Endothelial cells play a critical role in inflammation by responding to several endogenous and exogenous proinflammatory stimuli. The three most studied factors that provide inflammatory signals to endothelial cells are lipopolysaccharide (LPS), tumor necrosis factor (TNF)-a, and interleukin (IL)-1b; however, their effects on endothelial cells were thoroughly compared at the level of gene expression only. Therefore, our aim was to assess the differences in the signaling pathways, adhesion molecules, and cytokines induced by proinflammatory factors in human umbilical vein endothelial cells (HUVEC). In this study, we demonstrated that signaling of LPS was less effective than that of IL-1b, and was significantly slower than that of TNF-a and IL-1b, which can be partially explained by the special localization of Toll-like receptor 4 (TLR4). We showed that TLR4 is mainly localized in Golgi apparatus in HUVEC. The proinflammatory capacity of TNF-a was similar to that of IL-1b in inducing NF-jB nuclear translocation, while IL-1b was the strongest activator of MAPK pathways. Moreover, expression of E-selectin, IL-6, and IL-8 was induced most efficiently by IL-1b, while LPS and TNF-a had less effect, whereas we did not find such a difference in ICAM-1 and MCP-1 expression. Due to the higher induction of E-selectin and IL-8, IL-1b might have more important role in neutrophil recruitment than LPS and TNF-a. By above-mentioned parameters we identified a signaling and expression pattern for the three proinflammatory molecules. This pattern illustrates how complex a proinflammatory process can be, and may enable us to predict and compare the pathomechanism of various inflammatory diseases. '

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Proteins are essential elements for life. They are building blocks of all organisms and the operators of cellular functions. Humans produce a repertoire of at least 30,000 different proteins, each with a different role. Each protein has its own unique sequence and shape (native conformation) to fulfill its specific function. The appearance of incorrectly shaped (misfolded) proteins occurs on exposure to environmental changes. Protein misfolding and the subsequent aggregation is associated with various, often highly debilitating, diseases for which no sufficient cure is available yet. In the first part of this review we summarize the structural composition of proteins and the current knowledge of underlying forces that lead proteins to lose their native structure. In the second and third parts we describe the molecular and cellular mechanisms that are associated with protein misfolding in disease. Finally, in the last part we portray recent efforts to develop treatments for protein misfolding diseases.

Activation of T-helper cells is dependent upon the appropriate presentation of antigen-derived peptides on MHC class II molecules expressed on antigen presenting cells.In the current study we explored the repertoire of peptides presented on MHC class II molecules on human monocyte derived dendritic cells (moDCs) from four HLA-typed healthy donors. MHC class II-bound peptides could be routinely recovered from small cultures containing 5 ؋ 10 6 cells. A fraction of the identified peptides were derived from proteins localized in the plasma membrane, endosomes, and lysosomes, but the majority of peptides that were presented on MHC class II originate from other organelles. Subsequently, we studied the antigen-specific peptide repertoire after endocytosis of a soluble antigen. Blood coagulation factor VIII (FVIII) was chosen as the antigen since our current knowledge on MHC class II presented peptides derived from this immunogenic therapeutic protein is limited. Analysis of the total repertoire of MHC class II-associated peptides revealed that per individual sample 20 -50 FVIII-derived peptides were presented on FVIII-pulsed moDCs. Repertoires of FVIII-derived peptides eluted from moDCs derived from a panel of four HLA typed donors revealed that some MHC class II-presented FVIII peptides were presented by multiple donors, whereas the presentation of other FVIII peptides was donor-specific. In total 32 different core peptides were presented on FVIII-pulsed moDCs from four HLAtyped donors. Together our findings provide an unbiased approach to identify peptides that are presented by MHC class II on antigen-loaded moDCs from individual donors.

Objective-Protein misfolding diseases result from the deposition of insoluble protein aggregates that often contain fibrils called amyloid. Amyloids are found in Alzheimer disease, atherosclerosis, diabetes mellitus, and systemic amyloidosis, which are diseases where platelet activation might be implicated. Methods and Results-We induced amyloid properties in 6 unrelated proteins and found that all induced platelet aggregation in contrast to fresh controls. Amyloid-induced platelet aggregation was independent of thromboxane A 2 formation and ADP secretion but enhanced by feedback stimulation through these pathways. Treatments that raised cAMP (iloprost), sequestered Ca 2ϩ (BAPTA-AM) or prevented amyloid-platelet interaction (sRAGE, tissue-type plasminogen activator [tPA]) induced almost complete inhibition. Modulation of the function of CD36 (CD36 Ϫ/Ϫ mice), p38 MAPK (SB203580), COX-1 (indomethacin), and glycoprotein Ib␣ (Nk-protease, 6D1 antibody) induced Ϸ50% inhibition. Interference with fibrinogen binding (RGDS) revealed a major contribution of ␣ IIb ␤ 3 -independent aggregation (agglutination). Conclusions-Protein misfolding resulting in the appearance of amyloid induces platelet aggregation. Amyloid activates platelets through 2 pathways: one is through CD36, p38 MAPK , thromboxane A 2 -mediated induction of aggregation; the other is through glycoprotein Ib␣-mediated aggregation and agglutination. The platelet stimulating properties of amyloid might explain the enhanced platelet activation observed in many diseases accompanied by the appearance of misfolded proteins with amyloid. Key Words: amyloid Ⅲ platelet activation Ⅲ sRAGE Ⅲ tissue plasminogen activator Ⅲ CD36 Ⅲ glycoprotein Ib␣ P roteins typically adopt a well-defined 3-dimensional structure. There is now an increasing amount of evidence that abnormalities in this process have far reaching consequences for human health. Certain mutations and posttranslational modifications such as glycation and oxidation interfere with proper folding, resulting in protein misfolding, aggregation, and ultimately polymerization into insoluble fibrils called amyloid. 1,2 The term amyloidosis defines a group of systemic and localized diseases associated with the deposition of amyloid in different tissues. Alzheimer disease is caused by abnormal folding of amyloid-␤ and formation of amyloid-rich plaques that obstruct neurons and microvessels of the brain. 3 One has argued that these plaques cause the hyperreactivity of platelets observed in these patients as illustrated by P-selectin positive platelets and increased levels of urinary thromboxane A 2 metabolite. 4,5 An environmental risk factor for Alzheimer disease is Herpes Simplex virus. It contains glycoprotein B, a protein fragment that assembles into fibrils that are ultrastructurally indistinguishable from amyloid-␤. 6 Protein misfolding is not restricted to Alzheimer disease but is a common feature in the pathology of atherosclerosis, diabetes mellitus, and systemic amyloidosis. 7-9 Atherosclerotic plaques contain ...