Summary Glycosylceramides in mammalian species are thought to be present in the form of β-anomers. This conclusion was reinforced by the identification of only one glucosylceramide and one galactosylcerhamide synthase, both β-transferases, in mammalian genomes. Thus, the possibility that small amounts of α-anomers could be produced by an alternative enzymatic pathway, by an unfaithful enzyme, or spontaneously in unusual cellular compartments has not been examined in detail. We approached the question by taking advantage of the exquisite specificity of T and B lymphocytes and combined it with the specificity of catabolic enzymes of the sphingolipid pathway. Here, we demonstrate that mammalian immune cells produce constitutively very small quantities of α-glycosylceramides, which are the major endogenous ligands of natural killer T cells. Catabolic enzymes of the ceramide and glycolipid pathway tightly control the amount of these α-glycosylceramides. The exploitation of this pathway to manipulate the immune response will create new therapeutic opportunities.
Polyvalent interactions allow biological structures to exploit low-affinity ligand–receptor binding events to affect physiological responses. We describe here the use of bacteriophage Qβ as a multivalent platform for the display of polycationic motifs that act as heparin antagonists. Point mutations to the coat protein allowed us to generate capsids bearing the K16M, T18R, N10R, or D14R mutations; because 180 coat proteins form the capsid, the mutants provide a spectrum of particles differing in surface charge by as much as +540 units (K16M vs. D14R). Whereas larger poly-Arg insertions (for example, C-terminal Arg8) did not yield intact virions, it was possible to append chemically synthesized oligo-Arg peptides to stable wild-type (WT) and K16M platforms. Heparin antagonism by the particles was evaluated by using the activated partial thrombin time (aPTT) clotting assay; this revealed that T18R, D14R, and WT-(R8G2)95 were the most effective at disrupting heparin-mediated anticoagulation (>95 % inhibition). This activity agreed with measurements of ζ potential (ZP) and retention time on cation exchange chromatography for the genetic constructs, which distribute their added positive charge over the capsid surface (+180 and +360 for T18R and D14R relative to WT). The potent activity of WT-(R8G2)95, despite its relatively diminished overall surface charge is likely a consequence of the particle’s presentation of locally concentrated regions with high positive charge density that interact with heparin’s extensively sulfated domains. The engineered cationic capsids retained their ability to inhibit heparin at high concentrations and showed no anticlotting activity of the kind that limits the utility of antiheparin polycationic agents that are currently in clinical use.
Pre-treatment of B16 melanoma cells with recombinant interferon-gamma (IFN-gamma) markedly increased their lung-colonising capacity following i.v. injection into syngeneic mice as compared with control cells. A similar enhancement was observed following the injection of treated cells into athymic nude mice but not in athymic mice carrying the beige mutation. Pre-treatment of syngeneic mice with anti-asialo GM1 antibody effectively abrogated any interferon-induced increase in experimental metastatic activity. The same IFN-gamma treatment significantly increased resistance of B16 cells to splenic natural killer (NK) cell activity as determined by in vitro assays. IFN-alpha/beta pre-treatment of B16 cells decreased sensitivity to NK-cell-mediated lysis to a lesser extent than IFN-gamma and had no detectable effect upon the subsequent metastatic activity of the tumor cells. Class-I antigen expression was altered by these IFN treatments, with IFN-gamma causing dramatic increases in expression of H-2Db antigen, in a pattern consistent with the possibility that increased H-2 antigen expression on B16 cells led to decreased NK-cell sensitivity which was reflected by an increase in experimental metastatic capacity.
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