Publications

INTRODUCTION: The inverse correlation between prevalence of auto-immune disorders like the chronic neuro-inflammatory disease multiple sclerosis (MS) and the occurrence of helminth (worm) infections, suggests that the helminth-trained immune system is protective against auto-immunity. As monocytes are regarded as crucial players in the pathogenesis of auto-immune diseases, we explored the hypothesis that these innate effector cells are prime targets for helminths to exert their immunomodulatory effects. RESULTS: Here we show that soluble products of the porcine nematode Trichuris suis (TsSP) are potent in changing the phenotype and function of human monocytes by skewing classical monocytes into anti-inflammatory patrolling cells, which exhibit reduced trans-endothelial migration capacity in an in vitro model of the blood-brain barrier. Mechanistically, we identified the mannose receptor as the TsSP-interacting monocyte receptor and we revealed that specific downstream signalling occurs via protein kinase C (PKC), and in particular PKCδ. CONCLUSION: This study provides comprehensive mechanistic insight into helminth-induced immunomodulation, which can be therapeutically exploited to combat various auto-immune disorders.

Functional glycomics has been impeded by the lack of inexpensive enzymatic and mild chemical methods to acquire natural glycans in significant amounts. In this study, we have developed a new strategy we term "threshing and trimming" (TaT) to quickly obtain N-glycans from glycoproteins and animal tissues. TaT employs low-cost Pronase to degrade peptides and N-bromosuccinimide (NBS) to effect oxidative decarboxylation under very mild reaction conditions to generate homogeneous aglycon moieties as nitriles or aldehydes. These aglycons can be readily conjugated with fluorescent tags for profiling and functional study. TaT is an affordable alternative to expensive specialty enzymes and strong chemical treatment and unpleasant reagents, and should further drive the functional glycomics of N-glycans.

It has been known for many years that influenza viruses bind by their hemagglutinin surface glycoprotein to sialic acid (N-acetylneuraminic acid) on the surface of the host cell, and that avian viruses most commonly bind to sialic acid linked α2-3 to galactose while most human viruses bind to sialic acid in the α2-6 configuration. Over the past few years there has been a large increase in data on this binding due to technological advances in glycan binding assays, reverse genetic systems for influenza and in X-ray crystallography. The results show some surprising changes in binding specificity that do not appear to affect the ability of the virus to infect host cells.

Glycomics is a broad and emerging scientific discipline focused on defining the structures and functional roles of glycans in biological systems. The staggering complexity of the glycome, minimally defined as the repertoire of glycans expressed in a cell or organism, has resulted in many challenges that must be overcome; these are being addressed by new advances in mass spectrometry as well as by the expansion of genetic and cell biology studies. Conversely, identifying the specific glycan recognition determinants of glycan-binding proteins by employing the new technology of glycan microarrays is providing insights into how glycans function in recognition and signaling within an organism and with microbes and pathogens. The promises of a more complete knowledge of glycomes are immense in that glycan modifications of intracellular and extracellular proteins have critical functions in almost all biological pathways.

Glycan microarrays have become indispensable tools for studying protein-glycan interactions. Along with chemo-enzymatic synthesis, glycans isolated from natural sources have played important roles in array development and will continue to be a major source of glycans. N-glycans and O-glycans from glycoproteins, and glycans from glycosphingolipids (GSLs) can be released from corresponding glycoconjugates with relatively mature methods, although isolation of large numbers and quantities of glycans is still very challenging. Glycosylphosphatidylinositol (GPI) anchors and glycosaminoglycans (GAGs) are less represented on current glycan microarrays. Glycan microarray development has been greatly facilitated by bifunctional fluorescent linkers, which can be applied in a 'Shotgun Glycomics' approach to incorporate isolated natural glycans. Glycan presentation on microarrays may affect glycan binding by GBPs, often through multivalent recognition by the GBP.

Host immunity represents a complex array of factors that evolved to provide protection against potential pathogens. While many factors regulate host immunity, glycan binding proteins (GBPs) appear to play a fundamental role in orchestrating this process. In addition, GBPs also reside at the key interface between host and pathogen. While early studies sought to understand GBP glycan binding specificity, limitations in the availability of test glycans made it difficult to elucidate a detailed understanding of glycan recognition. Recent developments in glycan microarray technology revolutionized analysis of GBP glycan interactions with significant implications in understanding the role of GBPs in host immunity. In this review, we explore different glycan microarray formats with a focus on the impact of these arrays on understanding the binding specificity and function of GBPs involved in immunity.

Inflammation and infection downregulate the activity and expression of cytochrome P450s (P450s) and other drug metabolizing enzymes (DMEs) involved in hepatic drug clearance. Schistosoma mansoni infection was reported to cause a downregulation of hepatic P450-dependent activities in mouse liver, but little is known about the specific enzymes affected or whether phase II DMEs are also affected. Here we describe the effect of murine schistosomiasis on the expression of hepatic P450s, NADPH-cytochrome P450 reductase (Cpr), phase II drug metabolizing enzymes, and nuclear receptors at 30 and 45 days postinfection (dpi). Although the hepatic expression of some of these genes was altered at 30 dpi, we observed substantial changes in the expression of the majority of P450 mRNAs and proteins measured, Cpr protein, as well as many of the UDP-glucuronosyltransferases and sulfotransferases at 45 dpi. S. mansoni infection also altered nuclear receptor expression, inducing mRNA levels at 30 dpi and depressing levels at 45 dpi. S. mansoni evoked a T helper 2 (Th2) inflammatory response at 45 dpi, as indicated by the induction of hepatic Th2 cytokine mRNAs [interleukins 4, 5, and 13], whereas the hepatic proinflammatory response was relatively weak. Thus, chronic schistosomiasis markedly and selectively alters the expression of multiple DMEs, which may be associated with Th2 cytokine release. This would represent a novel mechanism of DME regulation in disease states. These findings have important implications for drug testing in infected mice, whereas the relevance to humans with schistosomiasis needs to be determined.

The recognition of sialic acids by two strains of minute virus of mice (MVM), MVMp (prototype) and MVMi (immunosuppressive), is an essential requirement for successful infection. To understand the potential for recognition of different modifications of sialic acid by MVM, three types of capsids, virus-like particles, wild type empty (no DNA) capsids, and DNA packaged virions, were screened on a sialylated glycan microarray (SGM). Both viruses demonstrated a preference for binding to 9-O-methylated sialic acid derivatives, while MVMp showed additional binding to 9-O-acetylated and 9-O-lactoylated sialic acid derivatives, indicating recognition differences. The glycans recognized contained a type-2 Galβ1-4GlcNAc motif (Neu5Acα2-3Galβ1-4GlcNAc or 3'SIA-LN) and were biantennary complex-type N-glycans with the exception of one. To correlate the recognition of the 3'SIA-LN glycan motif as well as the biantennary structures to their natural expression in cell lines permissive for MVMp, MVMi, or both strains, the N- and O-glycans, and polar glycolipids present in three cell lines used for in vitro studies, A9 fibroblasts, EL4 T lymphocytes, and the SV40 transformed NB324K cells, were analyzed by MALDI-TOF/TOF mass spectrometry. The cells showed an abundance of the sialylated glycan motifs recognized by the viruses in the SGM and previous glycan microarrays supporting their role in cellular recognition by MVM. Significantly, the NB324K showed fucosylation at the non-reducing end of their biantennary glycans, suggesting that recognition of these cells is possibly mediated by the Lewis X motif as in 3'SIA-Le(X) identified in a previous glycan microarray screen.

Carbohydrates and glycoconjugates have been shown to exert pro-inflammatory effects on the dendritic cells (DCs), supporting pathogen-induced innate immunity and antigen processing, as well as immunosuppressive effects in the tolerance to self-proteins. Additionally, the innate inflammatory response to implanted biomaterials has been hypothesized to be mediated by inflammatory cells interacting with adsorbed proteins, many of which are glycosylated. However, the molecular factors relevant for surface displayed glycoconjugate modulation of dendritic cell (DC) phenotype are unknown. Thus, in this study, a model system was developed to establish the role of glycan composition, density, and carrier cationization state on DC response. Thiol modified glycans were covalently bound to a model protein carrier, maleimide functionalized bovine serum albumin (BSA), and the number of glycans per BSA modulated. Additionally, the carrier isoelectric point was scaled from a pI of ∼4.0 to ∼10.0 using ethylenediamine (EDA). The DC response to the neoglycoconjugates adsorbed to wells of a 384-well plate was determined via a high throughput assay. The underlying trends in DC phenotype in relation to conjugate properties were elucidated via multivariate general linear models. It was found that glycoconjugates with more than 20 glycans per carrier had the greatest impact on the pro-inflammatory response from DCs, followed by conjugates having an isoelectric point above 9.5. Surfaces displaying terminal α1-2 linked mannose structures were able to increase the inflammatory DC response to a greater extent than did any other terminal glycan structure. The results herein can be applied to inform the design of the next generation of combination products and biomaterials for use in future vaccines and implanted materials.

The Tn antigen is a tumor-associated carbohydrate antigen that is not normally expressed in peripheral tissues or blood cells. Expression of this antigen, which is found in a majority of human carcinomas of all types, arises from a blockage in the normal O-glycosylation pathway in which glycans are extended from the common precursor GalNAcα1-O-Ser/Thr (Tn antigen). This precursor is generated in the Golgi apparatus on newly synthesized glycoproteins by a family of polypeptide α-N-acetylgalactosaminyltransferases (ppGalNAcTs) and then extended to the common core 1 O-glycan Galβ1-3GalNAcα1-O-Ser/Thr (T antigen) by a single enzyme termed the T-synthase (core 1 β3-galactosyltransferase or C1GalT). Formation of the active form of the T-synthase requires a unique molecular chaperone termed Cosmc, encoded by Cosmc on the X-chromosome (Xq24 in humans, Xc3 in mice). Cosmc resides in the endoplasmic reticulum (ER) and prevents misfolding, aggregation, and proteasome-dependent degradation of newly synthesized T-synthase. Loss of expression of active T-synthase or Cosmc can lead to expression of the Tn antigen, along with its sialylated version Sialyl Tn antigen as observed in several cancers. Both genetic and epigenetic pathways, in addition to potential metabolic regulation, can result in abnormal expression of the Tn antigen. Engineered expression of the Tn antigen by disruption of either C1GalT (T-syn) or Cosmc in mice is associated with a tremendous range of pathologies and engineered expression of the Tn antigen in mouse embryos leads to embryonic death. Studies indicate that many membrane glycoproteins expressing the Tn antigen and/or truncated O-glycans may be dysfunctional, due to degradation and/or misfolding. Thus, expression of normal O-glycans is associated with health and homeostasis whereas truncation of O-glycans, e.g. the Tn and/or Sialyl Tn antigens is associated with cancer and other pathologies.