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Abstract. There is great interest in developing efficient therapeutic cancer vaccines, as this type of therapy allows targeted killing of tumor cells as well as long-lasting immune protection. High levels of tumor-infiltrating CD8+ T cells are associated with better prognosis in many cancers, and it is expected that new generation vaccines will induce effective production of these cells. Epigenetic mechanisms can promote changes in host immune responses, as well as mediate immune evasion by cancer cells. Here, we focus on epigenetic modifications involved in both vaccine-adjuvant-generated T cell immunity and cancer immune escape mechanisms. We propose that vaccine-adjuvant systems may be utilized to induce beneficial epigenetic modifications and discuss how epigenetic interventions could improve vaccine-based therapies. Additionally, we speculate on how, given the unique nature of individual epigenetic landscapes, epigenetic mapping of cancer progression and specific subsequent immune responses, could be harnessed to tailor therapeutic vaccines to each patient.

Abstract. Stem cell differentiation depends on transcriptional activation driven by lineage‐specific regulators as well as changes in chromatin organization. However, the coordination of these events is poorly understood. Here, we show that T‐box proteins team up with chromatin modifying enzymes to drive the expression of the key lineage regulator, Eomes during endodermal differentiation of embryonic stem (ES) cells. The Eomes locus is maintained in a transcriptionally poised configuration in ES cells. During early differentiation steps, the ES cell factor Tbx3 associates with the histone demethylase Jmjd3 at the enhancer element of the Eomes locus to allow enhancer–promoter interactions. This spatial reorganization of the chromatin primes the cells to respond to Activin signalling, which promotes the binding of Jmjd3 and Eomes to its own bivalent promoter region to further stimulate Eomes expression in a positive feedback loop. In addition, Eomes activates a transcriptional network of core regulators of endodermal differentiation. Our results demonstrate that Jmjd3 sequentially associates with two T‐box factors, Tbx3 and Eomes to drive stem cell differentiation towards the definitive endoderm lineage.

Abstract. Cell-fate decisions and pluripotency are dependent on networks of key transcriptional regulators. Recent reports demonstrated additional functions of pluripotency-associated factors during early lineage commitment. The T-box transcription factor TBX3 has been implicated in regulating embryonic stem cell self-renewal and cardiogenesis. Here, we show that TBX3 is dynamically expressed during specification of the mesendoderm lineages in differentiating embryonic stem cells (ESCs) in vitro and in developing mouse and Xenopus embryos in vivo. Forced TBX3 expression in ESCs promotes mesendoderm specification by directly activating key lineage specification factors and indirectly by enhancing paracrine Nodal/Smad2 signaling. TBX3 loss-of-function analyses in the Xenopus underline its requirement for mesendoderm lineage commitment. Moreover, we uncovered a functional redundancy between TBX3 and Tbx2 during Xenopus gastrulation. Taken together, we define further facets of TBX3 actions and map TBX3 as an upstream regulator of the mesendoderm transcriptional program during gastrulation.

Summary. These observations indirectly add to the evidence for the hypothesis that elevated hepcidin levels may contribute to the development of atherosclerotic vascular disease, probably by causing macrophages to sequester iron, increasing oxidative stress and inducing their transformation into foam cells. Furthermore, our data in conjunction with those reported by Valenti et al. suggest that anti-hepcidin therapy might be beneficial in the prevention of atherosclerotic plaque forming in these patients.

Abstract. Monocyte infiltration across the endothelium is part of the innate immune response, however it may contribute to severity of chronic conditions. We have investigated the effects of iron on the cytokine-mediated recruitment of monocytes to the endothelium, using a physiological flow model and a monocyte transendothelial migration model. Under flow, iron loading to endothelial cells promoted an increased number of tumor necrosis factor-α-mediated firm arrest of human monocytes. Similarly, an increased number of firmly adhered monocytes were observed in conditions in which monocytes were iron-loaded, compared to the non-iron-loaded conditions. In both iron-loaded and non-iron-loaded conditions, blockade of the α4 and β2 integrins restored similar number and velocity of monocyte rolling, suggesting that iron did not modulate rolling interactions. However, with the integrin blockade, the number of firmly adhered cells remained higher in iron-loaded conditions than in control conditions, suggesting that iron could have modulated receptors other than the blocked integrins to promote firm arrest. Iron loading indeed upregulated expression of chemokine receptors, CC receptor-2 and CXC receptor-2, but not platelet endothelial cell adhesion molecule-1. This effect concomitantly promoted monocyte chemotactic protein-1-dependent transendothelial migration. In addition, iron-induced firm adhesion and transmigration were counteracted by iron chelation. These data reveal an immunomodulatory function of iron in the cascade of events of cytokine-mediated monocyte infiltration across endothelium, and therefore suggests the role for iron in inflammatory conditions underlying diseases like atherosclerosis and neurodegeneration.

Abstract. Epidemiological studies and experimental data suggest iron involvement in atherosclerosis. The relation between iron and atherosclerosis is complex and remains contradictory. In thalassemia patients, non transferrin bound iron (NTBI) and free hemoglobin (Hb) are present in plasma and may accelerate atherogenesis, but its progression may be inhibited by iron chelators. The mechanism whereby iron may stimulate atherogenesis has been intensively investigated. Non transferrin bound iron and sera from subjects with hemochromatosis induced endothelial activation with expression of vascular adhesion molecules and endothelial inflammatory chemokines. Such events could be inhibited by iron chelators and oxygen radical scavengers with intracellular activity. Iron chelators may be effective in preventing vascular damage in patients with high concentrations of NTBI as found in thalassemia.

Abstract. Heme oxygenase-1 (HO-1) contribution to iron homeostasis has been postulated, because it facilitates iron recycling by liberating iron mostly from heme catabolism. This enzyme also appears to be responsible for the resolution of inflammatory conditions. In a patient with HO-1 deficiency, inflammation and dysregulation of body iron homeostasis, including anemia and liver and kidney hemosiderosis, are evidenced. Here we postulated that HO-1 is critical in the regulation of ferroportin, the major cellular iron exporter, and hepcidin, the key regulator of iron homeostasis central in the pathogenesis of anemia of inflammation. Our current experiments in human THP-1 monocytic cells indicate a HO-1-induced iron-mediated surface-ferroportin expression, consistent with the role of HO-1 in iron recycling. Surprisingly, we observed low hepcidin levels in the HO-1-deficient patient, despite the presence of inflammation and hemosiderosis, both inducers of hepcidin. Instead, we observed highly increased soluble transferrin receptor levels. This suggests that the decreased hepcidin levels in HO-1 deficiency reflect the increased need for iron in the bone marrow due to the anaemia. Using human hepatoma cells, we demonstrate that HO-activity did not have a direct modulating effect on expression of HAMP, the gene that encodes for hepcidin. Therefore, we argue that the decreased iron recycling may, in part, have contributed to the low hepcidin levels. These findings indicate that dysregulation of iron homeostasis in HO-1 deficiency is the result of both defective iron recycling and erythroid activity-associated inhibition of hepcidin expression. This study therefore shows a crucial role for HO-1 in maintaining body iron balance.

Abstract. Hepcidin is a small liver-derived peptide central in the regulation of systemic iron homeostasis. Although the gene regulation has been extensively studied at transcriptional level, the corresponding effects on the production of bioactive peptide are largely unknown. We therefore applied a proteomics-based approach by combining immunocapture with time-of-flight mass spectrometry to characterize hepcidin-25 produced by hepatocyte-derived cell lines. Similar to its transcriptional regulation, mature hepcidin-25 was strongly secreted upon stimulation with BMPs and IL-6. The immunocaptured peptide down-modulated iron-exporter ferroportin on the monocyte/macrophage surface. Further mass spectrometry-based analyses indicated that hepcidin-25 in its bioactive conformation was very stable in serum and urine and not converted into its smaller isoforms. Hepcidin-25 was processed in the Golgi apparatus from its precursor, while the unprocessed prohepcidin was secreted only when furin-like protease activity was intracellularly inhibited. Furthermore, the amounts of hepatocytic secretion of hepcidin-25 are highly correlated with the gene transcript levels. An unexpected observation was the synergistic effect of BMPs and IL-6 on hepcidin-25 secretion, which points towards cross-talk between iron and inflammatory stimuli. The study underscores hepcidin-25 quantification as a valuable tool to unravel regulatory pathways in iron metabolism.

Abstract. Knowledge of hepcidin regulation is foremost gained by in vitro studies. We aimed to translate this knowledge into the human in vivo situation. Therefore, we measured serum markers as transferrin saturation (TS), soluble transferrin receptor (sTfR), and C-reactive protein (CRP) in parallel with hepcidin and prohepcidin in patients with iron metabolism disorders and controls. To assess sTfR as erythropoietic activity-associated factor in hepcidin regulation, we studied its influence on hepcidin expression in HepG2 cells. Results showed that sTfR highly associates with erythropoietic activity that strongly interfered with the iron store regulation of hepcidin. HepG2 expression results display an inverse association between hepcidin and sTfR. Inflammation was strongly related to increased hepcidin levels regardless of the iron store and erythropoietic activity status. In contrast, prohepcidin failed to correlate to any other parameter. In conclusion, these studies verify that previous conclusions based on in vitro studies on hepcidin regulation are also likely to apply to human patients. This is underscored by a simple algorithm, based on parameters reflecting the main regulating pathways, that accurately predict the actual measured hepcidin levels. Future studies are needed to validate the combined utility of this predictive algorithm together with actual measured hepcidin levels in clinical diagnosis.

Abstract. BACKGROUND: Chronic inflammation has been implemented in the pathogenesis of inflammatory diseases like atherosclerosis. Several pathogens like Chlamydia pneumoniae (Cp) and cytomegalovirus (CMV) result in inflammation and thereby are potentially artherogenic. Those infections could trigger endothelial activation, the starting point of the atherogenic inflammatory cascade. Considering the role of iron in a wide range of infection processes, the presence of iron may complicate infection-mediated endothelial activation. MATERIALS AND METHODS: Endothelial intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and endothelial selectin (E-selectin) expression were measured using flow cytometry, as an indication of endothelial activation. Cytotoxicity was monitored using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Immunostaining was applied to measure Cp and CMV infectivity to endothelial cells. RESULTS: An increased number of infected endothelial cells in a monolayer population leads to a raised expression of adhesion molecules of the whole cell population, suggesting paracrine interactions. Iron additively up-regulated Cp-induced VCAM-1 expression, whereas synergistically potentiated Cp-induced ICAM-1 expression. Together with CMV, iron also enhanced ICAM-1 and VCAM-1 expression. These iron effects were observed without modulation of the initial infectivity of both microorganisms. Moreover, the effects of iron could be reversed by intracellular iron chelation or radical scavenging, conforming modulating effects of iron on endothelial activation after infections. CONCLUSIONS: Endothelial response towards chronic infections depends on intracellular iron levels. Iron status in populations positive for Cp or CMV infections should be considered as a potential determinant for the development of atherosclerosis.

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Abstract. Nontransferrin-bound iron (NTBI) has been detected in iron overload diseases. This form of iron may exert pro-oxidant effects and modulate cellular function and inflammatory response. The present study has aimed to investigate the effects of serum NTBI on monocyte adherence to endothelium. Measured by a recently developed high-throughput fluorescence-based assay, serum NTBI was found to be higher in both homozygotes of HFE C282Y mutation of hereditary hemochromatosis (7.9+/-0.6 microM, n=9, P<0.001) and heterozygotes (4.0+/-0.5 microM, n=8, P<0.001), compared with controls (1.6+/-0.2 microM, n=21). The effects of these sera on monocyte adhesion and endothelial activation were examined. Adhesion of normal human monocytes to C282Y homozygote- and heterozygote-serum-treated human umbilical vein endothelial cells was higher (25.0+/-0.9 and 22.1+/-0.7%, respectively) compared with controls (17.6+/-0.5%, both P<0.001). For the three groups combined, the expression of adhesion molecules, ICAM-1, VCAM-1, and E-selectin, was positively correlated to NTBI levels but not to the inflammatory marker C-reactive protein. Furthermore, accumulation of intracellular labile iron and oxidative radicals within the cells due to NTBI was evidenced. Finally, counteraction of NTBI-induced endothelial activation was observed using iron chelators. These findings therefore identify a physiological function of NTBI in monocyte-endothelial interactions that may also contribute to the development of atherosclerosis and neurodegenerative diseases.

Abstract. OBJECTIVE: Elevated iron stores and high plasma iron concentration have been linked to an increased risk of atherosclerosis. Iron may thereby affect the interaction of monocytes to endothelium, an initial event in the formation of atherosclerotic plaques. METHODS AND RESULTS: Addition of 10 mumol/L non-transferrin-bound iron to the incubation medium caused a 2-fold increase in monocyte adhesion to human umbilical vein endothelial cells (HUVECs). A concordant increase in the expression of the following adhesion molecules was observed: vascular cell adhesion molecule-1, intercellular adhesion molecule-1, and endothelial selectin on HUVECs as well as very late antigen-4, and lymphocyte function-associated antigen-1 on monocytes. The inclusion of either deferiprone or salicylaldehyde isonicotinoylhydrazone counteracted these effects. Intracellular iron chelation by deferoxamine was completed only after 10 hours of incubation, shown by reversal of iron-quenched intracellular calcein signal, and concurrently the effects of iron were blunted. The membrane-impermeable chelator, diethylenetriamine pentaaceticacid, failed to negate iron effects, even after 48 hours of treatment. Furthermore, only membrane-permeable superoxide or hydroxyl radical scavengers were capable of preventing HUVEC activation by iron. CONCLUSIONS: Non-transferrin-bound iron increases the level of intracellular labile iron, which promotes monocyte recruitment to endothelium and may thereby contribute to the pathogenesis of atherosclerosis. Iron-induced adhesion molecule expression was observed, and this event may involve the production of oxygen radicals.

Abstract. Besides the fact that it is a vital element in life, iron may also participate in diverse pathological processes.It has been hypothesised that iron is involved in the development of atherosclerosis and related cardiovascular diseases. Several epidemiological studies as well as in vivo and in vitro experiments are in favour for this iron hypothesis, although some studies have yielded conflicting results. This review describes iron as a risk factor of atherosclerosis,through its involvement in the process of monocyte adhesion to endothelium, a crucial event of atherosclerotic plaque formation. Furthermore, the benefits of iron chelators in preventing this process are reviewed.

Summary. This report highlights key features of recent studies performed by NAMRU-2, LITBANGKES and collaborators in investigating acquired immunity to and innate protection against Plasmodium falciparum in Javanese transmigrant adults and children newly resident in malaria-endemic Papua, Indonesia. The findings show that the development of humoral immunity to defined pre-erythrocytic and erythrocytic antigens of P. falciparum is dependent not only on cumulative exposure, but also critically on age. The inability of children to generate adult-like anti-parasite immunity may be the key factor responsible for the poor clinical outcomes to chronic infection that occur in this age group. Furthermore,these findings may be critical not only for our understanding of how naturally acquired immunity to malaria develops in humans, but also for the development of effective malaria vaccination strategies

Abstract. A human gene has been reported that may encode the enzyme acetohydroxyacid synthase. Previously this enzyme was thought to be absent from animals although it is present in plants and many microorganisms. In plants, this enzyme is the target of a number of commercial herbicides and the use of these compounds may need to be reassessed if the human enzyme exists and proves to be susceptible to inhibition. Here we report the construction of several plasmid vectors containing the cDNA sequence for this protein, and their expression in Escherichia coli. High levels of expression were observed, but most of the protein proved to be insoluble. The small amounts of soluble protein contained little or no acetohydroxyacid synthase activity. Attempts to refold the insoluble protein were successful in so far as the protein became soluble. However, the refolded protein did not gain any acetohydroxyacid synthase activity. In vivo complementation tests of an E. coli mutant produced no evidence that the protein is active. Incorrect folding, or the lack of another subunit, may explain the data but we favor the interpretation that this gene does not encode an acetohydroxyacid synthase.

Abstract. Atherosclerotic heart disease has claimed the lives of millions of people each year in the Western world, and rapidly increases in prevalence in many other places. The disease results from cholesterolemia, coupled with a chronic inflammatory condition of the vascular wall that lead to vessel occlusion and various clinical manifestations.In 1981, Sullivan suggested possible benefits of iron depletion against coronary artery disease, from the observations where men and menopausal women were more prone to develop this disease. Following Sullivan’s proposal, basic and clinical data have begun to provide explanations for a link between iron and atherosclerosis (Chapter 1). This thesis has aimed to investigate the role of iron in the inflammatory events crucial in atherogenesis, especially in the course of phagocyte-endothelial cell interactions.In vitro studies described in this thesis reveals an immunomodulatory function of iron in inflammation. Chapter 2 and Chapter 3 show that NTBI promotes accumulation of intracellular labile iron and production of oxygen-derived free radicals, leading to cell activation. Activation of endothelial cells is a well-characterized phenotype that leads to endothelial dysfunction in vivo, which not only will initiate the development of atherosclerosis, but also plays a role at a critical late step of thrombosis, promoting vessel occlusion and acute cardiovascular events. In combination with chronic infections iron enhances infection-induced endothelial activation (Chapter 4), implying that in the presence of other stressors, iron may contribute significantly in aggravating atherogenesis. In addition, in Chapter 6, the drawbacks of EDTA chelation therapy particularly in inducing endothelial activation are described. The use of this alternative therapy for atherosclerosis therefore needs to be critically reconsidered, especially in regards to its effectiveness and safety. Furthermore, iron also promotes monocyte (Chapter 2 and Chapter 5) and neutrophil (Chapter 7) activation. An increased number of phagocyte infiltrates may thus complicate the progression of atherosclerotic vascular diseases. The infiltration of monocytes also play a role in neuroinflammation, stimulating the development of neurodegenerative diseases. Finally, Chapter 8 demonstrates the involvement of iron in enhancing the rate and the production of oxidised LDL cholesterol, especially through interactions with phagocyte-derived oxygen metabolites. Based on the findings in this thesis, iron depletion and iron chelation could be beneficial for such unfavourable conditions generated by iron. The identified modifying role of iron in inflammation described in this study (Chapter 9) offers an additional strategy for prevention and therapy of inflammatory diseases, like atherosclerotic vascular diseases and neurodegenerative diseases.

Summary. There is growing evidence for the role of iron in the development of atherosclerosis and related cardiovascular diseases. With all the possible confounding factors being taken into account, the epidemiological studies suggest a positive correlation between body iron level and this vascular disease. In vitro studies show several possible mechanisms in which iron may play a role in atherosclerosis. In vivo studies confirm the mechanisms of iron action in atherosclerotic plaque formation. The animal studies further validate the involvement of iron in this disease. Furthermore, elevated body iron level and NTBI have been observed in the carriers of hereditary hematochromatosis. The affected HFE gene has a prevalence of 10% in caucasian population. Together with the tendency of having iron overload in the modern world, many more people may thereby suffer from the risk of developing early cardiovascular disease.