Structure of Foot-and-mouth disease virus serotype A10 61 alone and complexed with oligosaccharide receptor: receptor conservation in the face of antigenic variation.

Foot-and-mouth disease viruses (FMDVs) target epithelial cells via integrin receptors, but can acquire the capacity to bind cell-surface heparan sulphate (or alternative receptors) on passage in cell culture. Vaccine viruses must be propagated in cell culture and, hence, some rationale for the selection of variants in this process is important. Crystal structures are available for type O, A and C viruses and also for a complex of type O strain O(1)BFS with heparin. The structure of FMDV A10(61) (a cell culture-adapted strain) complexed with heparin has now been determined. This virus has an RGSD motif in place of the otherwise conserved RGD integrin-binding motif and the potential to bind heparan sulphate (suggested by sequence analyses). FMDV A10(61) was closely similar in structure to other serotypes, deviating most in antigenic sites. The VP1 GH loop comprising the integrin-binding motif was disordered. Heparin bound at a similar site and in a similar conformation to that seen in the analogous complex with O(1)BFS, although the binding had a lower affinity and was more ionic

SULF2, a heparan sulfate endosulfatase, is present in the blood of healthy individuals and increases in cirrhosis

BACKGROUND: SULF2 is an extracellular sulfatase that acts on heparan sulfate proteoglycans and modulates multiple signaling pathways. It is normally bound to the cell surface but can be released into the medium of cultured cells. SULF2 is known to be increased in cirrhotic liver compared to healthy liver. We asked whether SULF2 protein was present in the blood of healthy controls and increased in patients with liver cirrhosis. METHODS: We devised a sandwich ELISA for SULF2 using 2 novel monoclonal antibodies (mAbs) and measured its levels in sera of normal individuals and cirrhosis patients. RESULTS: SULF2 was higher in cirrhosis patients (1460 ± 1160 pg/ml, N =34) than healthy individuals (728 ± 400 pg/ml, N =37). SULF2 levels increased with age in both healthy and patient groups. CONCLUSIONS: SULF2 may be a useful serologic biomarker for liver cirrhosis

The major determinant of the heparin binding of glial cell-line-derived neurotrophic factor is near the N-terminus and is dispensable for receptor binding

GDNF (glial cell-line-derived neurotrophic factor), and the closely related cytokines artemin and neurturin, bind strongly to heparin. Deletion of a basic amino-acid-rich sequence of 16 residues N-terminal to the first cysteine of the transforming growth factor β domain of GDNF results in a marked reduction in heparin binding, whereas removal of a neighbouring sequence, and replacement of pairs of other basic residues with alanine had no effect. The heparin-binding sequence is quite distinct from the binding site for the high affinity GDNF polypeptide receptor, GFRα1 (GDNF family receptor α1), and heparin-bound GDNF is able to bind GFRα1 simultaneously. The heparin-binding sequence of GDNF is dispensable both for GFRα1 binding, and for activity for in vitro neurite outgrowth assay. Surprisingly, the observed inhibition of GDNF bioactivity with the wild-type protein in this assay was still found with the deletion mutant lacking the heparin-binding sequence. Heparin neither inhibits nor potentiates GDNF–GFRα1 interaction, and the extracellular domain of GFRα1 does not bind to heparin itself, precluding heparin cross-bridging of cytokine and receptor polypeptides. The role of heparin and heparan sulfate in GDNF signalling remains unclear, but the present study indicates that it does not occur in the first step of the pathway, namely GDNF–GFRα1 engagement

Natural killer cell differentiation from hematopoietic stem cells: a comparative analysis of heparin- and stromal cell-supported methods

Item does not contain fulltextNatural killer (NK) cells differentiated from hematopoietic stem cells (HSCs) may have significant clinical benefits over NK cells from adult donors, including the ability to choose alloreactive donors and potentially more robust in vivo expansion. Stromal-based methods have been used to study the differentiation of NK cells from HSCs. Stroma and cytokines support NK cell differentiation, but may face considerable regulatory hurdles. A recently reported clinical-grade, heparin-based method could serve as an alternative. How the stromal-based and heparin-based approaches compare in terms of NK cell generating efficiency or function is unknown. We show that compared with heparin-based cultures, stroma significantly increases the yield of HSC-derived NK cells by differentiating less-committed progenitors into the NK lineage. NK cells generated by both approaches were similar for most NK-activating and -inhibiting receptors. Although both approaches resulted in a phenotype consistent with CD56(bright) stage IV NK cells, heparin-based cultures favored the development of CD56(+)CD16(+) cells, whereas stroma produced more NK cell immunoglobulin-like receptor-expressing NK cells, both of which are markers of terminal maturation. At day 21, stromal-based cultures demonstrated significantly more IL-22 production, and both methods yielded similar amounts of IFN-gamma production and cytotoxicity by day 35. These findings suggest that heparin-based cultures are an effective replacement for stroma and may facilitate clinical trials testing HSC-derived NK cells

ECM components guide IL-10 producing regulatory T-cell (TR1) induction from effector memory T-cell precursors

We describe a role for ECM as a biosensor for inflammatory microenvironments that plays a critical role in peripheral immune tolerance. We show that hyaluronan (HA) promotes induction of Foxp3- IL-10–producing regulatory T cells (TR1) from conventional T-cell precursors in both murine and human systems. This is, to our knowledge, the first description of an ECM component inducing regulatory T cells. Intact HA, characteristic of healing tissues, promotes induction of TR1 capable of abrogating disease in an IL-10–dependent mouse colitis model whereas fragmentary HA, typical of inflamed tissues, does not, indicating a decisive role for tissue integrity in this system. The TR1 precursor cells in this system are CD4(+)CD62L(−)FoxP3(−), suggesting that effector memory cells assume a regulatory phenotype when they encounter their cognate antigen in the context of intact HA. Matrix integrity cues might thereby play a central role in maintaining peripheral tolerance. This TR1 induction is mediated by CD44 cross-linking and signaling through p38 and ERK1/2. This induction is suppressed, also in a CD44-dependent manner, by osteopontin, a component of chronically inflamed ECM, indicating that CD44 signaling serves as a nexus for fate decisions regarding TR1 induction. Finally, we demonstrate that TR1 induction signals can be recapitulated using synthetic matrices. These results reveal important roles for the matrix microenvironment in immune regulation and suggest unique strategies for immunomodulation