Dendritic cell maturation results in pronounced changes in glycan expression affecting recognition by siglecs and galectins

Dendritic cells (DC) are the most potent APC in the organism. Immature dendritic cells (iDC) reside in the tissue where they capture pathogens whereas mature dendritic cells (mDC) are able to activate T cells in the lymph node. This dramatic functional change is mediated by an important genetic reprogramming. Glycosylation is the most common form of posttranslational modification of proteins and has been implicated in multiple aspects of the immune response. To investigate the involvement of glycosylation in the changes that occur during DC maturation, we have studied the differences in the glycan profile of iDC and mDC as well as their glycosylation machinery. For information relating to glycan biosynthesis, gene expression profiles of human monocyte-derived iDC and mDC were compared using a gene microarray and quantitative real-time PCR. This gene expression profiling showed a profound maturation-induced up-regulation of the glycosyltransferases involved in the expression of LacNAc, core 1 and sialylated structures and a down-regulation of genes involved in the synthesis of core 2 O-glycans. Glycosylation changes during DC maturation were corroborated by mass spectrometric analysis of N- and O-glycans and by flow cytometry using plant lectins and glycan-specific Abs. Interestingly, the binding of the LacNAc-specific lectins galectin-3 and -8 increased during maturation and up-regulation of sialic acid expression by mDC correlated with an increased binding of siglec-1, -2, and -7.     

Acid Suppression Therapy Does Not Predispose to Clostridium difficile Infection: The Case of the Potential Bias

Objective

An adverse effect of acid-suppression medications on the occurrence of Clostridium difficile infection (CDI) has been a common finding of many, but not all studies. We hypothesized that association between acid-suppression medications and CDI is due to the residual confounding in comparison between patients with infection to those without, predominantly from non-tested and less sick subjects. We aimed to evaluate the effect of acid suppression therapy on incidence of CDI by comparing patients with CDI to two control groups: not tested patients and patients suspected of having CDI, but with a negative test.

Methods

We conducted a case-control study of adult patients hospitalized in internal medicine department of tertiary teaching hospital between 2005–2010 for at least three days. Controls from each of two groups (negative for CDI and non-tested) were individually matched (1∶1) to cases by primary diagnosis, Charlson comorbidity index, year of hospitalization and gender. Primary outcomes were diagnoses of International Classification of Diseases (ICD-9)–coded CDI occurring 72 hours or more after admission.

Results

Patients with CDI were similar to controls with a negative test, while controls without CDI testing had lower clinical severity. In multivariable analysis, treatment by acid suppression medications was associated with CDI compared to those who were not tested (OR = 1.88, p-value = 0.032). Conversely, use of acid suppression medications in those who tested negative for the infection was not associated with CDI risk as compared to the cases (OR = 0.66; p = 0.059).

Conclusions

These findings suggest that the reported epidemiologic associations between use of acid suppression medications and CDI risk may be spurious. The control group choice has an important impact on the results. Clinical differences between the patients with CDI and those not tested and not suspected of having the infection may explain the different conclusions regarding the acid suppression effect on CDI risk.     

Functional role of astrocyte glutamate receptors and carbon monoxide in cerebral vasodilation response to glutamate

In newborn pigs, vasodilation of pial arterioles in response to glutamate is mediated via carbon monoxide (CO), a gaseous messenger endogenously produced from heme degradation by a heme oxygenase (HO)-catalyzed reaction. We addressed the hypothesis that ionotropic glutamate receptors (iGluRs), including N-methyl-D-aspartic acid (NMDA)- and 2-amino-3-(5-methyl-3-oxo-1,2-oxazol-4-yl) propanoic acid (AMPA)/kainate-type receptors, expressed in cortical astrocytes mediate glutamate-induced astrocyte HO activation that leads to cerebral vasodilation. Acute vasoactive effects of topical iGluR agonists were determined by intravital microscopy using closed cranial windows in anesthetized newborn pigs. iGluR agonists, including NMDA, (±)1-aminocyclopentane-cis-1,3-dicarboxylic acid (cis-ACPD), AMPA, and kainate, produced pial arteriolar dilation. Topical L-2-aminoadipic acid, a gliotoxin that selectively disrupts glia limitans, reduced vasodilation caused by iGluR agonists, but not by hypercapnia, bradykinin, or sodium nitroprusside. In freshly isolated and cultured cortical astrocytes constitutively expressing HO-2, iGluR agonists NMDA, cis-ACPD, AMPA, and kainate rapidly increased CO production two- to threefold. Astrocytes overexpressing inducible HO-1 had high baseline CO but were less sensitive to glutamate stimulation of CO production when compared with HO-2-expressing astrocytes. Glutamate-induced astrocyte HO-2-mediated CO production was inhibited by either the NMDA receptor antagonist (R)-3C4HPG or the AMPA/kainate receptor antagonist DNQX. Accordingly, either antagonist abolished pial arteriolar dilation in response to glutamate, NMDA, and AMPA, indicating functional interaction among various subtypes of astrocytic iGluRs in response to glutamate stimulation. Overall, these data indicate that the astrocyte component of the neurovascular unit is responsible for the vasodilation response of pial arterioles to topically applied glutamate via iGluRs that are functionally linked to activation of constitutive HO in newborn piglets.     

Galectin-1, -2, and -3 exhibit differential recognition of sialylated glycans and blood group antigens

Human galectins have functionally divergent roles, although most of the members of the galectin family bind weakly to the simple disaccharide lactose (Galbeta1-4Glc). To assess the specificity of galectin-glycan interactions in more detail, we explored the binding of several important galectins (Gal-1, Gal-2, and Gal-3) using a dose-response approach toward a glycan microarray containing hundreds of structurally diverse glycans, and we compared these results to binding determinants on cells. All three galectins exhibited differences in glycan binding characteristics. On both the microarray and on cells, Gal-2 and Gal-3 exhibited higher binding than Gal-1 to fucose-containing A and B blood group antigens. Gal-2 exhibited significantly reduced binding to all sialylated glycans, whereas Gal-1 bound alpha2-3- but not alpha2-6-sialylated glycans, and Gal-3 bound to some glycans terminating in either alpha2-3- or alpha2-6-sialic acid. The effects of sialylation on Gal-1, Gal-2, and Gal-3 binding to cells also reflected differences in cellular sensitivity to Gal-1-, Gal-2-, and Gal-3-induced phosphatidylserine exposure. Each galectin exhibited higher binding for glycans with poly-N-acetyllactosamine (poly(LacNAc)) sequences (Galbeta1-4GlcNAc)(n) when compared with N-acetyllactosamine (LacNAc) glycans (Galbeta1-4GlcNAc). However, only Gal-3 bound internal LacNAc within poly(LacNAc). These results demonstrate that each of these galectins mechanistically differ in their binding to glycans on the microarrays and that these differences are reflected in the determinants required for cell binding and signaling. The specific glycan recognition by each galectin underscores the basis for differences in their biological activities.     

Distribution of ecosystem C and N within contrasting vegetation types in a semiarid rangeland in the Great Basin,USA

Semiarid sagebrush ecosystems are being transformed by wildfire, rangeland improvement techniques, and exotic plant invasions, but the effects on ecosystem C and N dynamics are poorly understood. We compared ecosystem C and N pools to 1 m depth among historically grazed Wyoming big sagebrush, introduced perennial crested wheatgrass, and invasive annual cheatgrass communities, to examine whether the quantity and quality of plant inputs to soil differs among vegetation types. Natural abundance δ¹⁵N isotope ratios were used to examine differences in ecosystem N balance. Sagebrush-dominated sites had greater C and N storage in plant biomass compared to perennial or annual grass systems, but this was predominantly due to woody biomass accumulation. Plant C and N inputs to soil were greatest for cheatgrass compared to sagebrush and crested wheatgrass systems, largely because of slower root turnover in perennial plants. The organic matter quality of roots and leaf litter (as C:N ratios) was similar among vegetation types, but lignin:N ratios were greater for sagebrush than grasses. While cheatgrass invasion has been predicted to result in net C loss and ecosystem degradation, we observed that surface soil organic C and N pools were greater in cheatgrass and crested wheatgrass than sagebrush-dominated sites. Greater biomass turnover in cheatgrass and crested wheatgrass versus sagebrush stands may result in faster rates of soil C and N cycling, with redistribution of actively cycled N towards the soil surface. Plant biomass and surface soil δ¹⁵N ratios were enriched in cheatgrass and crested wheatgrass relative to sagebrush-dominated sites. Source pools of plant available N could become ¹⁵N enriched if faster soil N cycling rates lead to greater N trace gas losses. In the absence of wildfire, if cheatgrass invasion does lead to degradation of ecosystem function, this may be due to faster nutrient cycling and greater nutrient losses, rather than reduced organic matter inputs.     

Installation of O-glycan sulfation capacities in human HEK293 cells for display of sulfated mucins

The human genome contains at least 35 genes that encode Golgi sulfotransferases that function in the secretory pathway, where they are involved in decorating glycosaminoglycans, glycolipids, and glycoproteins with sulfate groups. Although a number of important interactions by proteins such as selectins, galectins, and sialic acid–binding immunoglobulin-like lectins are thought to mainly rely on sulfated O-glycans, our insight into the sulfotransferases that modify these glycoproteins, and in particular GalNAc-type O-glycoproteins, is limited. Moreover, sulfated mucins appear to accumulate in respiratory diseases, arthritis, and cancer. To explore further the genetic and biosynthetic regulation of sulfated O-glycans, here we expanded a cell-based glycan array in the human embryonic kidney 293 (HEK293) cell line with sulfation capacities. We stably engineered O-glycan sulfation capacities in HEK293 cells by site-directed knockin of sulfotransferase genes in combination with knockout of genes to eliminate endogenous O-glycan branching (core2 synthase gene GCNT1) and/or sialylation capacities in order to provide simplified substrates (core1 Galβ1–3GalNAcα1–O-Ser/Thr) for the introduced sulfotransferases. Expression of the galactose 3-O-sulfotransferase 2 in HEK293 cells resulted in sulfation of core1 and core2 O-glycans, whereas expression of galactose 3-O-sulfotransferase 4 resulted in sulfation of core1 only. We used the engineered cell library to dissect the binding specificity of galectin-4 and confirmed binding to the 3-O-sulfo-core1 O-glycan. This is a first step toward expanding the emerging cell-based glycan arrays with the important sulfation modification for display and production of glycoconjugates with sulfated O-glycans.     

Structure and bacterial receptor activity of a human salivary proline-rich glycoprotein

Using an overlay technique, we previously showed that the Gram-negative periodontal pathogen Fusobacterium nucleatum binds to a glycoprotein of Mr 89,000 (Prakobphol, A., Murray, P., and Fischer, S.J. (1987) Anal. Biochem. 164, 5-11) in the parotid saliva of some individuals. We now show that deglycosylation of the purified glycoprotein results in loss of receptor activity. Amino acid analysis of the protein core showed predominantly proline, glycine, and glutamic acid/glutamine, a characteristic of proline-rich glycoproteins (PRG). The amino terminus contained repeating sequences of Ser-Gln-Gly-Pro-Pro-Pro-Arg-Pro-Gly-Lys-Pro-Glu-Gly-Pro-Pro-Pro- Gln-Gly that had significant compositional and sequence homology to that encoded by exon 3 of the PRB3 gene. We analyzed the PRG oligosaccharides by a combination of mass spectrometry techniques and nuclear magnetic resonance spectroscopy. Twenty-seven highly fucosylated structures were identified. The most abundant was as follows (where Fuc is fucose). (formula; see text) To understand the structural basis of F. nucleatum binding, we screened glycolipids and neoglycolipids carrying carbohydrate structures related to those of the PRG for receptor activity; components with unsubstituted terminal lactosamine residues best supported adherence. Neoglycolipids constructed from PRG oligosaccharides were also receptors. Treatment with beta-galactosidase, but not alpha-fucosidase, abolished binding, suggesting that unsubstituted lactosamine units, including the 6-antenna of the major oligosaccharide, mediate F. nucleatum adherence.     

Novel polyfucosylated N-linked glycopeptides with blood group A, H, X, and Y determinants from human small intestinal epithelial cells

A novel type of N-linked glycopeptides representing a major part of the glycans in human small intestinal epithelial cells from blood group A and O individuals were isolated by gel filtrations and affinity chromatography on concanavalin A-Sepharose and Bandeiraea simplicifolia lectin I-Sepharose. Sugar composition, methylation analysis, 1H NMR spectroscopy of the underivatized glycopeptides and FAB-mass spectrometry and electron impact-mass spectrometry of the permethylated glycopeptides indicated a tri- and tetra-antennary structure containing an intersecting N-acetylglucosamine and an alpha (1----6)-linked fucose residue in the core unit for the majority of the glycans. In contrast to most glycopeptides of other sources, the intestinal glycopeptides were devoid of sialic acid, but contained 6-7 residues of fucose. The outer branches contained the following structures: Fuc alpha 1-2Gal beta 1-3GleNAc beta 1- (H type 1) Fuc alpha 1-2Gal beta 1-4GleNAc beta 1- (H type 2) Gal beta 1-4 (Fuc alpha 1-3)GlcNAc beta 1- (X) Fuc alpha 1-2Gal beta 1-4(Fuc alpha 1-3)GleNAc beta 1- (Y) GalNAc alpha 1-3(Fuc alpha 1-2)Gal beta 1-3GleNAc beta 1- (A type 1) GalNAc alpha 1-3(Fuc alpha 1-2)Gal beta 1-4GleNAc beta 1- (monofucosyl A type 2) GalNAc alpha 1-3(Fuc alpha 1-2)Gal beta 1-4 (Fuc alpha 1-3)GlcNAc beta 1- (trifucosyl A type 2) The blood group determinant structures were mainly of type 2, whereas glycolipids from the same cells contained mainly type 1 determinants. The polyfucosylated glycans represent a novel type of blood group active glycopeptides. The unique properties of the small intestinal glycopeptides as compared with glycopeptides of other tissue sources may be correlated with the specialized functional properties of the small intestinal epithelial cells.     

Surgery increases fetal plasma prostacyclin

Pulmonary arterial prostacyclin (as 6-keto-PGF_1α) concentrations of near term, fetakl lambs and goats were determined following fetal surgery and 24, 48, and 72 hrs later. Blood gases, pH, and arterial pressure were determined also. At the end of 2.5 hrs of surgery including exteriorization of the uterus and fetal thorocotomy, pulmonary arterial concentrations of 6-keto-PGF_1α was 948 ± 92 (SEM) pg/ml of blood. Twenty-four hrs later it had fallen to 435 ± 92 pg/ml and remained constant for the duration of monitoring. Maternal arterial 6-keto-PGF_1α concentration was much lower (105 ± 20 pg/ml of blood). No significance changes in fetal Pa_O2, Pa_CO2, pH, or arterial pressure were observed, although Pa_CO2 appeared to be elevated and pH reduced following surgery. These values normalized within 24 hrs. We conclude that surgical perturbation increases fetal arterial prostacyclin concentration. Increased prostacyclin levels are transient, reaching stable values within 24 hrs following completion of extensive surgery.