4,272 research outputs found
Modulation of endothelial hemostatic properties: an active role in the host response.
No full textAs the cells forming the luminal vascular surface, endothelium regulates both barrier function, and pro- and anticoagulant reactions. Endothelial cells can do this by controlling the expression of cell surface molecules, such as receptors that regulate the hemostatic balance and those that affect permeability across the endothelial monolayer. This regulation occurs in response to environmental stimuli, such as cytokines, which have a central role in inflammation, or glucose-modified proteins, which accumulate in the vasculature in aging and diabetes and are associated with vascular complications. The endothelial cell emerges as a dynamic regulator maintaining homeostasis in the quiescent state and contributing to the pathogenesis of vascular lesions in the stimulated state
Stern-judging: A simple, successful norm which promotes cooperation under indirect reciprocity.
Get PDFWe study the evolution of cooperation under indirect reciprocity, believed to constitute the biological basis of morality. We employ an evolutionary game theoretical model of multilevel selection, and show that natural selection and mutation lead to the emergence of a robust and simple social norm, which we call stern-judging. Under stern-judging, helping a good individual or refusing help to a bad individual leads to a good reputation, whereas refusing help to a good individual or helping a bad one leads to a bad reputation. Similarly for tit-for-tat and win-stay-lose-shift, the simplest ubiquitous strategies in direct reciprocity, the lack of ambiguity of stern-judging, where implacable punishment is compensated by prompt forgiving, supports the idea that simplicity is often associated with evolutionary success
Endothelium and regulation of coagulation
No full textAdvanced glycosylation end products (AGE) of proteins accumulate in the vasculature with diabetes and aging, and are thought to be associated with vascular complications. This led us to examine the interaction of AGE-BSA as a prototype of this class of nonenzymatically glycosylated proteins subjected to further processing, with endothelium. Incubation of 125I-AGE-BSA with cultured bovine endothelium resulted in time-dependent, saturable binding that was half-maximal at a concentration of approximately 100 nM. Although unlabeled normal BSA was not a competitor, unlabeled AGE-BSA was an effective competitor of 125I-AGE-BSA-endothelial cell interaction. In addition, AGE modification of two alternative proteins, hemoglobin and ribonuclease, rendered them inhibitors of 125I-AGE-BSA binding to endothelium, although the native, unmodified forms of these proteins were not. At 37 degrees C, binding of 125I-AGE-BSA or gold-labeled AGE-BSA was followed by internalization and subsequent segregation either to a lysosomal compartment or to the endothelial-derived matrix after transcytosis. Exposure of endothelium to AGE-BSA led to perturbation of two important endothelial cell homeostatic properties, coagulant and barrier function. AGE-BSA downregulated the anticoagulant endothelial cofactor thrombomodulin, and induced synthesis and cell surface expression of the procoagulant cofactor tissue factor over the same range of concentrations that resulted in occupancy of cell surface AGE-BSA binding sites. In addition, AGE-BSA increased endothelial permeability, resulting in accelerated passage of an inert macromolecular tracer, [3H]inulin, across the monolayer. These results indicate that AGE derivatives of proteins, potentially important constituents of pathologic vascular tissue, bind to specific sites on the endothelial cell surface and modulate central endothelial cell functions. The interaction of AGE-modified proteins with endothelium may play an important role in the early stages of increased vascular permeability, as well as vessel wall-related abnormalities of the coagulation system, characteristic of diabetes and aging
Studies of HLA-DM in Antigen Presentation and CD4+ T Cell Epitope Selection: A Dissertation
Get PDFAntigen presented to CD4+ T cells by major histocompatibility complex class II molecules (MHCII) plays a key role in adaptive immunity. Antigen presentation is initiated by the proteolytic cleavage of pathogenic or self proteins and loading of resultant peptides to MHCII. The loading and exchange of peptides to MHCII is catalyzed by a nonclassical MHCII molecule, HLA-DM (DM). It is well established that DM promotes peptide exchange in vitro and in vivo. However, the mechanism of DM-catalyzed peptide association and dissociation, and how this would affect epitope selection in human responses to infectious disease remain unclear. The work presented in this thesis was directed towards the understanding of mechanism of DM-mediated peptide exchange and its role in epitope selection. In Chapter II, I measured the binding affinity, intrinsic dissociation half-life and DM-mediated dissociation half-life for a large set of peptides derived from vaccinia virus and compared these properties to the peptide-specific CD4+ T cell responses. These data indicated that DM shapes the peptide repertoire during epitope selection by favoring the presentation of peptides with greater DM-mediated kinetic stability, and DM-susceptibility is a strong and independent factor governing peptide immunogenicity. In Chapter III, I computationally simulated peptide binding competition reactions and found that DM influences the IC50 (50% inhibition concentration) of peptides based on their susceptibility to DM, which was confirmed by experimental data. Therefore, I developed a novel fluorescence polarization-based method to measure DM-susceptibility, reported as a IC50 (change in IC50 in the absence and presence of DM). Traditional assays to measure DM-susceptibility based on differential peptide dissociation rates are cumbersome because each test peptide has to be individually labeled and multiple time point samples have to be collected. However, in this method developed here only single probe peptide has to be labeled and only single reading have to be done, which allows for fast and high throughput measure of DM-susceptibility for a large set of peptides. In Chapter IV, we generated a series of peptide and MHCII mutants, and investigated their interactions with DM. We found that peptides with non-optimal P1 pocket residues exhibit low MHCII affinity, low kinetic stability and high DM-susceptibility. These changes were accompanied with conformational alterations detected by surface plasmon resonance, gel filtration, dynamic light scattering, small-angle X-ray light scattering, antibody-binding, and nuclear magnetic resonance assays. Surprisingly, all these kinetic and conformational changes could be reversed by reconstitution with a more optimal P9 pocket residue. Taken together, our data demonstrated that conformation of MHCII-peptide complex constrained by interactions throughout the peptide binding groove is a key determinant of DM-susceptibility. B cells recognizing cognate antigen on the virion can internalize and process the whole virion for antigen presentation to CD4+ T cells specific for an epitope from any of the virion proteins. In turn, the epitope-specific CD4+ T cells provide intermolecular (also known as noncognate or heterotypic) help to B cells to generate antibody responses against any protein from the whole virion. This viral intermolecular help model in which CD4+ T cells provide help to B cells with different protein specificities was established in small size influenza virus, hepatitis B virus and viral particle systems. For large and complex pathogens such as vaccinia virus and bacteria, the CD4+ T cell-B cell interaction model may be complicated because B cells might not be able to internalize the large whole pathogen. Recently, a study in mice observed that CD4+ T cell help is preferentially provided to B cells with the same protein specificity to generate antibody responses against vaccinia virus. However, for larger pathogens such as vaccinia virus and bacteria the CD4+ T cell-B cell interaction model has yet to be tested in humans. In Chapter V, I measured in 90 recently vaccinated and 7 long-term vaccinia-immunized human donors the CD4+ T cell responses and antibody responses against four vaccinia viral proteins (A27L, A33R, B5R and L1R) known to be strongly targeted by cellular and humoral responses. We found that there is no direct linkage between antibody and CD4+ T cell responses against each protein. However, the presence of immune responses against these four proteins is linked together within donors. Taken together, our data indicated that individual viral proteins are not the primary recognition unit and CD4+ T cells provide intermolecular help to B cells to generate robust antibody responses against large and complicated vaccinia virus in humans.Immunology and Microbiolog
HLA-DM focuses on conformational flexibility around P1 pocket to catalyze peptide exchange
No full textPeptides presented by major histocompatibility complex class II (MHCII) molecules to CD4+ T cells play a central role in the initiation of adaptive immunity. This antigen presentation process is characterized by the proteolytic cleavage of foreign and self proteins, and loading of the resultant peptides onto MHCII molecules. Loading and exchange of antigenic peptides is catalyzed by a non-classical MHCII molecule, HLA-DM. The impact of HLA-DM on epitope selection has been appreciated for a long time. However, the molecular mechanism by which HLA-DM mediates peptide exchange remains elusive. Here, we review recent efforts in elucidating how HLA-DM works, highlighted by two recently solved co-structures of HLA-DM bound to HLA-DO (a natural inhibitor of HLA-DM), or to HLA-DR1 (a common MHCII). In light of these efforts, a model for HLA-DM action in which HLA-DM utilizes conformational flexibility around the P1 pocket of the MHCII-peptide complex to catalyze peptide exchange is proposed
Distinct Intracellular Compartments Involved in Invariant Chain Degradation and Antigenic Peptide Loading of Major Histocompatibility Complex (MHC) Class II Molecules
Get PDFMajor histocompatibility complex (MHC) class II molecules are transported to intracellular MHC class II compartments via a transient association with the invariant chain (Ii). After removal of the invariant chain, peptides can be loaded onto class II molecules, a process catalyzed by human leukocyte antigen-DM (HLA-DM) molecules. Here we show that MHC class II compartments consist of two physically and functionally distinct organelles. Newly synthesized MHC class II/Ii complexes were targeted to endocytic organelles lacking HLA-DM molecules, where Ii degradation occurred. From these organelles, class II molecules were transported to a distinct organelle containing HLADM, in which peptides were loaded onto class II molecules. This latter organelle was not directly accessible via fluid phase endocytosis, suggesting that it is not part of the endosomal pathway. Uptake via antigen-specific membrane immunoglobulin resulted however in small amounts of antigen in the HLA-DM positive organelles. From this peptide-loading compartment, class II–peptide complexes were transported to the plasma membrane, in part after transit through endocytic organelles. The existence of two separate compartments, one involved in Ii removal and the other functioning in HLA-DM–dependent peptide loading of class II molecules, may contribute to the efficiency of antigen presentation by the selective recruitment of peptide-receptive MHC class II molecules and HLA-DM to the same subcellular location
ELECTROCHEMICAL BEHAVIOR OF TIN IN THE NaCl SOLUTION CONCENTRATION 0.1 mol dm-3 : bachelor thesis
No full textIspitano je korozijsko ponašanje kositra u 0,1 mol dm -3 otopini NaCl metodama linearne polarizacije u uskom i širokom području potencijala oko potencijala otvorenog strujnog kruga te metodom elektrokemijske impedancijske spektroskopije tijekom 5 sati boravka elektrode u elektrolitu. Polarizacijske krivulje analizirane su metodom ekstrapolacije Tafelovih pravaca te metodom Stern-Geary i određeni su korozijski kinetički parametri. Impedancijski spektri kositra u elektrolitu modelirani su ekvivalentnim električnim krugom s dvije vremenske konstante.Corrosion behavior of tin in 0,1 mol dm -3 NaCl solution was tested using linear polarization methods in a narrow and wide potential range around the open circuit potential and electrochemical impedance spectroscopy during 5 hours of the electrode immersion in the electrolyte. The polarization curves were analyzed using Tafel line extrapolation method and the Stern-Geary method and the corrosion kinetic parameters were determined. The impedance spectra of tin in the electrolyte are modeled by an equivalent electrical circuit with two time constants
ELECTROCHEMICAL BEHAVIOR OF TIN IN THE NaCl SOLUTION CONCENTRATION 0.1 mol dm-3 : bachelor thesis
No full textIspitano je korozijsko ponašanje kositra u 0,1 mol dm -3 otopini NaCl metodama linearne polarizacije u uskom i širokom području potencijala oko potencijala otvorenog strujnog kruga te metodom elektrokemijske impedancijske spektroskopije tijekom 5 sati boravka elektrode u elektrolitu. Polarizacijske krivulje analizirane su metodom ekstrapolacije Tafelovih pravaca te metodom Stern-Geary i određeni su korozijski kinetički parametri. Impedancijski spektri kositra u elektrolitu modelirani su ekvivalentnim električnim krugom s dvije vremenske konstante.Corrosion behavior of tin in 0,1 mol dm -3 NaCl solution was tested using linear polarization methods in a narrow and wide potential range around the open circuit potential and electrochemical impedance spectroscopy during 5 hours of the electrode immersion in the electrolyte. The polarization curves were analyzed using Tafel line extrapolation method and the Stern-Geary method and the corrosion kinetic parameters were determined. The impedance spectra of tin in the electrolyte are modeled by an equivalent electrical circuit with two time constants
ELECTROCHEMICAL BEHAVIOR OF TIN IN THE NaCl SOLUTION CONCENTRATION 0.1 mol dm-3 : bachelor thesis
No full textIspitano je korozijsko ponašanje kositra u 0,1 mol dm -3 otopini NaCl metodama linearne polarizacije u uskom i širokom području potencijala oko potencijala otvorenog strujnog kruga te metodom elektrokemijske impedancijske spektroskopije tijekom 5 sati boravka elektrode u elektrolitu. Polarizacijske krivulje analizirane su metodom ekstrapolacije Tafelovih pravaca te metodom Stern-Geary i određeni su korozijski kinetički parametri. Impedancijski spektri kositra u elektrolitu modelirani su ekvivalentnim električnim krugom s dvije vremenske konstante.Corrosion behavior of tin in 0,1 mol dm -3 NaCl solution was tested using linear polarization methods in a narrow and wide potential range around the open circuit potential and electrochemical impedance spectroscopy during 5 hours of the electrode immersion in the electrolyte. The polarization curves were analyzed using Tafel line extrapolation method and the Stern-Geary method and the corrosion kinetic parameters were determined. The impedance spectra of tin in the electrolyte are modeled by an equivalent electrical circuit with two time constants
A novel method to measure HLA-DM-susceptibility of peptides bound to MHC class II molecules based on peptide binding competition assay and differential IC(50) determination
No full textHLA-DM (DM) functions as a peptide editor that mediates the exchange of peptides loaded onto MHCII molecules by accelerating peptide dissociation and association kinetics. The relative DM-susceptibility of peptides bound to MHCII molecules correlates with antigen presentation and immunodominance hierarchy, and measurement of DM-susceptibility has been a key effort in this field. Current assays of DM-susceptibility, based on differential peptide dissociation rates measured for individually labeled peptides over a long time base, are difficult and cumbersome. Here, we present a novel method to measure DM-susceptibility based on peptide binding competition assays performed in the presence and absence of DM, reported as a delta-IC(50) (change in 50% inhibition concentration) value. We simulated binding competition reactions of peptides with various intrinsic and DM-catalyzed kinetic parameters and found that under a wide range of conditions the delta-IC(50) value is highly correlated with DM-susceptibility as measured in off-rate assay. We confirmed experimentally that DM-susceptibility measured by delta-IC(50) is comparable to that measured by traditional off-rate assay for peptides with known DM-susceptibility hierarchy. The major advantage of this method is that it allows simple, fast and high throughput measurement of DM-susceptibility for a large set of unlabeled peptides in studies of the mechanism of DM action and for identification of CD4+ T cell epitopes.Immunology and Microbiolog
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