Cross Reactive Material 197 glycoconjugate vaccines contain privileged conjugation sites

Production of glycoconjugate vaccines involves the chemical conjugation of glycans to an immunogenic carrier protein such as Cross-Reactive-Material-197 (CRM197). Instead of using glycans from natural sources recent vaccine development has been focusing on the use of synthetically defined minimal epitopes. While the glycan is structurally defined, the attachment sites on the protein are not. Fully characterized conjugates and batch-to-batch comparisons are the key to eventually create completely defined conjugates. A variety of glycoconjugates consisting of CRM197 and synthetic oligosaccharide epitopes was characterised using mass spectrometry techniques. The primary structure was assessed by combining intact protein MALDI-TOF-MS, LC-MALDI-TOF-MS middle-down and LC-ESI-MS bottom-up approaches. The middle-down approach on CNBr cleaved glycopeptides provided almost complete sequence coverage, facilitating rapid batch-to-batch comparisons, resolving glycan loading and identification of side products. Regions close to the N- and C-termini were most efficiently conjugated.Full Tex

A remote secondary binding pocket promotes heteromultivalent targeting of DC-SIGN

Dendritic cells (DC) are antigen-presenting cells coordinating the interplay of the innate and the adaptive immune response. The endocytic C-type lectin receptors DC-SIGN and Langerin display expression profiles restricted to distinct DC subtypes and have emerged as prime targets for next-generation immunotherapies and anti-infectives. Using heteromultivalent liposomes copresenting mannosides bearing aromatic aglycones with natural glycan ligands, we serendipitously discovered striking cooperativity effects for DC-SIGN+ but not for Langerin+ cell lines. Mechanistic investigations combining NMR spectroscopy with molecular docking and molecular dynamics simulations led to the identification of a secondary binding pocket for the glycomimetics. This pocket, located remotely of DC-SIGN’s carbohydrate bindings site, can be leveraged by heteromultivalent avidity enhancement. We further present preliminary evidence that the aglycone allosterically activates glycan recognition and thereby contributes to DC-SIGN-specific cell targeting. Our findings have important implications for both translational and basic glycoscience, showcasing heteromultivalent targeting of DCs to improve specificity and supporting potential allosteric regulation of DC-SIGN and CLRs in general

Heme oxygenase-1 regulates cell proliferation via carbon monoxide-mediated inhibition of T-type Ca2+ channels

Induction of the antioxidant enzyme heme oxygenase-1 (HO-1) affords cellular protection and suppresses proliferation of vascular smooth muscle cells (VSMCs) associated with a variety of pathological cardiovascular conditions including myocardial infarction and vascular injury. However, the underlying mechanisms are not fully understood. Over-expression of Cav3.2 T-type Ca2+ channels in HEK293 cells raised basal [Ca2+]i and increased proliferation as compared with non-transfected cells. Proliferation and [Ca2+]i levels were reduced to levels seen in non-transfected cells either by induction of HO-1 or exposure of cells to the HO-1 product, carbon monoxide (CO) (applied as the CO releasing molecule, CORM-3). In the aortic VSMC line A7r5, proliferation was also inhibited by induction of HO-1 or by exposure of cells to CO, and patch-clamp recordings indicated that CO inhibited T-type (as well as L-type) Ca2+ currents in these cells. Finally, in human saphenous vein smooth muscle cells, proliferation was reduced by T-type channel inhibition or by HO-1 induction or CO exposure. The effects of T-type channel blockade and HO-1 induction were non-additive. Collectively, these data indicate that HO-1 regulates proliferation via CO-mediated inhibition of T-type Ca2+ channels. This signalling pathway provides a novel means by which proliferation of VSMCs (and other cells) may be regulated therapeutically

Employment of gene expression profiling to identify transcriptional regulators of hepatic stellate cells

Activated hepatic stellate cells (HSC) play a central role in scar formation that leads to liver fibrosis. The molecular mechanisms underlying this process are not fully understood. Microarray and bioinformatics analyses have proven to be useful in identifying transcription factors that regulate cellular processes such as cell differentiation. Using oligonucleotide microarrays, we performed transcriptional analyses of activated human HSC cultured on Matrigel-coated tissue culture dishes. Examination of microarray data following Matrigel-induced deactivation of HSC revealed a significant down-regulation of myocardin, an important transcriptional regulator in smooth and cardiac muscle development. Thus, gene expression profiling as well as functional assays of activated HSC have provided the first evidence of the involvement of myocardin in HSC activation

Impact of inactivity and exercise on the vasculature in humans

The effects of inactivity and exercise training on established and novel cardiovascular risk factors are relatively modest and do not account for the impact of inactivity and exercise on vascular risk. We examine evidence that inactivity and exercise have direct effects on both vasculature function and structure in humans. Physical deconditioning is associated with enhanced vasoconstrictor tone and has profound and rapid effects on arterial remodelling in both large and smaller arteries. Evidence for an effect of deconditioning on vasodilator function is less consistent. Studies of the impact of exercise training suggest that both functional and structural remodelling adaptations occur and that the magnitude and time-course of these changes depends upon training duration and intensity and the vessel beds involved. Inactivity and exercise have direct “vascular deconditioning and conditioning” effects which likely modify cardiovascular risk

Complex Regulation and Function of the Inflammatory Smooth Muscle Cell Phenotype in Atherosclerosis

Vascular smooth muscle cell (SMC) phenotypic modulation plays a key role in atherosclerosis and is classically defined as a switch from a ‘contractile’ phenotype to a ‘synthetic’ phenotype, whereby genes that define the contractile SMC phenotype are suppressed and proliferation and/or migratory mechanisms are induced. There is also evidence that SMCs may take on a ‘proinflammatory’ phenotype, whereby SMCs secrete cytokines and express cell adhesion molecules, e.g. IL-8, IL-6, and VCAM-1, respectively, which may functionally regulate monocyte and macrophage adhesion and other processes during atherosclerosis. Factors that drive the inflammatory phenotype are not limited to cytokines but also include hemodynamic forces imposed on the blood vessel wall and intimate interaction of endothelial cells with SMCs, as well as changes in matrix composition in the vessel wall. However, it is critical to recognize that our understanding of the complex interaction of these multiple signal inputs has only recently begun to shed light on mechanisms that regulate the inflammatory SMC phenotype, primarily through models that attempt to recreate this environment ex vivo. The goal of this review is to summarize our current knowledge in this area and identify some of the key unresolved challenges and questions requiring further study