Extending the in vivo persistence of synthetic glycoconjugates using a serum-protein binder.

Synthetic glycoconjugates are used in the development of vaccines and the design of inhibitors for glycan-protein interactions. The in vivo persistence of synthetic glycoconjugates is an important factor in their efficacy, especially when prolonged interactions with specific cell types may be required. In this study, we applied a strategy for non-covalent association of an active compound with serum proteins for extension of glycoconjugate half-life in serum. The small molecule, AG10, has previously been used to extend the half-life of small molecules through its high affinity for transthyretin (TTR), a serum protein. Using a tetravalent polyethylene glycol (PEG)-based scaffold we developed a synthetic strategy for glycoconjugates that allowed for controlled addition of multiple tags, such as a TTR affinity tag or fluorophore. We designed a version of AG10 modified at the pyrazole core, named GD10, amenable to our conjugation strategy and introduced to glycoconjugates using a tri-functional linker. This approach allowed for attachment of GD10 and fluorophore tags, as well as carbohydrate antigens. We then tested the influence of the GD10 tag on glycoconjugate half-life in vivo using a mouse model. Our results suggest that the combination of the GD10 tag and the PEG scaffold extended the half-life of glycoconjugates by as much as 10-fold when compared to proteins of similar molecular weight. The GD10 tag was able to extend the half-life of similar glycoconjugates by as much as 2-fold. We observed a role for the terminal saccharide residue of the carbohydrate antigen and confirmed that conjugates were able to penetrate multiple compartments in vivo including bone marrow, lymph nodes, and other organs. The introduction of the GD10 tag did not obstruct the ability of conjugates to interact with lectin receptors. We conclude that serum protein binders can be used to extend the persistence of glycoconjugates in vivo.

Imaging Tolerance Induction in Neonatal Mice: Hierarchical Interplay Between Allogeneic Adult and Neonatal Immune Cells.

BACKGROUND: In Medawar's murine neonatal tolerance model, injection of adult semiallogeneic lymphohematopoietic cells (spleen cells [SC] and bone marrow cells [BMC]) tolerizes the neonatal immune system. An eventual clinical application would require fully allogeneic (allo) cells, yet little is known about the complex in vivo/in situ interplay between those cells and the nonconditioned neonatal immune system. METHODS: To this end, labeled adult SC and BMC were injected into allogeneic neonates; interactions between donor and host cells were analyzed and modulated by systematic depletion/inactivation of specific donor and host immune effector cell types. RESULTS: Consistent with effector cell compositions, allo-SC and allo-SC/BMC each induced lethal acute graft-versus-host disease, whereas allo-BMC alone did so infrequently. CD8 T cells from SC inoculum appeared naïve, while those of BMC were more memory-like. Age-dependent, cell-type dominance defined the interplay between adult donor cells and the neonatal host immune system such that if the dominant adult effector type was removed, then the equivalent neonatal one became dominant. Depletion of donor/host peripheral T cells protected against acute graft-versus-host disease and prolonged heart allograft survival; peripheral CD8 T-cell depletion together with CD4 T cell-costimulation blockade induced more robust tolerance. CONCLUSIONS: This comprehensive study provides direct observation of the cellular interplay between allogeneic donor and host immune systems, adds to our previous work with semiallogeneic donor cells, and provides important insights for robust tolerance induction. Induction of transplant tolerance in neonates will likely require "crowd sourcing" of multiple tolerizing cell types and involve depletion of immune effector cells with costimulation blockade.

Peroxisome biogenesis occurs in an unsynchronized manner in close association with the endoplasmic reticulum in temperature-sensitive Yarrowia lipolytica Pex3p mutants.

Pex3p is a peroxisomal integral membrane protein required early in peroxisome biogenesis, and Pex3p-deficient cells lack identifiable peroxisomes. Two temperature-sensitive pex3 mutant strains of the yeast Yarrowia lipolytica were made to investigate the role of Pex3p in the early stages of peroxisome biogenesis. In glucose medium at 16 degrees C, these mutants underwent de novo peroxisome biogenesis and exhibited early matrix protein sequestration into peroxisome-like structures found at the endoplasmic reticulum-rich periphery of cells or sometimes associated with nuclei. The de novo peroxisome biogenesis seemed unsynchronized, with peroxisomes occurring at different stages of development both within cells and between cells. Cells with peripheral nascent peroxisomes and cells with structures morphologically distinct from peroxisomes, such as semi/circular tubular structures that immunostained with antibodies to peroxisomal matrix proteins and to the endoplasmic reticulum-resident protein Kar2p, and that surrounded lipid droplets, were observed during up-regulation of peroxisome biogenesis in cells incubated in oleic acid medium at 16 degrees C. These structures were not detected in wild-type or Pex3p-deficient cells. Their role in peroxisome biogenesis remains unclear. Targeting of peroxisomal matrix proteins to these structures suggests that Pex3p directly or indirectly sequesters components of the peroxisome biogenesis machinery. Such a role is consistent with Pex3p overexpression producing cells with fewer, larger, and clustered peroxisomes.