Chromatin interactions in differentiating keratinocytes reveal novel atopic dermatitis– and psoriasis-associated genes

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Sarcomeric determinants of striated muscle relaxation kinetics

Ca²⁺ is the primary regulator of force generation by cross-bridges in striated muscle activation and relaxation. Relaxation is as necessary as contraction and, while the kinetics of Ca²⁺-induced force development have been investigated extensively, those of force relaxation have been both studied and understood less well. Knowledge of the molecular mechanisms underlying relaxation kinetics is of special importance for understanding diastolic function and dysfunction of the heart. A number of experimental models, from whole muscle organs and intact muscle fibres down to single myofibrils, have been used to explore the cascade of kinetic events leading to mechanical relaxation. By using isolated myofibrils and fast solution switching techniques we can distinguish the sarcomeric mechanisms of relaxation from those of myoplasmic Ca²⁺ removal. There is strong evidence that cross-bridge mechanics and kinetics are major determinants of the time course of striated muscle relaxation whilst thin filament inactivation kinetics and cooperative activation of thin filament by cycling, force-generating cross-bridges do not significantly limit the relaxation rate. Results in myofibrils can be explained well by a simple two-state model of the cross-bridge cycle in which the apparent rate of the force generating transition is modulated by fast, Ca²⁺-dependent equilibration between off- and on-states of actin. Inter-sarcomere dynamics during the final rapid phase of full force relaxation are responsible for deviations from this simple model.     

T cell activation correlates with an increased proportion of antigen among the materials acquired from target cells

We have investigated the density of peptides required to elicit different biological responses in cytotoxic T lymphocytes (CTL), including trogocytosis (i.e., the phenomenon whereby the lymphocytes actively capture fragments of plasma membrane from those cells with which they establish an immune synapse). We have used two separate mouse models of CTL recognising defined peptides presented by MHC class I molecules. In both systems, triggering of cytotoxicity and capture of membrane components reached saturation with low densities of ligand. On the other hand, down-modulation of cell-surface levels of TCR, induction of IFN-gamma production and detection of peptide captured required much higher ligand densities. Interestingly, fratricide (i.e., killing between CTL sharing the same specificity), a mechanism proposed to account for CTL exhaustion, was detected only at antigen concentrations still well above that second threshold leading to full blown activation. Taken together, our results show that the different thresholds that govern the elicitation of different CTL functions correlate with different proportions of antigen among the target cell components being captured via trogocytosis.     

Fluorescence polarization assay by flow cytometry

Fluorescence polarization measurement on cell suspensions provides a highly sensitive means for detecting subtle changes in the cells, such as occur early after lymphocyte activation or on malignant transformation. We review here the principles of fluorescence polarization, its measurement by a commercially available flow cytometer and application of such assays especially in cellular immunology.     

Heterogeneity in the complement-dependent bacteriolysis within the species of Borrelia burgdorferi

Sixteen Borrelia burgdorferi strains, including all three species, were compared in a colorimetric bactericidal assay for their ability to escape the complement-dependent bacteriolysis on incubation in normal human serum free of specific antibodies (NHS). The species B. afzelii was found to be serum resistant (EB1, EB3, FEM1, FEM2, Pko), whereas strains of the species B. garinii were found to be serum sensitive (1/B29, G1, G2, PSth, PBr, PTrob). Six strains, mainly B. burgdorferi sensu stricto, were only partially sensitive (Z25, 297, B31, PKa-I, PBi). All strains activated the complement cascade in NHS, whereas only four strains (G1, G2, PBr, PSth) could activate complement in the presence of EGTA-Mg. After complement activation, covalently bound C3 fragments (C3b, iC3b) were detected on serum-sensitive as well as serum-resistant borrelial strains. Heterogeneity, however, was observed between serum-resistant and serum-sensitive strains with respect to deposition of C6 and C9. Whereas serum-sensitive strains were strongly positive for C6 and C9 and were, therefore, killed by the terminal complement complex (TCC), serum-resistant strains were devoid of C6 and C9 on their cell surface. The serum resistance may, therefore, be due to an absent or only transient formation of TCC on the bacterial surface. Received: 17 September 1996     

Protective effect of retinoic acid on interleukin-1 beta-induced cytotoxicity of pancreatic beta-cells

Cytokines produced by immune cells in pancreatic islets infiltrating are important mediators of beta-cell destruction in insulin-dependent diabetes mellitus. In this study, the effects of retinoic acid (RA) on cytokine-induced beta-cell dysfunction were examined. RA significantly protected interleukin-1 beta (IL-1) and interferon-gamma (IFN-gamma)-mediated cytotoxicity of rat insulinoma cell (RINm5F), and also reduced in IL-1 and IFN-gamma-induced nitric oxide (NO) production, which correlated well with reduced levels of the inducible form of NO synthase (iNOS) mRNA and protein. The molecular mechanism, by which RA inhibited iNOS gene expression, appeared to involve the inhibition of NF-kappa B activation. Our results suggest possible therapeutic value of RA for the prevention of diabetes mellitus progression.     

Dissecting natural killer cell activation pathways through analysis of genetic mutations in human and mouse

Summary:  Natural killer (NK) cell cytotoxicity is mediated by multiple germ line-encoded activating receptors that recognize specific ligands expressed by tumor cells and virally infected cells. These activating receptors are opposed by NK inhibitory receptors, which recognize major histocompatibility complex class I molecules on potential targets, raising the threshold for NK cell activation. Once an abnormal cell has been detected, NK cells are the sentinel source of cytolytic mediators, such as granzymes and perforins, as well as interferon-γ, which can polarize the immune response to a T-helper 1 cell type. Activation signals are transmitted by adhesion-dependent pathways, immunoreceptor tyrosine-based activation motif (ITAM)-dependent pathways, DAP10 ITAM-independent pathways, and by signaling through immunoreceptor tyrosine-based switch motifs. These pathways activate downstream signaling partners to trigger NK cell cytotoxicity. Some of these downstream molecules are unique to the various pathways, and some of these molecules are shared. Because of the complexity of signals involved in NK cell–target cell interaction, the generation of mice with targeted mutations in signaling molecules involved in adhesion, activation, or inhibition is essential for a precise dissection of the mechanisms regulating NK cell effector functions. Here we review recent advances in the genetic analysis of the signaling pathways that mediate NK cell killing.     

Induction of NO synthesis in macrophages by Newcastle disease virus is associated with activation of nuclear factor-{kappa}B

Newcastle disease virus (NDV) has received much attention recentlybecause of its non-specific immune stimulating potential andits various anti-tumor activities. Here we describe that NDVinduces synthesis of NO and causes an activation of nuclearfactor-kB (NF-kB) In murine macrophages. These reactions werepart of an activation process which included also stimulationof adenosine deaminase and inhibition of 5'-nucleotidase. NDV-mediatedNO synthesis and NF-kB activation were blocked by an antioxidant(butylated hydroxyanisole), by an inhibitor of protein tyrosinekinase (genistein) and of protein kinase A (H-89), but not byan inhibitor of protein kinase C (staurosporin). These datasuggest that signalling requirements of NF-kB activation andNO production in NDV-treated macrophages are similar.     

Efficacy of HIV-specific and 'antibody-independent' mechanisms for complement activation by HIV-infected cells.

Previous studies in this laboratory have shown that efficient activation of complement (C) on HIV isolates and HIV-infected cells requires the binding of specific anti-HIV antibodies, while other investigators have observed 'antibody-independent' C activation. In an attempt to clarify these disparate findings, we investigated the effect of several variables on C activation by HIV-infected cells using flow cytometric analysis of C3 deposition. Antibody-mediated C activation using pooled sera from infected persons or human MoAbs directed against the V3 region of gp120 was always substantially higher than activation without antibody. Normal human serum (NHS) from a subset of HIV antibody-negative donors did, however, induce low levels of C3 deposition. Differences in C3 activation between the various NHS did not correlate with total haemolytic C levels or mannose-binding protein (MBP) levels. IgM isolated from NHS that induced high levels of C activation was at least partly responsible for the 'antibody-independent' C activation. Although there appeared to be a correlation between NHS that induced C activation and the presence of anti-blood type B IgM, absorption of anti-B did not abrogate the C3 deposition. Additionally, MoAb to the B antigen did not induce C3 deposition. These studies show that IgM in sera from HIV-uninfected donors can induce C3 deposition on HIV-infected cells, but that specific antibody-dependent C activation is substantially more efficient. Therefore, 'antibody-independent' C activation on HIV-infected cells may, in some cases, be more accurately described as HIV-cross-reactive antibody-dependent C activation.