Oral Immune Defense against Chronic Hyperplastic Candidosis

Candida yeast species are widespread opportunistic microbes, which are usually innocent opportunists unless the systemic or local defense system of the host becomes compromised. When they adhere on a fertile substrate such as moist and warm, protein-rich human mucosal membrane or biomaterial surface, they become activated and start to grow pseudo and real hyphae. Their growth is intricately guided by their ability to detect surface defects (providing secure hiding , thigmotropism) and nutrients (source of energy, chemotropism). The hypothesis of this work was that body mobilizes both non-specific and specific host defense against invading candidal cells and that these interactions involve resident epithelial cells, rapidly responding non-specific protector neutrophils and mast cells as well as the antigen presenting and responding den-dritic cell lymphocyte plasma cell system. It is supposed that Candida albicans, as a result of dar-winistic pressure, has developed or is utilizing strategies to evade these host defense reactions by e.g. adhering to biomaterial surfaces and biofilms. The aim of the study was to assess the host defense by taking such key molecules of the anti-candidal defense into focus, which are also more or less characteristic for the main cellular players in candida-host cell interactions. As a model for candidal-host interaction, sections of chronic hyperplastic candidosis were used and compared with sections of non-infected leukoplakia and healthy tissue. In this thesis work, neutrophil-derived anti-candidal α-defensin was found in the epithelium, not only diffusely all over in the epithelium, but as a strong α-defensin-rich superficial front probably able to slow down or prevent penetration of candida into the epithelium. Neutrophil represents the main host defence cell in the epithelium, to which it can rapidly transmigrate from the circulation and where it forms organized multicellular units known as microabscesses (study I). Neutrophil chemotactic inter-leukin-8 (IL-8) and its receptor (IL-8R) were studied and were surprisingly also found in the candidal cells, probably helping the candida to keep away from IL-8- and neutrophil-rich danger zones (study IV). Both leukocytes and resident epithelial cells contained TLR2, TLR4 and TLR6 receptors able to recognize candidal structures via utilization of receptors similar to the Toll of the banana fly. It seems that candida can avoid host defence via stimulation of the candida permissive TLR2 instead of the can-dida injurious TLR4 (study V). TLR also provides the danger signal to the immune system without which it will not be activated to specifically respond against candidal antigens. Indeed, diseased sites contained receptor activator of nuclear factor kappa B ligand (RANKL; II study), which is important for the antigen capturing, processing and presenting dendritic cells and for the T lymphocyte activation (study III). Chronic hyperplastic candidosis provides a disease model that is very useful to study local and sys-temic host factors, which under normal circumstances restrain C. albicans to a harmless commensal state, but failure of which in e.g. HIV infection, cancer and aging may lead to chronic infection.

Molecular basis of the kidney filtration barrier : Role of the nephrin protein complex

The kidney filtration barrier consists of fenestrated endothelial cell layer, glomerular basement membrane and slit diaphragm (SD), the specialized junction between glomerular viscelar epithelial cells (podocytes). Podocyte injury is associated with the development of proteinuria, and if not reversed the injury will lead to permanent deterioration of the glomerular filter. The early events are characterized by disruption of the integrity of the SD, but the molecular pathways involved are not fully understood. Congenital nephrotic syndrome of the Finnish type (CNF) is caused by mutations in NPHS1, the gene encoding the SD protein nephrin. Lack of nephrin results in loss of the SD and massive proteinuria beginning before birth. Furthermore, nephrin expression is decreased in acquired human kidney diseases including diabetic nephropathy. This highlights the importance of nephrin and consequently SD in regulating the kidney filtration function. However, the precise molecular mechanism of how nephrin is involved in the formation of the SD is unknown. This thesis work aimed at clarifying the role of nephrin and its interaction partners in the formation of the SD. The purpose was to identify novel proteins that associate with nephrin in order to define the essential molecular complex required for the establishment of the SD. The aim was also to decipher the role of novel nephrin interacting proteins in podocytes. Nephrin binds to nephrin-like proteins Neph1 and Neph2, and to adherens junction protein P-cadherin. These interactions have been suggested to play a role in the formation of the SD. In this thesis work, we identified densin as a novel interaction partner for nephrin. Densin was localized to the SD and it was shown to bind to adherens junction protein beta-catenin. Furthermore, densin was shown to behave in a similar fashion as adherens junction proteins in cell-cell contacts. These results indicate that densin may play a role in cell adhesion and, therefore, may contribute to the formation of the SD together with nephrin and adherens junction proteins. Nephrin was also shown to bind to Neph3, which has been previously localized to the SD. Neph3 and Neph1 were shown to induce cell adhesion alone, whereas nephrin needed to trans-interact with Neph1 or Neph3 from the opposite cell surface in order to make cell-cell contacts. This was associated with the decreased tyrosine phosphorylation of nephrin. These data extend the current knowledge of the molecular composition of the nephrin protein complex at the SD and also provide novel insights of how the SD may be formed. This thesis work also showed that densin was up-regulated in the podocytes of CNF patients. Neph3 was up-regulated in nephrin deficient mouse kidneys, which share similar podocyte alterations and lack of the SD as observed in CNF patients podocytes. These data suggest that densin and Neph3 may have a role in the formation of morphological alterations in podocytes detected in CNF patients. Furthermore, this thesis work showed that deletion of beta-catenin specifically from adult mouse podocytes protected the mice from the development of adriamycin-induced podocyte injury and proteinuria compared to wild-type mice. These results show that beta-catenin play a role in the adriamycin induced podocyte injury. Podocyte injury is a hallmark in many kidney diseases and the changes observed in the podocytes of CNF patient share characteristics with injured podocytes observed in chronic kidney diseases. Therefore, the results obtained in this thesis work suggest that densin, Neph3 and beta-catenin participate in the molecular pathways which result in morphological alterations commonly detected in injured podocytes in kidney diseases.

Studies on immune activation in rheumatoid arthritis and reactive arthritis

Rheumatoid arthritis (RA) is an autoimmune disease characterized by synovitis, progressive joint destruction, and disability. Reactive arthritis (ReA) is a sterile joint inflammation following a distant mucosal infection. The clinical course of these diseases is variable and cannot be predicted with reasonable accuracy by clinical and laboratory markers. The predictive value of circulating soluble interleukin-2 receptor (sIL-2R), a marker of lymphocyte activation, measured by Immulite® automated immunoassay analyzer, was evaluated in two cohorts of RA patients. In 175 patients with active early RA randomized to treatment with either on disease-modifying antirheumatic drug (DMARD) or a combination of 3 DMARDs and prednisolone, low baseline sIL-2R level predicted remission after 6 months in patients treated with a single DMARD. In 24 patients with active RA refractory to DMARDs, low baseline sIL-2R level predicted rapid clinical response to treatment with infliximab, an anti-tumour necrosis factor antibody. Furthermore, in a cohort of 26 patients with acute ReA, high baseline sIL-2R level predicted remission after 6 months. Levels of circulating soluble E-selectin (sE-selectin), a marker of endothelial activation, were measured annually by enzyme-linked immunosorbent assay (ELISA) in a cohort of 85 patients with early RA. During a five-year follow-up, sE-selectin levels were associated with activity and outcome of RA. The levels of neutrophil and monocyte CD11b/CD18 expression measured by flow cytometry, and circulating levels of sE-selectin measured by ELISA, and procalcitonin by immunoluminometric assay, were compared in 28 patients with acute ReA and 16 patients with early RA. The levels of the markers were comparable in ReA, RA, and healthy control subjects. In conlusion, sIL-2R may provide a new predictive marker in early RA treated with a single DMARD and refractory RA treated with infliximab. In addition, sIL-2R level predicts remission in acute ReA.

Copper Diffusion Barrier Deposition on Integrated Circuit Devices by Atomic Layer Deposition Technique

Transfer from aluminum to copper metallization and decreasing feature size of integrated circuit devices generated a need for new diffusion barrier process. Copper metallization comprised entirely new process flow with new materials such as low-k insulators and etch stoppers, which made the diffusion barrier integration demanding. Atomic Layer Deposition technique was seen as one of the most promising techniques to deposit copper diffusion barrier for future devices. Atomic Layer Deposition technique was utilized to deposit titanium nitride, tungsten nitride, and tungsten nitride carbide diffusion barriers. Titanium nitride was deposited with a conventional process, and also with new in situ reduction process where titanium metal was used as a reducing agent. Tungsten nitride was deposited with a well-known process from tungsten hexafluoride and ammonia, but tungsten nitride carbide as a new material required a new process chemistry. In addition to material properties, the process integration for the copper metallization was studied making compatibility experiments on different surface materials. Based on these studies, titanium nitride and tungsten nitride processes were found to be incompatible with copper metal. However, tungsten nitride carbide film was compatible with copper and exhibited the most promising properties to be integrated for the copper metallization scheme. The process scale-up on 300 mm wafer comprised extensive film uniformity studies, which improved understanding of non-uniformity sources of the ALD growth and the process-specific requirements for the ALD reactor design. Based on these studies, it was discovered that the TiN process from titanium tetrachloride and ammonia required the reactor design of perpendicular flow for successful scale-up. The copper metallization scheme also includes process steps of the copper oxide reduction prior to the barrier deposition and the copper seed deposition prior to the copper metal deposition. Easy and simple copper oxide reduction process was developed, where the substrate was exposed gaseous reducing agent under vacuum and at elevated temperature. Because the reduction was observed efficient enough to reduce thick copper oxide film, the process was considered also as an alternative method to make the copper seed film via copper oxide reduction.

Causes and consequences of variation in nestling immune function

Defence against pathogens is a vital need of all living organisms that has led to the evolution of complex immune mechanisms. However, although immunocompetence the ability to resist pathogens and control infection has in recent decades become a focus for research in evolutionary ecology, the variation in immune function observed in natural populations is relatively little understood. This thesis examines sources of this variation (environmental, genetic and maternal effects) during the nestling stage and its fitness consequences in wild populations of passerines: the blue tit (Cyanistes caeruleus) and the collared flycatcher (Ficedula albicollis). A developing organism may face a dilemma as to whether to allocate limited resources to growth or to immune defences. The optimal level of investment in immunity is shaped inherently by specific requirements of the environment. If the probability of contracting infection is low, maintaining high growth rates even at the expense of immune function may be advantageous for nestlings, as body mass is usually a good predictor of post-fledging survival. In experiments with blue tits and haematophagous hen fleas (Ceratophyllus gallinae) using two methods, methionine supplementation (to manipulate nestlings resource allocation to cellular immune function) and food supplementation (to increase resource availability), I confirmed that there is a trade-off between growth and immunity and that the abundance of ectoparasites is an environmental factor affecting allocation of resources to immune function. A cross-fostering experiment also revealed that environmental heterogeneity in terms of abundance of ectoparasites may contribute to maintaining additive genetic variation in immunity and other traits. Animal model analysis of extensive data collected from the population of collared flycatchers on Gotland (Sweden) allowed examination of the narrow-sense heritability of PHA-response the most commonly used index of cellular immunocompetence in avian studies. PHA-response is not heritable in this population, but is subject to a non-heritable origin (presumably maternal) effect. However, experimental manipulation of yolk androgen levels indicates that the mechanism of the maternal effect in PHA-response is not in ovo deposition of androgens. The relationship between PHA-response and recruitment was studied for over 1300 collared flycatcher nestlings. Multivariate selection analysis shows that it is body mass, not PHA-response, that is under direct selection. PHA-response appears to be related to recruitment because of its positive relationship with body mass. These results imply that either PHA-response fails to capture the immune mechanisms that are relevant for defence against pathogens encountered by fledglings or that the selection pressure from parasites is not as strong as commonly assumed.

Immune response to insulin and changes in the gut immune system in children with or at risk for type 1 diabetes

Type 1 diabetes (T1D) is considered to be an autoimmune disease. The cause of T1D is the destruction of insulin-producing β-cells in the pancreatic islets. The autoimmune nature of T1D is characterized by the presence of autoreactive T-cells and autoantibodies against β-cell molecules. Insulin is the only β-cell-specific autoantigen associated with T1D but the insulin autoantibodies (IAAs) are difficult to measure with proper sensitivity. T-cell assays for detection of autoreactive T-cells, such as insulin-specific T-cells, have also proven to be difficult to perform. The genetic risk of T1D is associated with the HLA gene region but the environmental factors also play an important role. The most studied environmental risk factors of T1D are enteroviruses and cow's milk which both affect the immune system through the gut. One hypothesis is that the insulin-specific immune response develops against bovine insulin in cow's milk during early infancy and later spreads to include human insulin. The aims of this study were to determine whether the separation of immunoglobulin (Ig)G from plasma would improve the sensitivity of the IAA assay and how insulin treatment affects the cellular immune response to insulin in newly diagnosed patients. Furthermore, the effect of insulin concentration in mother's breast milk on the development of antibodies to dietary insulin in the child was examined. Small intestinal biopsies were also obtained from children with T1D to characterize any immunological changes associated with T1D in the gut. The isolation of the IgG fraction from the plasma of T1D patients negative for plasma IAA led to detectable IAA levels that exceeded those in the control children. Thus the isolation of IgG may improve the sensitivity of the IAA assay. The effect of insulin treatment on insulin-specific T-cells was studied by culturing peripheral blood mononuclear cells with insulin. The insulin stimulation induced increased expression of regulatory T-cell markers, such as Foxp3, in those patients treated with insulin than in patients examined before initiating insulin treatment. This finding suggests that insulin treatment in patients with T1D stimulates regulatory T-cells in vivo and this may partly explain the difficulties in measuring autoantigen-specific T-cell responses in recently diagnosed patients. The stimulation of regulatory T-cells by insulin treatment may also explain the remission period often seen after initiating insulin treatment. In the third study we showed that insulin concentration in mother's breast milk correlates inversely with the levels of bovine insulin-specific antibodies in those infants who were exposed to cow's milk proteins in their diet, suggesting that human insulin in breast milk induces tolerance to dietary bovine insulin. However, in infants who later developed T1D-associated autoantibodies, the insulin concentration in their mother's breast milk was increased. This finding may indicate that in those children prone to β-cell autoimmunity, breast milk insulin does not promote tolerance to insulin. In the small intestinal biopsies the presence of several immunological markers were quantified with the RT-PCR. From these markers the expression of the interleukin (IL)-18 cytokine was significantly increased in the gut in patients with T1D compared with children with celiac disease or control children. The increased IL-18 expression lends further support for the hypothesis that the gut immune system is involved in the pathogenesis of T1D.

Microbe-induced Innate Immune Responses in Human Dendritic Cells

Two types of antigen-presenting cells (APCs), macrophages and dendritic cells (DCs), function at the interface of innate and adaptive immunity. Through recognition of conserved microbial patterns, they are able to detect the invading pathogens. This leads to activation of signal transduction pathways that in turn induce gene expression of various molecules required for immune responses and eventually pathogen clearance. Cytokines are among the genes induced upon detection of microbes. They play an important role in regulating host immune responses during microbial infection. Chemotactic cytokines, chemokines, are involved in migratory events of immune cells. Cytokines also promote the differentiation of distinct T cell responses. Because of the multiple roles of cytokines in the immune system, the cytokine network needs to be tightly regulated. In this work, the induction of innate immune responses was studied using human primary macrophages or DCs as cell models. Salmonella enterica serovar Typhimurium served as a model for an intracellular bacterium, whereas Sendai virus was used in virus experiments. The starting point of this study was that DCs of mouse origin had recently been characterized as host cells for Salmonella. However, only little was known about the immune responses initiated in Salmonella-infected human DCs. Thus, cellular responses of macrophages and DCs, in particular the pattern of cytokine production, to Salmonella infection were compared. Salmonella-induced macrophages and DCs were found to produce multiple cytokines including interferon (IFN) -gamma, which is conventionally produced by T and natural killer (NK) cells. Both macrophages and DCs also promoted the intracellular survival of the bacterium. Phenotypic maturation of DCs as characterized by upregulation of costimulatory and human leukocyte antigen (HLA) molecules, and production of CCL19 chemokine, were also detected upon infection with Salmonella. Another focus of this PhD work was to unravel the regulatory events controlling the expression of cytokine genes encoding for CCL19 and type III IFNs, which are central to DC biology. We found that the promoters of CCL19 and type III IFNs contain similar regulatory elements that bind nuclear factor kappaB (NF-kappaB) and interferon regulatory factors (IRFs), which could mediate transcriptional activation of the genes. The regulation of type III IFNs in virus infection resembled that of type I IFNs a cytokine class traditionally regarded as antiviral. The induction of type I and type III IFNs was also observed in response to bacterial infection. Taken together, this work identifies new details about the interaction of Salmonella with its phagocytic host cells of human origin. In addition, studies provide information on the regulatory events controlling the expression of CCL19 and the most recently identified IFN family genes, type III IFN genes.

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.

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.