin 10 (IL10) of the recipients were associated with AR. In addition, certain IL10 gene polymorphisms of the donors were associated with the incidence of cytomegalovirus infection and occurrence of later infection in a subpopulation of recipients. Further, polymorphisms in genes related to the risk of thrombosis and those of certain cytokines were not associated with the occurrence of thrombosis, infarction, AR or graft survival. In the study of biomarkers for AR, whole blood samples were prospectively collected from adult kidney Tx patients. With real-time quantitative PCR (RT-QPCR) gene expression quantities of CD154 and ICOS differentiated the patients with AR from those without, but not from the patients with other causes of graft dysfunction. Biomarkers for SCR were studied in paediatric kidney Tx patients. We used RT-QPCR to quantify the gene expression of immunological candidate genes in a low-density array format. In addition, we used RT-QPCR to validate the results of the microarray analysis. No gene marker differentiated patients with SCR from those without SCR. This research demonstrates the lack of robust markers among polymorphisms or biomarkers in investigated genes that could be included in routine analysis in a clinical laboratory. In genetic studies, kidney Tx can be regarded as a complex trait, i.e. several environmental and genetic factors may determine its outcome. A number of currently unknown genetic factors probably influence the results of Tx.

Prediction of Candidate Primary Immunodeficiency Disease Genes Using a Support Vector Machine Learning Approach

Screening and early identification of primary immunodeficiency disease (PID) genes is a major challenge for physicians. Many resources have catalogued molecular alterations in known PID genes along with their associated clinical and immunological phenotypes. However, these resources do not assist in identifying candidate PID genes. We have recently developed a platform designated Resource of Asian PDIs, which hosts information pertaining to molecular alterations, protein-protein interaction networks, mouse studies and microarray gene expression profiling of all known PID genes. Using this resource as a discovery tool, we describe the development of an algorithm for prediction of candidate PID genes. Using a support vector machine learning approach, we have predicted 1442 candidate PID genes using 69 binary features of 148 known PID genes and 3162 non-PID genes as a training data set. The power of this approach is illustrated by the fact that six of the predicted genes have recently been experimentally confirmed to be PID genes. The remaining genes in this predicted data set represent attractive candidates for testing in patients where the etiology cannot be ascribed to any of the known PID genes.

Associations of serum perfluoroalkyl acid levels with T-helper cell-specific cytokines in children: By gender and asthma status

Perfluoroalkyl acids (PFAAs) are a group of common chemicals that ubiquitously exist in wildlife and humans. Experimental data suggest that they may alter T-lymphocyte functioning in situ by preferentially enhancing the development of T-helper 2 (TH2)- and inhibiting TH1-lymphocyte development and might increase allergic inflammation, but few human studies have been conducted. To evaluate the association between serum PFAAs concentrations and T-lymphocyte-related immunological markers of asthma in children, and further to assess whether gender modified this association, 231 asthmatic children and 225 non-asthmatic control children from Northern Taiwan were recruited into the Genetic and Biomarker study for Childhood Asthma. Serum concentrations of ten PFAAs and levels of TH1 [interferon (IFN)-γ, interleukin (IL)-2] and TH2 (IL-4 and IL-5) cytokines were measured. The results showed that asthmatics had significantly higher serum PFAAs concentrations compared with the healthy controls. When stratified by gender, a greater number of significant associations between PFAAs and asthma outcomeswere found in males than in females. Among males, adjusted odds ratios for asthma among those with the highest versus lowest quartile of PFAAs exposure ranged from 2.59 (95% CI: 1.14, 5.87) for the perfluorobutanesulfonate (PFBS) to 4.38 (95% CI: 2.02, 9.50) for perfluorooctanesulfonate (PFOS); and serum PFAAs were associated positively with TH2 cytokines and inversely with TH1 cytokines among male asthmatics. Among females, no significant associations between PFAAs and TH2 cytokines could be detected. In conclusion, increased serum PFAAs levels may promote TH cell dysregulation and alter the availability of key TH1 and TH2 cytokines, ultimately contributing to the development of asthma that may differentially impact males to a greater degree than females. These results have potential relevance in asthma prevention.

Studies of Immune Regulation in Type 1 Diabetes

Type 1 diabetes (T1D) is considered to be an autoimmune disease. In T1D insulin producing pancreatic β cells are destroyed. The disease process begins years before the clinical diagnosis of T1D. During the pathogenesis of T1D, pancreatic islets are infiltrated by cells of the immune system and T-lymphocytes are considered to be the main mediators of the β-cell destruction. In children with an active β-cell destruction process, autoantibodies against β-cell antigens appear in the blood. Individuals at increased risk of developing T1D can often be identified by detecting serum autoantibodies against β-cell antigens. Immunological aberrancies associated with T1D are related to defects in the polarization of T cells and in the function of regulatory mechanisms. T1D has been considered as an organ-specific autoimmune disease mediated by uncontrolled Th1-responses. In human T1D, the evidence for the role of over-expression of cytokines promoting cytotoxicity is controversial. For the past 15 years, regulatory T cells (Tregs) have been recognized as having a key role in the initiation and maintenance of tolerance, limiting harmful autoantigen-specific inflammation processes. It is possible that, if regulatory mechanisms fail to be initiated, the subtle inflammation targeting β cells lead to insulitis and eventually to overt T1D in some individuals. In the present thesis, we studied the induction of Tregs during the generation of T-cell responses in T1D. The results suggest that the generation of regulatory mechanisms and effector mechanisms upon T-cell activation is aberrant in children with T1D. In our studies, an in vitro cytotoxic environment inhibited the induction of genes associated with regulatory functions upon T-cell activation. We also found T1D patients to have an impaired cytotoxic response against coxsackievirus B4. Ineffective virus clearance may increase the apoptosis of β cells, and thus the risk of β-cell specific autoimmunity, due to the increased presentation of β-cell-derived peptides by APCs to T cells in pancreatic lymph nodes. Recently, a novel T helper cell subset called Th17 has been discovered. Animal models have associated Th17 cells and especially co-producers of IL-17 and IFN-γ with the pathogenesis of T1D. We aimed to characterize the role of Th17 immunity in human T1D. We demonstrated IL-17 activation to be a major alteration in T1D patients in comparison to healthy children. Moreover, alterations related to the FOXP3-mediated regulatory mechanisms were associated with the IL-17 up-regulation seen in T1D patients. These findings may have therapeutic implications for the treatment and prevention of T1D.

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.

Damp building moulds: Assessment of sensitization in patients and studies into mechanisms of airway inflammation using experimental models

Exposure to water-damaged buildings and the associated health problems have evoked concern and created confusion during the past 20 years. Individuals exposed to moisture problem buildings report adverse health effects such as non-specific respiratory symptoms. Microbes, especially fungi, growing on the damp material have been considered as potential sources of the health problems encountered in these buildings. Fungi and their airborne fungal spores contain allergens and secondary metabolites which may trigger allergic as well as inflammatory types of responses in the eyes and airways. Although epidemiological studies have revealed an association between damp buildings and health problems, no direct cause-and-effect relationship has been established. Further knowledge is needed about the epidemiology and the mechanisms leading to the symptoms associated with exposure to fungi. Two different approaches have been used in this thesis in order to investigate the diverse health effects associated with exposure to moulds. In the first part, sensitization to moulds was evaluated and potential cross-reactivity studied in patients attending a hospital for suspected allergy. In the second part, one typical mould known to be found in water-damaged buildings and to produce toxic secondary metabolites was used to study the airway responses in an experimental model. Exposure studies were performed on both naive and allergen sensitized mice. The first part of the study showed that mould allergy is rare and highly dependent on the atopic status of the examined individual. The prevalence of sensitization was 2.7% to Cladosporium herbarum and 2.8% to Alternaria alternata in patients, the majority of whom were atopic subjects. Some of the patients sensitized to mould suffered from atopic eczema. Frequently the patients were observed to possess specific serum IgE antibodies to a yeast present in the normal skin flora, Pityrosporum ovale. In some of these patients, the IgE binding was partly found to be due to binding to shared glycoproteins in the mould and yeast allergen extracts. The second part of the study revealed that exposure to Stachybotrys chartarum spores induced an airway inflammation in the lungs of mice. The inflammation was characterized by an influx of inflammatory cells, mainly neutrophils and lymphocytes, into the lungs but with almost no differences in airway responses seen between the satratoxin producing and non-satratoxin producing strain. On the other hand, when mice were exposed to S. chartarum and sensitized/challenged with ovalbumin the extent of the inflammation was markedly enhanced. A synergistic increase in the numbers of inflammatory cells was seen in BAL and severe inflammation was observed in the histological lung sections. In conclusion, the results in this thesis imply that exposure to moulds in water damaged buildings may trigger health effects in susceptible individuals. The symptoms can rarely be explained by IgE mediated allergy to moulds. Other non-allergic mechanisms seem to be involved. Stachybotrys chartarum is one of the moulds potentially responsible for health problems. In this thesis, new reaction models for the airway inflammation induced by S. chartarum have been found using experimental approaches. The immunological status played an important role in the airway inflammation, enhancing the effects of mould exposure. The results imply that sensitized individuals may be more susceptible to exposure to moulds than non-sensitized individuals.