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Plasma N-glycome composition associates with chronic low back pain
Abstract
Background:
Low back pain (LBP) is the symptom of a group of syndromes with heterogeneous underlying mechanisms and molecular pathologies, making treatment selection and patient prognosis very challenging. Moreover, symptoms and prognosis of LBP are influenced by age, gender, occupation, habits, and psychological factors. LBP may be characterized by an underlying inflammatory process. Previous studies indicated a connection between inflammatory response and total plasma N-glycosylation. We wanted to identify potential changes in total plasma N-glycosylation pattern connected with chronic low back pain (CLBP), which could give an insight into the pathogenic mechanisms of the disease.
Methods:
Plasma samples of 1128 CLBP patients and 760 healthy controls were collected in clinical centers in Italy, Belgium and Croatia and used for N-glycosylation profiling by hydrophilic interaction ultra-performance liquid chromatography (HILIC-UPLC) after N-glycans release, fluorescent labeling and clean-up. Observed N-glycosylation profiles have been compared with a cohort of 126 patients with acute inflammation that underwent abdominal surgery.
Results:
We have found a statistically significant increase in the relative amount of high-branched (tri-antennary and tetra-antennary) N-glycan structures on CLBP patients' plasma glycoproteins compared to healthy controls. Furthermore, relative amounts of disialylated and trisialylated glycan structures were increased, while high-mannose and glycans containing bisecting N-acetylglucosamine decreased in CLBP.
Conclusions:
Observed changes in CLBP on the plasma N-glycome level are consistent with N-glycosylation changes usually seen in chronic inflammation.
General significance:
To our knowledge, this is a first large clinical study on CLBP patients and plasma N-glycome providing a new glycomics perspective on potential disease pathology.
... Concurrently, as information technology advances, metabolomics may provide insights into physiologic conditions and aberrant processes. Notably, recent findings emphasize the pivotal influence of specific metabolites in the pathogenesis of LBP [8][9][10]. For instance, meta-analyses have identified an association between significant reductions in 25(OH)D concentrations and a variety of chronic pain conditions [9]. ...
... Plasma protein glycosylation is a complex process which has interplay with specific SNPs already known to be associated with chronic pain states. 65,66 Trbojevic-Akmacic et al. reported that plasma of chronic low back pain patients had an increase in high-branched N-glycan structures with concomitant decrease in high-mannose and glycans containing bisecting N-acetylglucosamine. 67 Similar, Freidin et al. were able to demonstrate that changes in immunoglobulin G glycans that either promote or block antibody-dependent cell-mediated cytotoxicity were associated with chronic low back pain. 68 ...
Genetic risk factors for chronic postsurgical pain in adults have been established, but little is known whether the same associations exist in children. It is even less clear how much influence single nucleotide polymorphisms can exert on the phenotypic expression of chronic postsurgical pain in children in general. To this effect, a search was made for original articles which met the following criteria: evaluation of postsurgical pain in children with known genetic mutations or, conversely, evaluation of atypical pain trajectories of postsurgical children assessing for possible genetic mutations that may explain the phenotype. All titles and abstracts retrieved were reviewed for suitability for inclusion. The references of the selected articles were also checked for additional relevant papers. To assess the transparency and quality of the genetic studies both STrengthening the REporting of Genetic Association studies scores and Q-Genie scores were applied. Overall, there is a paucity of information regarding the link between genetic mutations and eventual chronic postsurgical pain development although there is some information on acute postoperative pain. Evidence has shown that the contribution of genetic risk factors to chronic postsurgical pain development appears to be minor, with its clinical relevance yet to be described. More advanced techniques in systems biology (proteomics, transcriptomics) suggest promising avenues for investigating the disease.
... Increasing complexity of the plasma glycome in COPD is driven mainly through significant augmentation of relative abundance of tetragalactosylated, tri-and tetrasialylated, and antennary fucosylated glycans. Similar changes in plasma glycome have been marked in several other conditions with inflammatory component, such as type 2 diabetes [27] or low back pain [28], as well as in acute systemic inflammation [21]. The common denominator of all these conditions is the increased branching of the glycans, driven mostly by increase in galactosylated and sialylated species, which the aforementioned studies reported. ...
... The glycomics profiles of patients with cLBP reveal a role for inflammation in the pathophysiology of the disease. Trbojevic-Akmacic et al. reported that the plasma of cLBP patients had a relative overall increase in high-branched N-glycan structures and a relative decrease in high-mannose and glycans containing bisecting N-acetylglucosamine [62]. These metabolite changes are consistent with those previously reported in chronic inflammation [63,64]. ...
Purpose
Chronic low back pain (cLBP) is a common health condition worldwide and a leading cause of disability with an estimated lifetime prevalence of 80–90% in industrialized countries. However, we have had limited success in treating cLBP likely due to its non-specific heterogeneous nature that goes beyond detectable anatomical changes. We propose that omics technologies as precision medicine tools are well suited to provide insight into its pathophysiology and provide diagnostic markers and therapeutic targets. Therefore, in this review, we explore the current state of omics technologies in the diagnosis and classification of cLBP. We identify factors that may serve as markers to differentiate between acute and chronic cases of low back pain (LBP). Finally, we also discuss some challenges that must be overcome to successfully apply precision medicine to the diagnosis and treatment of cLBP.
Methods
A literature search for the current applications of omics technologies to chronic low back pain was performed using the following search terms— “back pain,” “low back pain,” “proteomics,” “transcriptomics”, “epigenomics,” “genomics,” “omics.” We reviewed molecular markers identified from 35 studies which hold promise in providing information regarding molecular insights into cLBP.
Results
GWAS studies have found evidence for the role of single nucleotide polymorphisms (SNPs) associated with pain pathways in individuals with cLBP. Epigenomic modifications in patients with cLBP have been found to be enriched among genes involved in immune signaling and inflammation. Transcriptomics profiles of patients with cLBP show multiple lines of evidence for the role of inflammation in cLBP. The glycomics profiles of patients with cLBP are similar to those of patients with inflammatory conditions. Proteomics and microbiomics show promise but have limited studies currently.
Conclusion
Omics technologies have identified associations between inflammatory and pain pathways in the pathophysiology of cLBP. However, in order to integrate information across the range of studies, it is important for the field to identify and adopt standardized definitions of cLBP and control patients. Additionally, most papers have applied a single omics method to a sampling of cLBP patients which have yielded limited insight into the pathophysiology of cLBP. Therefore, we recommend a multi-omics approach applied to large global consortia for advancing subphenotyping and better management of cLBP, via improved identification of diagnostic markers and therapeutic targets.
... Other low-branched glycosylation features, such as monogalactosylated, disialylated, and monosialylated glycan structures exhibit the positive trend in their levels likewise, although not statistically significant. Elevated levels of complex high-branched and concomitant lower levels of low-branched glycan structures were found in many different chronic diseases, such as chronic obstructive pulmonary disease [56], chronic low back pain [57], T2D [35], and ovarian cancer [58], and are commonly related to chronic inflammation. Moreover, the same patterns of glycan branching as a response to a disease have been related to the specific serum proteins. ...
Aberrant plasma protein glycosylation is associated with a wide range of diseases, including diabetes, cardiovascular, and immunological disorders. To investigate plasma protein glycosylation alterations due to weight loss and successive weight-maintenance diets, 1850 glycomes from participants of the Diogenes study were analyzed using Ultra-High-Performance Liquid Chromatography (UHPLC). The Diogenes study is a large dietary intervention study in which participants were subjected to a low-calorie diet (LCD) followed by one of five different weight-maintenance diets in a period of 6 months. The most notable alterations of the plasma glycome were 8 weeks after the subjects engaged in the LCD; a significant increase in low-branched glycan structures, accompanied by a decrease in high-branched glycan structures. After the LCD period, there was also a significant rise in N-glycan structures with antennary fucose. Interestingly, we did not observe significant changes between different diets, and almost all effects we observed immediately after the LCD period were annulled during the weight-maintenance diets period.
... 5 monitoring of disease course [28] or response to treatment [82]. Moreover, metabolomics analysis have previously shown variations of metabolites in various biological fluids in patients with chronic painful disorders such as fibromyalgia [15,26,36,59,61], musculoskeletal pain [53,67,88,99], complex regional pain syndrome [2,65], vestibulodynia [49] and in orofacial pain such as temporo-mandibular disorder in women. ...
The pathophysiology of primary Burning mouth syndrome (BMS) remains controversial. Targeted analyses or "omics" approach of saliva provide diagnostic or pathophysiological biomarkers. This pilot study's primary objective was to explore the pathophysiology of BMS through a comparative analysis of the salivary metabolome among 26 BMS female cases and 25 age and sex matched controls. Secondary objectives included comparative analyses of inflammatory cytokines, neuro-inflammation markers and steroid hormones among cases and controls, and among BMS patients according to their clinical characteristics. Salivary metabolome, neuro-inflammatory markers, cytokines and steroids were respectively analysed by liquid chromatography coupled to mass sperctrometry, ELISA and protease activity assay, and multiparametric Luminex method. Among the 166 detected metabolites, univariate analysis did not find any discriminant metabolite between groups. Supervised multivariate analysis divided patients into two groups with an accuracy of 60% but did not allow significant discrimination (permutation test, p=0.35). Among the metabolites contributing to the model, three belonging to the tyrosine pathway (L-dopa, L-tyrosine and tyramine) were involved in the discrimination between cases and controls, and among BMS patients according to their levels of pain. Among the detectable molecules, levels of cytokines, steroid hormones and neuro-inflammatory markers did not differ between cases and controls and were not associated with characteristics of BMS patients. These results do not support the involvement of steroid hormones, inflammatory cytokines or inflammatory neurogenic mediators in the pathophysiology of pain in BMS, whereas the observed shift in tyrosine metabolism may indicate an adaptative response to chronic pain or an impaired dopaminergic transmission.
... Protein N-glycosylation is involved in a multitude of biological processes 8 . Accordingly, changes in N-glycosylation patterns have been associated with aging 9 and a wide range of diseases, including Parkinson's disease 10 , lower back pain 11 , rheumatoid arthritis 12 , ulcerative colitis 13 , Crohn's disease 13 , type 2 diabetes 14 and cancer [15][16][17] . However, for most of these conditions, it still remains to be clarified whether the disease causes changes in N-glycosylation or vice-versa. ...
Post-translational modifications diversify protein functions and dynamically coordinate their signalling networks, influencing most aspects of cell physiology. Nevertheless, their genetic regulation or influence on complex traits is not fully understood. Here, we compare the genetic regulation of the same PTM of two proteins – glycosylation of transferrin and immunoglobulin G (IgG). By performing genome-wide association analysis of transferrin glycosylation, we identify 10 significantly associated loci, 9 of which were not reported previously. Comparing these with IgG glycosylation-associated genes, we note protein-specific associations with genes encoding glycosylation enzymes (transferrin - MGAT5, ST3GAL4, B3GAT1; IgG - MGAT3, ST6GAL1), as well as shared associations (FUT6, FUT8). Colocalisation analyses of the latter suggest that different causal variants in the FUT genes regulate fucosylation of the two proteins. Glycosylation of these proteins is thus genetically regulated by both shared and protein-specific mechanisms.
... Much of what we know from large cohort studies of human N-glycosylation was learned from studies of glycome of blood plasma (plasma N-glycome), that is the spectrum of glycans attached to the blood plasma glycoproteins; and from the N-glycome of the immunoglobulin G (IgG glycome). In particular, association with changes in plasma N-glycome or IgG glycome has been found for Parkinson's disease (Russell et al. 2017), low back pain (Trbojević- Akmačić et al. 2018), rheumatoid arthritis (Gudelj et al. 2018b), ulcerative colitis, Crohn's disease (Trbojević Akmačić et al. 2015), type 2 diabetes (Lemmers et al. 2017), and others (see Gudelj et al. 2018a for a review). It was demonstrated that IgG glycosylation strongly changes with age (Krištić et al. 2014). ...
Although changes in protein glycosylation are observed in a wide range of diseases and pathological states, the examples of use of glycans as biomarkers and therapeutic targets are limited. This is not in small part because the understanding of human glycome regulation in vivo is incomplete and fragmented. Combination of human glycomics and genomics offers a powerful "data-driven hypotheses" approach to dissect the complex human glycobiology in vivo in an agnostic manner.In this chapter we review a decade of quantitative genetic studies of human N-glycome, including studies of its heritability and gene-mapping via genome-wide association studies (GWASs). We show that GWASs of human N-glycome start revealing regulators of the biochemical network of N-glycosylation. Some of these regulators demonstrate pleiotropic effects on human disease, especially autoimmune and inflammatory. We emphasize the use of in silico functional methods and multi-omics approaches to prioritize functional mechanisms to be further validated in laboratory experiments. This combined approach will lead to better understanding of mechanisms of regulation of human protein glycosylation and will provide a rich source of etiologic insight, therapeutic interventions, and biomarkers.
... In the future, it will progressively be more critical and routine to consider genomic biomarkers (9) that will be helpful not only for predicting patients at greater risk of developing chronic pain subsequent to acute episode, but also for leading physicians to a better understanding of patients' pain pathophysiology and helping them to choose the optimal treatment (10). As LBP is a dynamic process with several environmental factors that can interplay in modifying this disease, genomics will hopefully become more effective than genetics in guiding physicians in phenotyping patients and choosing the appropriate treatments (11). ...
Low back pain continues to be a major clinical challenge with high direct and indirect societal costs. It is a complex disease with complex pathophysiology both for acute and chronic low back pain.
Although there is consistent evidence about multidisciplinary treatment of low back pain, several different approaches and techniques are proposed, with different results often conflicting among them. In fact, even though the multidisciplinary approach is widely accepted, it is generally applied in different steps involving only one health care providing for each approach. This approach not only does not guarantee a real multidisciplinary vision of this disease but also lacks evaluation of the dynamic changes of the disease according to real patients’ needs.
In our hospital setting we have developed a “simultaneous multidisciplinary care” of low back pain patients in order to overcome these problems and to satisfy all patients’ needs by evaluating and treating all problems causing and related to low back pain. Starting from the existing literature we propose our approach as a new pathway to treat low back patients with a simultaneous multidisciplinary approach.
... In addition, alterations in plasma N-glycation levels in CLBP patients are consistent with common n-glycation changes during chronic inflammation. 81 Based on these reports, systemic and/or local inflammatory processes are partly responsible for the progression of CLBP in LBP patients. ...
Chronic low back pain (CLBP), lasting >3 months, is the end result of multiple pathogenic factors. Unfortunately, little is known about CLBP pathogenesis, which limits its advancements in clinical therapy and disease management. This paper summarizes the known pathological axes of CLBP, involving both peripheral and central systems. In particular, this paper details injurious nerve stimulation, inflammation-induced peripheral pathway, and central sensitization. Lumbar components, such as intervertebral disc (IVD), facet joints, muscles, fascia, ligaments, and joint capsules, contain pain receptors called nociceptors. Degeneration of the aforementioned lumbar components activates inflammatory pathways, which can directly damage nerves, lower nociceptor threshold to fire action potentials (AP), and cause pain. Additionally, damaged lumbar IVDs and endplates can also lead to the pathologic invasion of nerve growth and innervation, followed by the compression of herniated IVDs on nerve roots, thereby causing traumatic neuropathic pain. The central mechanism of CLBP involves alteration of the sensory processing of the brain and malfunction of the descending pain modulatory system, which facilitates pain amplification in the center nervous system (CNS). Lastly, abnormalities in the brain biochemical metabolism, activation of glial cells, and subsequent inflammation also play important roles in CLBP development. Taken together, inflammation plays an important role in both peripheral and central sensitization of CLBP. Due to the heterogeneity of CLBP, its pathological mechanism remains complex and difficult to understand. Therefore, it is a worthy field for future research into the subcomponents of CLBP pathogenesis, in order to distinguish the specific form of the disease, identify its origins, and develop corresponding highly effective comprehensive therapy against CLBP.
... In a large multicentre study on chronic low back pain, Trbojević et al hypothesized the presence of an underlying inflammatory process via plasma N-glycosylation and analysed the plasma of 1,128 patients and 760 healthy controls (Trbojević-Akmačić et al., 2018). A cohort of 126 patients with acute inflammation undergoing abdominal surgery served as a further control group. ...
Background and Objective
Metabolomics deals with the identification and quantification of small molecules (metabolites) in biological samples. As metabolite levels can reflect normal or altered metabolic pathways, their measurement provides information to improve the understanding, diagnosis and management of diseases. Despite its immense potential, metabolomics applications to pain research have been sparse. This paper describes current metabolomics techniques, reviews published human metabolomics pain research and compares successful metabolomics research in other areas of medicine with the goal of highlighting opportunities offered by metabolomics to advance pain medicine.
Databases and Data Treatment
Non‐systematic review.
Results
Our search identified 19 studies that adopted a metabolomics approach in: fibromyalgia (7), chronic widespread pain (4), other musculoskeletal pain conditions (5), neuropathic pain (1), complex regional pain syndrome (1) and pelvic pain (1). The studies used either mass spectrometry or nuclear magnetic resonance. Most are characterized by small sample sizes. Some consistency has been found for alterations in glutamate and testosterone metabolism, and metabolic imbalances caused by the gut microbiome.
Conclusions
Metabolomics research in chronic pain is in its infancy. Most studies are at the pilot stage. Metabolomics research has been successful in other areas of medicine. These achievements should motivate investigators to expand metabolomics research to improve the understanding of the basic mechanisms of human pain, as well as provide tools to diagnose, predict and monitor chronic pain conditions. Metabolomics research can lead to the identification of biomarkers to support the development and testing of treatments, thereby facilitating personalized pain medicine.
... The metabolic profile of plasma was analyzed in patients with chronic low back pain (CLBP) (n = 1128) and healthy controls (n = 760) via hydrophilic interaction ultra-performance liquid chromatography [113]. Significantly increased concentrations of tri-branched and tetra-branched N-glycan arrangements on the glycoproteins of CLBP patients compared to glycoproteins of controls. ...
Central sensitization syndromes are a collection of frequently painful disorders that contribute to decreased quality of life and increased risk of opiate abuse. Although these disorders cause significant morbidity, they frequently lack reliable diagnostic tests. As such, technologies that can identify key moieties in central sensitization disorders may contribute to the identification of novel therapeutic targets and more precise treatment options. The analysis of small molecules in biological samples through metabolomics has improved greatly and may be the technology needed to identify key moieties in difficult to diagnose diseases. In this review, we discuss the current state of metabolomics as it relates to central sensitization disorders. From initial literature review until Feb 2020, PubMed, Embase, and Scopus were searched for applicable studies. We included cohort studies, case series, and interventional studies of both adults and children affected by central sensitivity syndromes. The majority of metabolomic studies addressing a CSS found significantly altered metabolites that allowed for differentiation of CSS patients from healthy controls. Therefore, the published literature overwhelmingly supports the use of metabolomics in CSS. Further research into these altered metabolites and their respective metabolic pathways may provide more reliable and effective therapeutics for these syndromes.
... Increasing complexity of the plasma glycome in COPD is driven mainly through significant augmentation of relative abundance of tetragalactosylated, tri-and tetrasialylated, and antennary fucosylated glycans. Similar changes in plasma glycome have been marked in several other conditions with inflammatory component, such as type 2 diabetes [27] or low back pain [28], as well as in acute systemic inflammation [21]. The common denominator of all these conditions is the increased branching of the glycans, driven mostly by increase in galactosylated and sialylated species, which the aforementioned studies reported. ...
Background: Chronic obstructive pulmonary disease (COPD) is a complex condition, whose diagnosis requires spirometric assessment. However, considering its heterogeneity, subjects with similar spirometric parameters do not necessarily have the same functional status. To overcome this limitation novel biomarkers for COPD have been investigated. Therefore, we aimed to explore the potential value of N-glycans as COPD biomarkers and to examine the individual variation of plasma protein and immunoglobulin G (IgG) glycosylation profiles in subjects with COPD and healthy controls.
Methods: Both the total plasma protein and IgG N-glycome have been profiled in the total of 137 patients with COPD and 95 matching controls from Croatia. Replication cohort consisted of 61 subjects with COPD and 148 controls recruited at another Croatian medical centre.
Results: Plasma protein N-glycome in COPD subjects exhibited significant decrease in low branched and conversely, an increase in more complex glycan structures (tetragalactosylated, trisialylated, tetrasialylated and antennary fucosylated glycoforms). We also observed a significant decline in plasma monogalactosylated species, and the same change replicated in IgG glycome. N-glycans also showed value in distinguishing subjects in different COPD GOLD stages, where the relative abundance of more complex glycan structures increased as the disease progressed. Glycans also showed statistically significant associations with the frequency of exacerbations and demonstrated to be affected by smoking, which is the major risk factor for COPD development.
Conclusions: This study showed that complexity of glycans associates with COPD, mirroring also the disease severity. Moreover, changes in N-glycome associate with exacerbation frequency and are affected by smoking. In general, this study provided new insights into plasma protein and IgG N-glycome changes occurring in COPD and pointed out potential novel markers of the disease progression and severity.
Keywords: N-glycosylation, COPD, Biomarkers, Plasma glycoproteins, Immunoglobulin G
... Since Hex isoenzymes have been identified in various bodily fluids (Perez, Prieto, & Tutor, 2007;Waszkiewicz et al., 2013), extracellular PMP formation cannot be entirely ruled out. Regardless, PMP levels are generally low in mammalian blood plasma and in other bodily fluids as evidenced by glycomics experiments performed under normal and pathophysiological conditions (Kim et al., 2017;Reiding et al., 2017;Trbojevic-Akmacic et al., 2018;Yang et al., 2017b). This indicates that PMPs either reside (and function) mostly in the peripheral tissue or are rapidly eliminated if secreted into circulation. ...
Paucimannosidic proteins (PMPs) are bioactive glycoproteins carrying truncated α‐ or β‐mannosyl‐terminating asparagine (N)‐linked glycans widely reported across the eukaryotic domain. Our understanding of human PMPs remains limited, despite findings documenting their existence and association with human disease glycobiology. This review comprehensively surveys the structures, biosynthetic routes and functions of PMPs across the eukaryotic kingdoms with the aim of synthesising an improved understanding on the role of protein paucimannosylation in human health and diseases. Convincing biochemical, glycoanalytical and biological data detail a vast structural heterogeneity and fascinating tissue‐ and subcellular‐specific expression of PMPs within invertebrates and plants, often comprising multi‐α1,3/6‐fucosylation and β1,2‐xylosylation amongst other glycan modifications and non‐glycan substitutions e.g. O‐methylation. Vertebrates and protists express less‐heterogeneous PMPs typically only comprising variable core fucosylation of bi‐ and trimannosylchitobiose core glycans. In particular, the Manα1,6Manβ1,4GlcNAc(α1,6Fuc)β1,4GlcNAcβAsn glycan (M2F) decorates various human neutrophil proteins reportedly displaying bioactivity and structural integrity demonstrating that they are not degradation products. Less‐truncated paucimannosidic glycans (e.g. M3F) are characteristic glycosylation features of proteins expressed by human cancer and stem cells. Concertedly, these observations suggest the involvement of human PMPs in processes related to innate immunity, tumorigenesis and cellular differentiation. The absence of human PMPs in diverse bodily fluids studied under many (patho)physiological conditions suggests extravascular residence and points to localised functions of PMPs in peripheral tissues. Absence of PMPs in Fungi indicates that paucimannosylation is common, but not universally conserved, in eukaryotes. Relative to human PMPs, the expression of PMPs in plants, invertebrates and protists is more tissue‐wide and constitutive yet, similar to their human counterparts, PMP expression remains regulated by the physiology of the producing organism and PMPs evidently serve essential functions in development, cell–cell communication and host–pathogen/symbiont interactions. In most PMP‐producing organisms, including humans, the N‐acetyl‐β‐hexosaminidase isoenzymes and linkage‐specific α‐mannosidases are glycoside hydrolases critical for generating PMPs via N‐acetylglucosaminyltransferase I (GnT‐I)‐dependent and GnT‐I‐independent truncation pathways. However, the identity and structure of many species‐specific PMPs in eukaryotes, their biosynthetic routes, strong tissue‐ and development‐specific expression, and diverse functions are still elusive. Deep exploration of these PMP features involving, for example, the characterisation of endogenous PMP‐recognising lectins across a variety of healthy and N‐acetyl‐β‐hexosaminidase‐deficient human tissue types and identification of microbial adhesins reactive to human PMPs, are amongst the many tasks required for enhanced insight into the glycobiology of human PMPs. In conclusion, the literature supports the notion that PMPs are significant, yet still heavily under‐studied biomolecules in human glycobiology that serve essential functions and create structural heterogeneity not dissimilar to other human N‐glycoprotein types. Human PMPs should therefore be recognised as bioactive glycoproteins that are distinctly different from the canonical N‐glycoprotein classes and which warrant a more dedicated focus in glycobiological research.
... Glycoproteins comprise various enzymes, hormones, cytokines, receptors, immunoglobulins, structural, adhesion and other protein molecules, and altered glycosylation is increasingly recognized to be implicated in human pathologies. In particular, association with changes in total plasma protein Nglycome composition or immunoglobulin G (IgG) glycosylation has been found for Parkinson's disease (10), low back pain (11), rheumatoid arthritis (12), ulcerative colitis, Crohn's disease (13) and type 2 diabetes (14). Beyond that, aberrant glycosylation is involved in key pathological steps of tumor development and is even considered a new hallmark of cancer (15)(16)(17). ...
Glycosylation is a common post-translational modification of proteins. Glycosylation is associated with a number of human diseases. Defining genetic factors altering glycosylation may provide a basis for novel approaches to diagnostic and pharmaceutical applications. Here we report a genome-wide association study of the human blood plasma N-glycome composition in up to 3811 people measured by Ultra Performance Liquid Chromatography (UPLC) technology. Starting with the 36 original traits measured by UPLC, we computed an additional 77 derived traits leading to a total of 113 glycan traits. We studied associations between these traits and genetic polymorphisms located on human autosomes. We discovered and replicated 12 loci. This allowed us to demonstrate an overlap in genetic control between total plasma protein and IgG glycosylation. The majority of revealed loci contained genes that encode enzymes directly involved in glycosylation (FUT3/FUT6, FUT8, B3GAT1, ST6GAL1, B4GALT1, ST3GAL4, MGAT3 and MGAT5) and a known regulator of plasma protein fucosylation (HNF1A). However, we also found loci that could possibly reflect other more complex aspects of glycosylation process. Functional genomic annotation suggested the role of several genes including DERL3, CHCHD10, TMEM121, IGH and IKZF1. The hypotheses we generated may serve as a starting point for further functional studies in this research area.
Biomarkers are commonly recognized as objective indicators of a medical state or clinical outcome and have been widely used as clinical and diagnostic tools and surrogate endpoints in many pathological conditions. In the context of intervertebral disc (IVD) and associated back pain, also known as degenerative disc disease (DDD), the use of biomarkers has been poorly explored. DDD is currently diagnosed using imaging techniques and subjective pain scales, limiting an objective association between DDD and pain levels, as well as an evaluation of disease progression. There is a need for objective and reliable measurements for DDD, pain and pathology progression. DDD predictors could also help clinicians in deciding on the optimal treatment for distinct patient groups. This review addresses the current candidate biomarkers in DDD, including imaging, genetic, metabolite and protein‐based parameters, both at the tissue and systemic levels, that may become a major advance in the diagnosis and prognosis of the disease, as well as in the management of therapeutic approaches to DDD.
Background
Chronic pain is a significant public health problem in the United States, affecting approximately 100 million people. Yet there is a lack of robust biomarkers for clinical use in chronic pain conditions. Downstream effects of environmental, genomic, and proteomic variations in individuals with chronic pain conditions can be identified and quantified using a metabolomic approach.
Aim/Design
The purpose of this systematic review was to examine the literature for reports of potential metabolomic signatures associated with chronic pain conditions.
Methods
We searched relevant electronic databases for published studies that used various metabolomic approaches to investigate chronic pain conditions among subjects of all ages.
Results
Our search identified a total of 586 articles, 18 of which are included in this review. The reviewed studies used metabolomics to investigate fibromyalgia ( n = 5), osteoarthritis ( n = 4), migraine ( n = 3), musculoskeletal pain ( n = 2), and other chronic pain conditions ( n = 1/condition). Results show that several known and newly identified metabolites differ in individuals with chronic pain conditions compared to those without these conditions. These include amino acids (e.g., glutamine, serine, and phenylalanine) and intermediate products (e.g., succinate, citrate, acetylcarnitine, and N-acetylornithine) of pathways that metabolize various macromolecules.
Conclusion
Though more high-quality research is needed, this review provides insights into potential biomarkers for future metabolomics studies in people with chronic pain conditions.
N-glycosylation profile of total human plasma proteins could be a useful biomarker for various pathological states. Reliable high-throughput methods for such profiling have been developed. However, studies of relative importance of genetic and environmental factors in regulation of plasma N glycome are scarce. The aim of our study was to determine the role of genetic factors in phenotypic variation of plasma N glycan profile through the estimates of its heritability. Thirty-nine total plasma N glycome traits were analysed in 2816 individuals from the TwinsUK dataset. For the majority of the traits, high heritability estimates (>50%) were obtained pointing at a significant contribution of genetic factors in plasma N-glycome variation, especially for glycans mostly attached to immunoglobulins. We have also found several structures with higher environmental contribution to their variation.
Background
The Global Burden of Diseases, Injuries, and Risk Factors Study 2017 (GBD 2017) includes a comprehensive assessment of incidence, prevalence, and years lived with disability (YLDs) for 354 causes in 195 countries and territories from 1990 to 2017. Previous GBD studies have shown how the decline of mortality rates from 1990 to 2016 has led to an increase in life expectancy, an ageing global population, and an expansion of the non-fatal burden of disease and injury. These studies have also shown how a substantial portion of the world's population experiences non-fatal health loss with considerable heterogeneity among different causes, locations, ages, and sexes. Ongoing objectives of the GBD study include increasing the level of estimation detail, improving analytical strategies, and increasing the amount of high-quality data.
Methods
We estimated incidence and prevalence for 354 diseases and injuries and 3484 sequelae. We used an updated and extensive body of literature studies, survey data, surveillance data, inpatient admission records, outpatient visit records, and health insurance claims, and additionally used results from cause of death models to inform estimates using a total of 68 781 data sources. Newly available clinical data from India, Iran, Japan, Jordan, Nepal, China, Brazil, Norway, and Italy were incorporated, as well as updated claims data from the USA and new claims data from Taiwan (province of China) and Singapore. We used DisMod-MR 2.1, a Bayesian meta-regression tool, as the main method of estimation, ensuring consistency between rates of incidence, prevalence, remission, and cause of death for each condition. YLDs were estimated as the product of a prevalence estimate and a disability weight for health states of each mutually exclusive sequela, adjusted for comorbidity. We updated the Socio-demographic Index (SDI), a summary development indicator of income per capita, years of schooling, and total fertility rate. Additionally, we calculated differences between male and female YLDs to identify divergent trends across sexes. GBD 2017 complies with the Guidelines for Accurate and Transparent Health Estimates Reporting.
Findings
Globally, for females, the causes with the greatest age-standardised prevalence were oral disorders, headache disorders, and haemoglobinopathies and haemolytic anaemias in both 1990 and 2017. For males, the causes with the greatest age-standardised prevalence were oral disorders, headache disorders, and tuberculosis including latent tuberculosis infection in both 1990 and 2017. In terms of YLDs, low back pain, headache disorders, and dietary iron deficiency were the leading Level 3 causes of YLD counts in 1990, whereas low back pain, headache disorders, and depressive disorders were the leading causes in 2017 for both sexes combined. All-cause age-standardised YLD rates decreased by 3·9% (95% uncertainty interval [UI] 3·1–4·6) from 1990 to 2017; however, the all-age YLD rate increased by 7·2% (6·0–8·4) while the total sum of global YLDs increased from 562 million (421–723) to 853 million (642–1100). The increases for males and females were similar, with increases in all-age YLD rates of 7·9% (6·6–9·2) for males and 6·5% (5·4–7·7) for females. We found significant differences between males and females in terms of age-standardised prevalence estimates for multiple causes. The causes with the greatest relative differences between sexes in 2017 included substance use disorders (3018 cases [95% UI 2782–3252] per 100 000 in males vs s1400 [1279–1524] per 100 000 in females), transport injuries (3322 [3082–3583] vs 2336 [2154–2535]), and self-harm and interpersonal violence (3265 [2943–3630] vs 5643 [5057–6302]).
Interpretation
Global all-cause age-standardised YLD rates have improved only slightly over a period spanning nearly three decades. However, the magnitude of the non-fatal disease burden has expanded globally, with increasing numbers of people who have a wide spectrum of conditions. A subset of conditions has remained globally pervasive since 1990, whereas other conditions have displayed more dynamic trends, with different ages, sexes, and geographies across the globe experiencing varying burdens and trends of health loss. This study emphasises how global improvements in premature mortality for select conditions have led to older populations with complex and potentially expensive diseases, yet also highlights global achievements in certain domains of disease and injury.
Purpose: To evaluate the impact of age and gender on the international classification of functioning, disability and health (ICF)-based assessment for chronic low back pain.
Methods: Two hundred forty-four chronic low back pain patients (52% female) with a mean age of 49 years (SD =17.64) were interviewed with the comprehensive ICF core set for activities and participation, and environmental factors. After conducting explorative factor analysis, the impact of age and gender on the different factors was analyzed using analyzes of variances.
Results: Results revealed that older patients experienced more limitations within “self-care and mobility” and “walking” but less problems with “transportation” compared to younger patients. Older or middle-aged low back pain patients further perceived more facilitation through “architecture and products for communication”, “health services”, and “social services and products for mobility” than younger patients. Regarding gender differences, women reported more restriction in “housework” than men. An interaction effect between age and gender was found for “social activities and recreation” with young male patients reporting the highest impairment.
Conclusions: The study demonstrated that the comprehensive ICF core set classification for chronic low back pain is influenced by age and gender. This impact is relevant for ICF-based assessments in clinical practice, and should be considered in intervention planning for rehabilitative programs.
• Implications for rehabilitation
• It is important to consider age and gender differences when classifying with the ICF.
• The intervention planning based on the ICF should focus on improvement of bodily functioning and mobility in older patients, facilitation of household activities in women, consideration of work-life balance and recreation (e.g., through mindfulness based stress reduction), and reduction of dissatisfaction with rehabilitation in younger patients.
• It is important to offer patients the opportunity to participate in intervention planning based on the ICF.
• For intervention planning professionals should bear in mind the resource-oriented approach of the ICF (e.g., facilitation through environmental factors), and a collaboration with other professionals.
Chronic low back pain (CLBP) is one of the most common medical conditions, ranking as the greatest contributor to global disability and accounting for huge societal costs based on the Global Burden of Disease 2010 study.
Large genetic and -omics studies provide a promising avenue for the screening, development and validation of biomarkers useful for personalized diagnosis and treatment (precision medicine). Multicentre studies are needed for such an effort, and a standardized and homogeneous approach is vital for recruitment of large numbers of participants among different centres (clinical and laboratories) to obtain robust and reproducible results. To date, no validated standard operating procedures (SOPs) for genetic/-omics studies in chronic pain have been developed.
In this study, we validated an SOP model that will be used in the multicentre (5 centres) retrospective “PainOmics” study, funded by the European Community in the 7th Framework Programme, which aims to develop new biomarkers for CLBP through three different -omics approaches: genomics, glycomics and activomics.
The SOPs describe the specific procedures for (1) blood collection, (2) sample processing and storage, (3) shipping details and (4) cross-check testing and validation before assays that all the centres involved in the study have to follow.
Multivariate analysis revealed the absolute specificity and homogeneity of the samples collected by the five centres for all genetics, glycomics and activomics analyses.
The SOPs used in our multicenter study have been validated. Hence, they could represent an innovative tool for the correct management and collection of reliable samples in other large-omics-based multicenter studies.
Low back pain (LBP) is one of the major disabling health conditions among older adults aged 60 years or older. While most causes of LBP among older adults are non-specific and self-limiting, seniors are prone to develop certain LBP pathologies and/or chronic LBP given their age-related physical and psychosocial changes. Unfortunately, no review has previously summarized/discussed various factors that may affect the effective LBP management among older adults. Accordingly, the objectives of the current narrative review were to comprehensively summarize common causes and risk factors (modifiable and non-modifiable) of developing severe/chronic LBP in older adults, to highlight specific issues in assessing and treating seniors with LBP, and to discuss future research directions. Existing evidence suggests that prevalence rates of severe and chronic LBP increase with older age. As compared to working-age adults, older adults are more likely to develop certain LBP pathologies (e.g., osteoporotic vertebral fractures, tumors, spinal infection, and lumbar spinal stenosis). Importantly, various age-related physical, psychological, and mental changes (e.g., spinal degeneration, comorbidities, physical inactivity, age-related changes in central pain processing, and dementia), as well as multiple risk factors (e.g., genetic, gender, and ethnicity), may affect the prognosis and management of LBP in older adults. Collectively, by understanding the impacts of various factors on the assessment and treatment of older adults with LBP, both clinicians and researchers can work toward the direction of more cost-effective and personalized LBP management for older people.
Background
Physical activity in leisure time seems to reduce the risk of low back pain, but it is not known whether occupational activity, as recorded in a representative working population, produces a higher or lower risk.
Objective
To study associations between physical activity level at work and risk of chronic low back pain.
Methods
Associations were examined in a Norwegian prospective study using data from the HUNT2 and HUNT3 surveys carried out in the whole county of Nord-Trøndelag. Participants were 7580 women and 7335 men who supplied information about physical activity level at work. Levels considered were sedentary work, work involving walking but no heavy lifting, work involving walking and heavy lifting, and particularly strenuous physical work. Nobody in the cohort was affected by chronic low back pain at baseline. After 11 years, participants reported whether they suffered from chronic low back pain. Generalized linear modelling with adjustment for potential confounders was applied to assess associations with risk factors.
Results
In age-adjusted analyses both women and men showed statistically significant associations between physical activity at work and risk of chronic low back pain, suggesting positive relationships. For particularly strenuous physical work the relative risk of chronic low back pain was 1.30 (95% CI: 1.00–1.71) in women and 1.36 (95% CI 1.17–1.59) in men, compared to sedentary work. Women still showed a general association with activity level after adjustment for education, leisure time physical activity, BMI, smoking and occupational category. In men, the higher risk was only maintained for particularly strenuous work.
Conclusion
In this cohort, women had a higher risk of chronic low back pain with work involving walking and heavy lifting or particularly strenuous work, compared to sedentary work. Men participating in particularly strenuous work also experienced a higher risk of chronic low back pain.
Key Points
Human platelets, endothelial cells, and plasma proteins are extensively O-glycosylated, with >1123 O-glycosites identified in this study. O-glycosites can be classified into functional subgroups; one important function includes the protection from proteolytic processing.
Background: Low back pain (LBP) is a very frequent condition, affecting most people at some point throughout their life. This cross-sectional study was aimed to investigate a selected panel of cytokines and inflammatory biomarkers in patients with or without LBP.
Methods: The study population consisted of 104 patients diagnosed with LBP (52 non-persistent and 52 persistent) and 52 healthy subjects with no LBP. Blood samples were collected for assessment of adiponectin, leptin, monocyte chemoattractant protein-1 (MCP-1) and C reactive protein (CRP). The duration of LBP was categorized as “no pain”, “non-persistent LBP” and “persistent LBP”.
Results: Higher values of CRP and lower concentrations of both leptin and MCP-1 were found in LBP patients compared to controls, whereas adiponectin did not differ among groups. MCP-1 was also lower in patients with non-persistent than in those with persistent LBP. Age, leptin (relative risk, 11.8; 95% CI, 3.9–35.8) and MCP-1 (relative risk, 2.7; 95% CI, 1.7–4.4) were independently associated with presence and duration of LBP. The combination of age, leptin and MCP-1 predicted 61% of the risk of LBP duration. The area under the curve of MCP-1 for distinguishing persistent from non-persistent LBP was 0.65 (95% CI, 0.54–0.76).
Conclusions: The results of our study suggest that leptin and MCP-1 may be promising biomarkers for diagnosis of acute LBP and its risk to become chronic.
Introduction
Chronic low back pain (CLBP) produces considerable direct costs as well as indirect burdens for society, industry and health systems. CLBP is characterised by heterogeneity, inclusion of several pain syndromes, different underlying molecular pathologies and interaction with psychosocial factors that leads to a range of clinical manifestations. There is still much to understand in the underlying pathological processes and the non-psychosocial factors which account for differences in outcomes. Biomarkers that may be objectively used for diagnosis and personalised, targeted and cost-effective treatment are still lacking. Therefore, any data that may be obtained at the ‘-omics’ level (glycomics, Activomics and genome-wide association studies—GWAS) may be helpful to use as dynamic biomarkers for elucidating CLBP pathogenesis and may ultimately provide prognostic information too. By means of a retrospective, observational, case-cohort, multicentre study, we aim to investigate new promising biomarkers potentially able to solve some of the issues related to CLBP.
Methods and analysis
The study follows a two-phase, 1:2 case–control model. A total of 12 000 individuals (4000 cases and 8000 controls) will be enrolled; clinical data will be registered, with particular attention to pain characteristics and outcomes of pain treatments. Blood samples will be collected to perform -omics studies. The primary objective is to recognise genetic variants associated with CLBP; secondary objectives are to study glycomics and Activomics profiles associated with CLBP.
Ethics and dissemination
The study is part of the PainOMICS project funded by European Community in the Seventh Framework Programme. The study has been approved from competent ethical bodies and copies of approvals were provided to the European Commission before starting the study. Results of the study will be reviewed by the Scientific Board and Ethical Committee of the PainOMICS Consortium. The scientific results will be disseminated through peer-reviewed journals.
Trial registration number
NCT02037789; Pre-results.
Systemic inflammation participates to the complex healing process occurring after major surgery, thus directly affecting the surgical outcome and patient recovery. Total plasma N-glycome might be an indicator of inflammation after major surgery, as well as an anti-inflammatory therapy response marker, since protein glycosylation plays an essential role in the inflammatory cascade. Therefore, we assessed the effects of surgery on the total plasma N-glycome and the association with self-administration of postoperative morphine in two cohorts of patients that underwent major abdominal surgery. We found that plasma N-glycome undergoes significant changes one day after surgery and intensifies one day later, thus indicating a systemic physiological response. In particular, we observed the increase of bisialylated biantennary glycan, A2G2S[3,6]2, 12 hours after surgery, which progressively increased until 48 postoperative hours. Most changes occurred 24 hours after surgery with the decrease of most core-fucosylated biantennary structures, as well as the increase in sialylated tetraantennary and FA3G3S[3,3,3]3 structures. Moreover, we observed a progressive increase of sialylated triantennary and tetraantennary structures two days after surgery, with a concomitant decrease of the structures containing bisecting N-acetylglucosamine along with bi- and trisialylated triantennary glycans. We did not find any statistically significant association between morphine consumption and plasma N-glycome.
Low back pain (LBP) is a common debilitating condition which aetiology and pathogenesis are poorly understood. We carried out a first so far analysis of associations between LBP and plasma IgG N-glycome in a sample of 4511 twins from TwinsUK database assessed for LBP, lumbar disc degeneration (LDD) as its possible cause, and IgG-glycan levels. Using weighted correlation network analysis, we established a correlation between LBP and glycan modules featured by glycans that either promote or block antibody-dependent cell-mediated cytotoxicity (ADCC). The levels of four glycan traits representing two of those modules were statistically significantly different in monozygotic twins discordant for LBP. Also, the trend to higher prevalence of systemic inflammatory disorders was shown for twins with low level of fucosylated glycans and high level of non-fucosylated glycans. Core fucosylation of IgG is a “safety switch” reducing ADCC, thus our results suggest the involvement of ADCC and associated inflammation in pathogenesis of LBP. No correlation between LDD scores and glycans was found assuming that the inflammation may not be a part of LDD. These data provide a new insight into understanding the complex pathophysiology of LBP and suggest glycan levels as a possible biomarker for inflammation-related subtypes of LBP.
Background:
Immunoglobulin G (IgG) effector functions are regulated by the composition of glycans attached to a conserved N-glycosylation site in the Fc part. Intrathecal production of IgG, especially IgG1, is a hallmark of multiple sclerosis (MS), but nothing is known about IgG Fc glycosylation in MS and in cerebrospinal fluid (CSF) in general.
Methods:
We applied mass spectrometry of tryptic Fc glycopeptides to analyze IgG Fc glycosylation (sialylation, galactosylation, fucosylation, and bisecting N-acetylglucosamine (GlcNAc)) in 48 paired CSF and serum samples from adult patients with MS or a first demyelinating event highly suggestive of MS (designated as MS cases), and from healthy volunteers and patients with other non-inflammatory diseases (control group). p values were adjusted for multiple testing.
Results:
Our experiments revealed four main results. First, IgG1 glycosylation patterns were different in CSF vs. serum, in the MS group and even in control donors without intrathecal IgG synthesis. Second, in MS patients vs. controls, IgG1 glycosylation patterns were altered in CSF, but not in serum. Specifically, in CSF from the MS group, bisecting GlcNAc were elevated, and afucosylation and galactosylation were reduced. Elevated bisecting GlcNAc and reduced galactosylation are known to enhance IgG effector functions. Third, hypothesis-free regression analysis revealed that alterations of afucosylation and bisecting GlcNAc in CSF from MS cases peaked 2-3 months after the last relapse. Fourth, CSF IgG1 glycosylation correlated with the degree of intrathecal IgG synthesis and CSF cell count.
Conclusions:
The CNS compartment as well as the inflammatory milieu in MS affect IgG1 Fc glycosylation. In MS, the CSF IgG1 glycosylation has features that enhance Fc effector functions.
The spectrum of all glycan structures - the glycome - is immense. In humans, its size is orders of magnitude greater than the number of proteins that are encoded by the genome, one percent of which encodes proteins that make, modify, localize or bind sugar chains, which are known as glycans. In the past decade, over 30 genetic diseases have been identified that alter glycan synthesis and structure, and ultimately the function of nearly all organ systems. Many of the causal mutations affect key biosynthetic enzymes, but more recent discoveries point to defects in chaperones and Golgi-trafficking complexes that impair several glycosylation pathways. As more glycosylation disorders and patients with these disorders are identified, the functions of the glycome are starting to be revealed.
Glycosylation is the most abundant and complex protein modification, and can have a profound structural and functional effect on the conjugate. The oligosaccharide fraction is recognized to be involved in multiple biological processes, and to affect proteins physical properties, and has consequentially been labeled a critical quality attribute of biopharmaceuticals. Additionally, due to recent advances in analytical methods and analysis software, glycosylation is targeted in the search for disease biomarkers for early diagnosis and patient stratification. Biofluids such as saliva, serum or plasma are of great use in this regard, as they are easily accessible and can provide relevant glycosylation information. Thus, as the assessment of protein glycosylation is becoming a major element in clinical and biopharmaceutical research, this review aims to convey the current state of knowledge on the N-glycosylation of the major plasma glycoproteins alpha-1-acid glycoprotein, alpha-1-antitrypsin, alpha-1B-glycoprotein, alpha-2-HS-glycoprotein, alpha-2-macroglobulin, antithrombin-III, apolipoprotein B-100, apolipoprotein D, apolipoprotein F, beta-2-glycoprotein 1, ceruloplasmin, fibrinogen, immunoglobulin (Ig) A, IgG, IgM, haptoglobin, hemopexin, histidine-rich glycoprotein, kininogen-1, serotransferrin, vitronectin, and zinc-alpha-2-glycoprotein. In addition, the less abundant immunoglobulins D and E are included because of their major relevance in immunology and biopharmaceutical research. Where available, the glycosylation is described in a site-specific manner. In the discussion, we put the glycosylation of individual proteins into perspective and speculate how the individual proteins may contribute to a total plasma N-glycosylation profile determined at the released glycan level.
Electronic supplementary material
The online version of this article (doi:10.1007/s10719-015-9626-2) contains supplementary material, which is available to authorized users.
Background:
The majority of human proteins are being modified by covalent attachment of complex oligosaccharides--glycans. Both glycans and polypeptide parts of a protein contribute to its structure and function, but contrary to polypeptide that is defined by the sequence of nucleotides in the corresponding gene, glycans are shaped by complex dynamic interactions between hundreds of enzymes, transcription factors, ion channels and other proteins.
Scope of review:
An overview of current knowledge about the importance of N-glycans in normal human physiology and disease mechanisms, exemplified by IgG N-glycans.
Major conclusions:
Recent technological development enabled systematic analysis of glycome composition in large epidemiological cohorts and clinical studies. However, the majority of these studies is still missing any glycomic component, and consequently also lacks this layer of biological information. Individual variation in glycosylation is potentially important for individualized disease risk, disease course and response to therapy. Evidence in support of this hypothesis is accumulating, but further studies are needed to enable understanding of the role of changes in protein glycosylation in disease.
General significance:
Glycans are involved in virtually all physiological processes. Inter-individual variation in glycome composition is large, and these differences associate with disease risk, disease course and the response to therapy. This article is part of a Special Issue entitled "Glycans in personalised medicine" Guest Editor: Professor Gordan Lauc.
Glycans attached to the Fc part of immunoglobulin G (IgG) are important modulators of IgG effector functions. Inter-individual differences in IgG glycome composition are large and they strongly associate with different inflammatory and autoimmune diseases. IKZF1, HLA-DQ2A/B and BACH2 genetic loci that affect IgG glycome composition show pleiotropy with systemic lupus erythematosus (SLE), indicating potentially causative role of aberrant IgG glycosylation in SLE. The main objective of this first large multicentre case/control study was to determine whether SLE is associated with altered IgG glycosylation.
Using UPLC analysis of released and 2AB-labelled glycans, the composition of IgG glycome was analysed in 251 SLE patients and 252 matching controls of Latin American mestizo origin (discovery cohort) and in two independent replication cohorts of different ethnicity (108 cases and 193 controls from Trinidad, and 107 cases and 200 controls from China).
Multiple statistically significant differences in the IgG glycome composition between patients and controls were observed. The most significant changes included decreased galactosylation and sialylation of IgG (that regulate pro- and anti-inflammatory action of IgG) as well as decreased core-fucose and increased bisecting GlcNAc (that affects antibody dependent cellular cytotoxicity).
IgG glycome in SLE patients is significantly altered in a way that decreases immunosuppressive action of circulating immunoglobulins. The magnitude of observed changes associated with the intensity of the disease, indicating that aberrant IgG glycome composition, or changes in IgG glycosylation may be an important molecular mechanism in SLE. This article is protected by copyright. All rights reserved.
© 2015, American College of Rheumatology.
Glycosylation affects structure, folding, and function of numerous proteins. Aberrant glycosylation has been shown to be associated with different diseases. A wide range of analytical methods is available for glycan analysis of antibodies (mainly IgG), but analysis of plasma glycans is less established due to additional challenges encountered with higher complexity of the sample. Here we describe development and optimization of a high-throughput sample preparation method for hydrophilic interaction liquid chromatography and ultra-performance liquid chromatography analysis of plasma N-glycans. Clean-up of labeled glycans was found to be the largest source of variation, and we tested cellulose, silica gel, Bio-Gel, and hydrophilic GHP filter as stationary phases for solid-phase extraction. All stationary phases were shown to be suitable for purification of labeled glycans, but GHP filter plate in combination with cold 96% acetonitrile had the highest reproducibility and was easiest to work with. The method was further optimized with Plackett - Burman screening design and validated in terms of analysis of major step variation and between-day and between-person variation. The developed method is fast, cost-effective, and easy to perform, and it has very good reproducibility during long period of time, enabling the detection of biological variability of the plasma N-glycome.
Background:
Glycobiology is an underexplored research area in inflammatory bowel disease (IBD), and glycans are relevant to many etiological mechanisms described in IBD. Alterations in N-glycans attached to the immunoglobulin G (IgG) Fc fragment can affect molecular structure and immunological function. Recent genome-wide association studies reveal pleiotropy between IBD and IgG glycosylation. This study aims to explore IgG glycan changes in ulcerative colitis (UC) and Crohn's disease (CD).
Methods:
IgG glycome composition in patients with UC (n = 507), CD (n = 287), and controls (n = 320) was analyzed by ultra performance liquid chromatography.
Results:
Statistically significant differences in IgG glycome composition between patients with UC or CD, compared with controls, were observed. Both UC and CD were associated with significantly decreased IgG galactosylation (digalactosylation, UC: odds ratio [OR] = 0.71; 95% confidence interval [CI], 0.5-0.9; P = 0.01; CD: OR = 0.41; CI, 0.3-0.6; P = 1.4 × 10) and significant decrease in the proportion of sialylated structures in CD (OR = 0.46, CI, 0.3-0.6, P = 8.4 × 10). Logistic regression models incorporating measured IgG glycan traits were able to distinguish UC and CD from controls (UC: P = 2.13 × 10 and CD: P = 2.20 × 10), with receiver-operator characteristic curves demonstrating better performance of the CD model (area under curve [AUC] = 0.77) over the UC model (AUC = 0.72) (P = 0.026). The ratio of the presence to absence of bisecting GlcNAc in monogalactosylated structures was increased in patients with UC undergoing colectomy compared with no colectomy (FDR-adjusted, P = 0.05).
Conclusions:
The observed differences indicate significantly increased inflammatory potential of IgG in IBD. Changes in IgG glycosylation may contribute to IBD pathogenesis and could alter monoclonal antibody therapeutic efficacy. IgG glycan profiles have translational potential as IBD biomarkers.
Recovery after cardiac surgery is a complex process that has to compensate for both individual variability and extensive tissue damage in the context of systemic inflammation. Protein glycosylation is essential in many steps of the inflammatory cascade, but due to technological limitations the role of individual variation in glycosylation in systemic inflammation has not been addressed until now. We analysed composition of the total plasma and IgG N-glycomes in 107 patients undergoing cardiac surgery. In nearly all individuals plasma N-glycome underwent the same pattern of changes in the first 72 h, revealing a general mechanism of glycosylation changes. To the contrary, changes in the IgG glycome were very individualized. Bi-clustering analysis revealed the existence of four distinct patterns of changes. One of them, characterized by a rapid increase in galactosylated glycoforms, was associated with nearly double mortality risk measured by EuroSCORE II. Our results indicate that individual variation in IgG glycosylation changes during acute systemic inflammation associates with increased mortality risk and indicates new avenues for the development of personalized diagnostic and therapeutic approach.
Heterogeneity and latent variables are now widely recognized as major sources of bias and variability in high-throughput
experiments. The most well-known source of latent variation in genomic experiments are batch effects—when samples are processed
on different days, in different groups or by different people. However, there are also a large number of other variables that
may have a major impact on high-throughput measurements. Here we describe the sva package for identifying, estimating and removing unwanted sources of variation in high-throughput experiments. The sva package supports surrogate variable estimation with the sva function, direct adjustment for known batch effects with the ComBat function and adjustment for batch and latent variables in prediction problems with the fsva function.
Availability: The R package sva is freely available from http://www.bioconductor.org.
Contact: jleek{at}jhsph.edu
Supplementary information: Supplementary data are available at Bioinformatics online.
The identification of serum biomarkers has lead to improvements in the detection and diagnosis of cancer, and combinations of these biomarkers have increased further their sensitivity and specificity. Glycosylation is the most common PTM of secreted proteins and the identification of novel serum glyco-biomarkers has become a topic of increasing interest because the glycan processing pathways are frequently disturbed in cancer cells. A future goal is to combine current biomarkers with glyco-biomarkers to yield further improvements. Well characterised N-glycosylation changes in the serum glycome of cancer patients include changes in the levels of tri- and tetra-antennary glycan structures, sialyl Lewis X epitopes and agalactosylated bi-antennary glycans. Several of these glycosylated markers have been linked to chronic inflammatory diseases, promoting questions about the links between inflammation and cancer. In this review, the glycoproteins which display these glycan epitopes, the glycosyl transferases which can generate them, their potential functions and their use as biomarkers are evaluated.
All immunoglobulin G molecules carry N-glycans, which modulate their biological activity. Changes in N-glycosylation of IgG associate with various diseases and affect the activity of therapeutic antibodies and intravenous immunoglobulins. We have developed a novel 96-well protein G monolithic plate and used it to rapidly isolate IgG from plasma of 2298 individuals from three isolated human populations. N-glycans were released by PNGase F, labeled with 2-aminobenzamide and analyzed by hydrophilic interaction chromatography with fluorescence detection. The majority of the structural features of the IgG glycome were consistent with previous studies, but sialylation was somewhat higher than reported previously. Sialylation was particularly prominent in core fucosylated glycans containing two galactose residues and bisecting GlcNAc where median sialylation level was nearly 80%. Very high variability between individuals was observed, approximately three times higher than in the total plasma glycome. For example, neutral IgG glycans without core fucose varied between 1.3 and 19%, a difference that significantly affects the effector functions of natural antibodies, predisposing or protecting individuals from particular diseases. Heritability of IgG glycans was generally between 30 and 50%. The individual's age was associated with a significant decrease in galactose and increase of bisecting GlcNAc, whereas other functional elements of IgG glycosylation did not change much with age. Gender was not an important predictor for any IgG glycan. An important observation is that competition between glycosyltransferases, which occurs in vitro, did not appear to be relevant in vivo, indicating that the final glycan structures are not a simple result of competing enzymatic activities, but a carefully regulated outcome designed to meet the prevailing physiological needs.
The metafor package provides functions for conducting meta-analyses in R. The package includes functions for fitting the meta-analytic fixed- and random-effects models and allows for the inclusion of moderators variables (study-level covariates) in these models. Meta-regression analyses with continuous and categorical moderators can be conducted in this way. Functions for the Mantel-Haenszel and Peto's one-step method for meta-analyses of 2 x 2 table data are also available. Finally, the package provides various plot functions (for example, for forest, funnel, and radial plots) and functions for assessing the model fit, for obtaining case diagnostics, and for tests of publication bias.
Glycan heterogeneity was shown to be associated with numerous diseases and glycan analysis has a great diagnostic potential. Recently, we reported high biological variability of human plasma N-glycome at the level of population. The observed variations were larger than changes reported to be associated with some diseases; thus, it was of great importance to examine the temporal constancy of human N-glycome before glycosylation changes could be routinely analyzed in diagnostic laboratories. Plasma samples were taken from 12 healthy individuals. The blood was drawn on seven occasions during 5 days. N-Linked glycans, released from plasma proteins, were separated using hydrophilic interaction high-performance liquid chromatography into 16 groups (GP1-GP16) and quantified. The results showed very small variation in all glycan groups, indicating very good temporal stability of N-glycome in a single individual. Coefficients of variation from 1.6% for GP8 to 11.4% for GP1 were observed. The average coefficient of variation was 5.6%. These variations were comparable to those observed when analytical procedure was tested for its precision. Good stability of plasma N-glycome in healthy individuals implies that glycosylation is under significant genetic control. Changes observed in glycan profiles are consequence of environmental influences and physiologic responses and therefore have a significant diagnostic potential.
Inflammatory diseases are accompanied by numerous changes at the site of inflammation as well as many systemic physiological and biochemical changes. In the past two decades more and more attention is being paid to changes in glycosylation and in this review we describe some of the changes found on main serum proteins (alpha1-acid glycoprotein, immunoglobulin G, immunoglobulin A, transferrin, haptoglobin, alpha2-macroglobulin, C-reactive protein, and others). Molecular background and physiological importance of most of these changes are yet to be discovered, but it is evident that glycosylation plays an important role in the inflammatory response. Maybe the greatest value of these changes currently lays in their potential diagnostic and prognostic usage, either in combination with current diagnostic markers or on their own. However, determining glycan structures is still technically too complex for most clinical laboratories and further efforts have to be made to develop simple analytical tools to study changes in glycosylation.
This study is a prospective cross-sectional analytic study.
The authors determined the prevalence and clinical features of patients with pain stemming from the lumbar zygapophysial joints.
Previous studies have demonstrated a wide range of prevalence for zygapophysial joint pain and conflicting results with regard to clinical signs.
One hundred and seventy-six consecutive patients with chronic low back pain were investigated with a series of screening zygapophysial joint blocks using lignocaine and confirmatory blocks using bupivacaine.
Forty-seven percent of patients had a definite or greater response to the screening injection at one or more levels but only 15% had a 50% or greater response to a confirmatory block. Response to zygapophysial joint injection was not associated with any single clinical feature or set of clinical features.
The zygapophysial joint is an important source of pain but the existence of a "facet syndrome" must be questioned.
Background and aims:
Based on a transdiagnostic approach, this study assesses the impact of cognitive and emotional processes (difficulties in emotional regulation, impulsiveness, rumination and somatosensory amplification) on the psychological risk factors of chronic low-back pain.
Methods:
The study was carried out with 256 patients with chronic low-back pain. All the variables were assessed through a booklet of 10 validated questionnaires. Multiple regression analysis and moderation analysis were performed.
Results:
Predictors included in multiple regression models explain 3%-42% (adjusted R(2)) of the variance in psychological risk factors. Moreover, analyses reveal a significant moderator effect of somatosensory amplification on the link between fear-avoidance beliefs linked to work and pain intensity (F(3;250)=12.33; p=.00), of somatosensory amplification and brooding on the link between depression and functional repercussions (FR) on everyday life (F(3;252)=13.36; p=.000; F(1;252)=12.42; p=.00), of the reflection dimension of rumination on the link between the helplessness dimension of catastrophizing and FRs on sociability (F(3;252)=37.02; p=.00). There is also a moderation analysis with a significant trend concerning the lack of emotional awareness and the difficulties in controlling impulsive behaviours.
Conclusions:
Our results indicate an important role of some dimensions of difficulties in emotional regulation, somatosensory amplification and rumination in the increase in negative affects and dysfunctional beliefs, and in the links between those psychological risk factors and pain/disability.
Implications:
This study identifies some cognitive and emotional dysregulations substantially involved in work-related chronic pain. This contribute to put in place psychotherapeutic protocols to tackle these deficits and dysregulations in a relevant way.
Ultra-performance liquid chromatography (UPLC) is the established technology for accurate analysis of IgG Fc N-glycosylation due to its superior sensitivity, resolution, speed, and its capability to provide branch-specific information of glycan species. Correct and cost-efficient preprocessing of chromatographic data is the major prerequisite for subsequent analyses ranging from inference of structural isomers to biomarker discovery and prediction of humoral immune response from characterized changes in glycosylation. The complexity of glycomic chromatograms poses a number of challenges for developing automated data annotation and quantitation algorithms, which frequently necessitated manual or semi-manual approaches to preprocessing, most notably to peak detection and integration. Such procedures are meticulous and time-consuming, and may be a source of confounding due to their dependence on human labelers. Although liquid chromatography is a mature field and a number of methods have been developed for automatic peak detection outside the area of glycomics analysis, we found that hardly any of them are suitable for automatic integration of UPLC glycomic profiles without substantial modifications. In this chapter, we illustrate practical challenges of automatic peak detection of UPLC glycomics chromatograms. We outline a robust, semi-supervised method ACE (Automatic Chromatogram Extraction) for automated alignment and detection of glycan peaks in chromatograms, developed by Pharmatics Limited (UK) in collaboration with Genos Limited (Croatia). Application of the tool requires minimal human interference, which results in a significant reduction in the time and cost of IgG glycomics signal integration using Waters Acquity UPLC instrument (Milford, MA, USA) in several human cohorts with blind technical replicas.
Immunoglobulin G1 (IgG1) is the most abundant circulating human antibody and also the scaffold for many therapeutic monoclonal antibodies (mAbs). The destruction of IgG-coated targets by cell-mediated pathways begins with an interaction between the IgG Fc region and multiple varieties of membrane-bound Fc γ receptors (FcγRs) on the surface of leukocytes. This interaction requires the presence of an asparagine-linked (N-)glycan on the Fc, and variations in the N-glycan composition can affect the affinity of CD16A binding (an FcγR). Contemporary efforts to glycoengineer mAbs focus on increasing CD16A affinity, and thus treatment efficacy, but it is unclear how these changes affect affinity for the other FcγRs. Here, we measure binding of the extracellular Fc-binding domains for human CD16A and B, CD32A, B and C, and CD64 to six well-defined IgG1 Fc glycoforms that cover ∼85% of the pool of human IgG1 Fc glycoforms. Core α1–6 fucosylation showed the greatest changes with CD16B (8.5-fold decrease), CD16A (3.9-fold decrease) and CD32B/C (1.8-fold decrease), but did not affect binding to CD32A. Adding galactose to the non-reducing termini of the complex-type, biantennary glycan increased affinity for all CD16s and 32s tested by 1.7 fold. Sialylation did not change the affinity of core-fucosylated Fc, but increased the affinity of afucosylated Fc slightly by an average of 1.16 fold for all CD16s and CD32s tested. The effects of fucose and galactose modification are additive, suggesting the contributions of these residues to Fc γ receptor affinity are independent.
Unlabelled:
High interindividual variability in postoperative opioid consumption is related to genetic and environmental factors. We tested the association between morphine consumption, postoperative pain, and single nucleotide polymorphisms (SNPs) within opioid receptor μ 1 (OPRM1), catechol-O-methyltransferase (COMT), uridine diphosphate glucose-glucuronosyltransferase-2B7, and estrogen receptor (ESR1) gene loci to elucidate genetic prediction of opioid consumption. We analyzed 20 SNPs in 201 unrelated Caucasian patients who underwent abdominal surgery and who were receiving postoperative patient-controlled analgesia-administered morphine. Morphine consumption and pain intensity were dependent variables; age and sex were covariates. A haplotype of 7 SNPs in OPRM1 showed significant additive effects on opioid consumption (P = .007); a linear regression model including age and 9 SNPs in ESR1, OPRM1, and COMT explained the highest proportion of variance of morphine consumption (10.7%; P = .001). The minimal model including 3 SNPs in ESR1, OPRM1, and COMT explained 5% of variance (P = .007). We found a significant interaction between rs4680 in COMT and rs4986936 in ESR1 (P = .007) on opioid consumption. SNPs rs677830 and rs540825 of OPRM1 and rs9340799 of ESR1 were nominally associated with pain Numeric Rating Scale scores. Combinations of genetic variants within OPRM1, COMT, and ESR1 better explain variability in morphine consumption than single genetic variants. Our results contribute to the development of genetic markers and statistical models for future diagnostic tools for opioid consumption/efficacy.
Perspective:
This article presents the efforts dedicated to detect correlations between the genetic polymorphisms and the clinical morphine effect self-administered by patients using a patient-controlled analgesia pump after major surgery. The clinical effect is expressed in terms of morphine consumption and pain scores. REGISTERED ON CLINICALTRIALS.GOV: NCT01233752.
Asparagine(N)297-linked glycosylation of immunoglobulin G (IgG) Fc is required for binding to FcγRIIa, IIb, and IIIa, although it is unclear how it contributes. We found the quaternary structure of glycosylated Fc was indistinguishable from aglycosylated Fc, indicating that N-glycosylation does not maintain relative Fc Cγ2/Cγ3 domain orientation. However, the conformation of the C'E loop, which contains N297, was significantly perturbed in the aglycosylated Fc variant. The conformation of the C'E loop as measured with a range of Fc variants shows a strong correlation with FcγRIIIa affinity. These results indicate that the primary role of the IgG1 Fc N-glycan is to stabilize the C'E loop through intramolecular interactions between carbohydrate and amino acid residues, and preorganize the FcγRIIIa interface for optimal binding affinity. The features that contribute to the capacity of the IgG1 Fc N-glycan to restrict protein conformation and tune binding affinity are conserved in other antibodies including IgG2-IgG4, IgD, IgE, and IgM.
Copyright © 2015 Elsevier Ltd. All rights reserved.
An improved separation of the human serum N-glycome using hydrophilic interaction chromatography (HILIC) technology with UPLC is described, where more than 140 N-glycans were assigned. Using this technique serum samples from 107 healthy controls and 62 newly diagnosed breast cancer patients were profiled. The most statistically significant alterations were observed in cancer patients compared to healthy controls: an increase in sialylation, branching and outer arm-fucosylation and a decrease in high mannosylated and biantennary core-fucosylated glycans. In the controls and cases combined systemic features were analysed; serum estradiol was associated with increase in digalactosylated glycans, higher mammographic density was associated with increase in biantennary digalactosylated glycans and with decrease in trisialylated and in outer arm-fucosylated glycans. Furthermore, particular glycans were altered in some features of the breast carcinomas - bisected biantennary non-fucosylated glycans were decreased in patients with progesterone receptor positive tumours and core-fucosylated biantennary bisected monogalactosylated glycans were decreased in patients with the TP53 mutation. Systemic features show more significant associations with the serum N-glycome than do the features of the breast carcinomas. In conclusion, the UPLC-based glycan analysis technique described here reveals highly significant differences between healthy women and breast cancer patients. Significant associations with breast carcinoma and systemic features are described.
Psychological factors including catastrophizing thoughts are believed to influence the development of chronic low back pain.
To assess the prognostic importance of catastrophizing as a coping strategy in patients with low back pain.
Systematic Review PATIENT SAMPLE: Patients with low back pain.
Work related outcomes and perceived measures including return to work, pain and disability.
In September 2012, the following databases were searched: BIOSIS, CINAHL, Cochrane Library, Embase, OTSeeker, PeDRO, PsycInfo, Medline, Scopus, and Web of Science. To ensure completeness of the search, a hand search and a search of bibliographies was conducted and all relevant references included. All observational studies investigating the prognostic value of catastrophizing in patients with low back pain were eligible. Included were studies with 100 and more patients and follow-up of at least three months. Excluded were studies with poor methodological quality, short follow-up duration, and small sample size. This study was not funded and the authors have no conflict of interest to declare.
1473 references were retrieved, and 706 references remained after the removal of duplicates. For 77 references, the full text was assessed and 19 publications based on 16 studies were included. Of four studies that investigated work-related outcomes, two found catastrophizing to be associated with work status. Most studies that investigated self-reported outcome measures (n= 8, 66%) found catastrophizing to be associated with pain and disability at follow-up in acute, subacute, and chronic low back pain patients. In most studies that applied cut-off values, patients identified as high catastrophizers experienced a worse outcome compared to low catastrophizers (n=5, 83%).
There is some evidence that catastrophizing as a coping strategy might lead to delayed recovery. The influence of catastrophizing in patients with low back pain is not fully established and should be further investigated. Of particular importance is the establishment of cut-off levels for identifying patients at risk.
Sacroiliac (SI) joint pain is a challenging condition af- fecting 15% to 25% of patients with axial low back pain, for which there is no standard long-term treatment. Re- cent studies have demonstrated that historical and physical examination findings and radiological imag- ing are insufficient to diagnose SI joint pain. The most commonly used method to diagnose the SI joint as a pain generator is with small-volume local anesthetic blocks, although the validity of this practice remains unproven. In the present review I provide a compre- hensive review of the anatomy, function, and mecha- nisms of injury of the SI joint, along with a systematic assessment of its diagnosis and treatment. (Anesth Analg 2005;101:1440-53)
1-acid glycoprotein (AGP) is a serum acute phase glycoprotein which possesses five N-linked complex type heteroglycan side chains which may be present as bi-, tri- and tetraantennary structures. Depending upon the content of biantennary structure on AGP, up to four glycoforms of AGP are present in serum. These glycoforms can be easily estimated in body fluids by means of crossed affinity-immunoelectrophoresis (CAIE) with the lectin, Concanavalin A (Con A). Con A selectively binds biantennary structures; the more biantennary structures on AGP, the stronger the binding. In acute inflammation, a relative increase of AGP glycoforms with biantennary units is observed - a type I glycosylation change. In some chronic inflammatory states there is an relative decrease of AGP glycoforms with biantennary heteroglycans — a type II glycosylation change. Moreover, in certain other states such as pregnancy, estrogen administration or liver damage, type II glycosylation changes are also seen. A detailed analysis of the clinical applications of the assessment of AGP glycoforms in sera of patients with rheumatic diseases, AIDS and various types of cancers is presented. Accumulated data shows that AGP glycoforms may be very useful in the detection of intercurrent infections in the course of rheumatoid arthritis, systemic lupus erythematosus, or myeloblastic leukaemia, and in the detection of secondary infections in human immunodeficiency virus infected individuals. AGP glycoforms are also very useful in differentiation between various forms of trophoblastic disease and are helpful in monitoring the treatment of these patients. Finally, AGP glycoforms provide valuable information for differentiation between primary and secondary liver cancer.
Facet joints have been described as an important source of low back pain. The value of medial branch blocks in the diagnosis
of facet joint mediated pain is considered important. However, the therapeutic value of medial branch blocks has not been
determined.
This study was designed to evaluate the duration of relief obtained and therapeutic value following controlled medial branch
blocks with or without adjuvant agents Sarapin (High Chemical Company, Levittown, PA) and Depo-medrol (Pharmacia and Upjohn
Company, Kalamazoo, MI). The study population consisted of 180 consecutive patients seen in a single pain management practice,
divided into three groups with 60 patients in each group. Group I was treated with local anesthetic only, Group II with the
addition of Sarapin, and Group III with the addition of Depo-medrol along with Sarapin. The prevalence of facet joint pain
in chronic low back pain was determined as 36%, with a false-positive rate of 25%. Comparison of duration of relief in days
with each block in the three groups showed that the relief was significantly superior in Group III compared with Group I and
Group II, whereas Group II was superior to Group I.
Several different chromatographic methods and a lectin-based assay have been compared for the quantitation of oligosaccharides released from immunoglobulin G (IgG). The analysis of a series of IgG samples purified from the serum of rheumatoid arthritis patients was carried out by these methods to evaluate the percentage of the glycoforms having 0, 1 or 2 galactose residues (G0, G1 and G2) in order to (a) identify the method that can be most widely used for quantitation, (b) accurately define the range of G0 values found in patients with rheumatoid arthritis, and (c) make available a series of characterised standards for distribution to clinical chemistry laboratories. The chromatographic methods involved: release of oligosaccharides by glycoamidase A after protease digestion followed by HPLC analysis of aminopyridine derivatives on reverse phase and normal phase columns; hydrazinolysis treatment with exoglycosidases (G0 mix) and Biogel P4 chromatography of 2-aminobenzamide (2-AB) derivatives; hydrazinolysis and weak anion exchange or normal phase HPLC of 2-AB derivatives; release of oligosaccharides by PNGase F and either Biogel P4 chromatography of 2-AB derivatives or HPAEC-PAD analysis of native oligosaccharides. The G0 values given by these methods compared favourably with each other and a dot blot assay of denatured IgG interaction with Ricinus communis agglutinin and Bandeiraea simplicifolia lectin II. The HPLC and HPAEC methods give additional information that may be important in less routine assays.
A rapid glycomic profiling method is described wherein N-glycans from plasma samples individually labelled with aniline, 2-aminobenzamide and 2-aminoacridone are mixed, co-injected and separated in the same HILIC-fluorescence run. Transfer of the multiplexed method to UPLC-fluorescence permits an increase in sample throughput from 24 to 864 plasma samples per day.
Glycan structures attached to the CH2 domain of the Fc region of immunoglobulin G (IgG) are essential for specific effector functions but their role in modulating
clearance is less clear. Clearance is of obvious importance for therapeutic monoclonal antibodies (Mabs) as it directly impacts
efficacy. Here, we study the impact of Fc glycan structure on the clearance of four therapeutic human IgGs (one IgG1 and three
IgG2s) in humans. The therapeutic IgGs were affinity purified from serum samples from human pharmacokinetic studies, and changes
to the glycan profile over time were determined by peptide mapping employing high-resolution mass spectrometry. Relative levels
of high-mannose 5 (M5) glycan decreased as a function of circulation time, whereas other glycans remained constant. These
results demonstrate that therapeutic IgGs containing Fc high-mannose glycans are cleared more rapidly in humans than other
glycan forms. The quantitative effect of this on pharmacokinetic area under the curve was calculated and shown to be relatively
minor for three of the four molecules studied, but, depending on the dosing regimen and the relative level of the high-mannose
glycan, this can also have significant impact. High-mannose content of therapeutic Mabs should be considered an important
product quality attribute which may affect pharmacokinetic properties of therapeutic antibodies.
#### Summary points
Back pain is the leading cause of occupational disability in the world and the most common cause of missed workdays. As the population ages and our lives become more sedentary, this situation is unlikely to change. We aim here to provide an evidence based overview of low back pain aimed at primary care physicians.
The most frequently quoted epidemiological studies cite lifetime adult prevalence rates varying from 50% to 80%, and point prevalence rates from 15% to 30%.1 Yet even these statistics may underestimate the problem. A recent prospective study of 154 reserve soldiers with no prior history of back pain found that 64% of this low risk group developed at least moderate back pain over an 18 month period when queried monthly.w1 This suggests that reported prevalence rates may be a function of the type and frequency of surveillance.
### History
Box 1 lists the causes of low back pain. The origin of pain can be broadly classified as mechanical, neuropathic, or secondary to another cause. Mechanical back pain implies that the source of pain is in the spine or its supporting structures. Neuropathic back pain denotes the presence of symptoms that stem from irritation of a nerve root(s).
#### Box 1 Common causes of low back pain*
##### Mechanical (80-90%)
Plasma glycans were analyzed in 1008 individuals to evaluate variability and heritability, as well as the main environmental determinants that affect glycan structures. By combining HPLC analysis of fluorescently labeled glycans with sialidase digestion, glycans were separated into 33 chromatographic peaks and quantified. A high level of variability was observed with the median ratio of minimal to maximal values of 6.17 and significant age- and gender-specific differences. Heritability estimates for individual glycans varied widely, ranging from very low to very high. Glycome-wide environmental determinants were also detected with statistically significant effects of different variables including diet, smoking and cholesterol levels.
Microheterogeneity of acute phase proteins frequently differs in acute and chronic types of inflammation. However, it is unknown whether these changes depend on the duration of the inflammation in a given disease. We therefore investigated the microheterogeneity of alpha 1-acid glycoprotein (AGP) in sera from patients with acute and chronic bacterial infection in comparison to rheumatoid arthritis and ankylosing spondylitis. In acute bacterial infection Con A-reactivity of AGP was significantly elevated. By contrast, AGP in chronic bacterial infection showed the same glycosylation pattern as rheumatoid arthritis and ankylosing spondylitis being characterized by a decreased reactivity to Con A. Serial measurements in individual patients with bacterial infections showed a transition from the initially elevated to decreased reactivity to Con A as the disease became chronic.
At least 6 N-acetylglucosaminyltransferases (GlcNAc-T I, II, III, IV, V and VI) are involved in initiating the synthesis of the various branches found in complex asparagine-linked oligosaccharides (N-glycans), as indicated below: GlcNAc beta 1-6 GlcNAc-T V GlcNAc beta 1-4 GlcNAc-T VI GlcNAc beta 1-2Man alpha 1-6 GlcNAc-T II GlcNAc beta 1-4Man beta 1-4-R GlcNAc T III GlcNAc beta 1-4Man alpha 1-3 GlcNAc-T IV GlcNAc beta 1-2 GlcNAc-T I where R is GlcNAc beta 1-4(+/- Fuc alpha 1-6)GlcNAcAsn-X. HPLC was used to study the substrate specificities of these GlcNAc-T and the sequential pathways involved in the biosynthesis of highly branched N-glycans in hen oviduct (I. Brockhausen, J.P. Carver and H. Schachter (1988) Biochem. Cell Biol. 66, 1134-1151). The following sequential rules have been established: GlcNAc-T I must act before GlcNAc-T II, III and IV; GlcNAc-T II, IV and V cannot act after GlcNAc-T III, i.e., on bisected substrates; GlcNAc-T VI can act on both bisected and non-bisected substrates; both Glc-NAc-T I and II must act before GlcNAc-T V and VI; GlcNAc-T V cannot act after GlcNAc-T VI. GlcNAc-T V is the only enzyme among the 6 transferases cited above which can be essayed in the absence of Mn2+. In studies on the possible functional role of N-glycan branching, we have measured GlcNAc-T III in pre-neoplastic rat liver nodules (S. Narasimhan, H. Schachter and S. Rajalakshmi (1988) J. Biol. Chem. 263, 1273-1281). The nodules were initiated by administration of a single dose of carcinogen 1,2-dimethyl-hydrazine.2 HCl 18 h after partial hepatectomy and promoted by feeding a diet supplemented with 1% orotic acid for 32-40 weeks. The nodules had significant GlcNAc-T III activity (1.2-2.2 nmol/h/mg), whereas the surrounding liver, regenerating liver 24 h after partial hepatectomy and control liver from normal rats had negligible activity (0.02-0.03 nmol/h/mg). These results suggest that GlcNAc-T III is induced at the pre-neoplastic stage in liver carcinogenesis and are consistent with the reported presence of bisecting GlcNAc residues in N-glycans from rat and human hepatoma gamma-glutamyl transpeptidase and their absence in enzyme from normal liver of rats and humans (A. Kobata and K. Yamashita (1984) Pure Appl. Chem. 56, 821-832).
alpha 1-acid glycoprotein (AGP) is a serum acute phase glycoprotein which possesses five N-linked complex type heteroglycan side chains which may be present as bi-, tri- and tetraantennary structures. Depending upon the content of biantennary structure on AGP, up to four glycoforms of AGP are present in serum. These glycoforms can be easily estimated in body fluids by means of crossed affinity-immunoelectrophoresis (CAIE) with the lectin, Concanavalin A (Con A). Con A selectively binds biantennary structures; the more biantennary structures on AGP, the stronger the binding. In acute inflammation, a relative increase of AGP glycoforms with biantennary units is observed-a type I glycosylation change. In some chronic inflammatory states there is an relative decrease of AGP glycoforms with biantennary heteroglycans-a type II glycosylation change. Moreover, in certain other states such as pregnancy, estrogen administration or liver damage, type II glycosylation changes are also seen. A detailed analysis of the clinical applications of the assessment of AGP glycoforms in sera of patients with rheumatic diseases, AIDS and various types of cancers is presented. Accumulated data shows that AGP glycoforms may be very useful in the detection of intercurrent infections in the course of rheumatoid arthritis, systemic lupus erythematosus, or myeloblastic leukaemia, and in the detection of secondary infections in human immunodeficiency virus infected individuals. AGP glycoforms are also very useful in differentiation between various forms of trophoblastic disease and are helpful in monitoring the treatment of these patients. Finally, AGP glycoforms provide valuable information for differentiation between primary and secondary liver cancer.
The glycosylation of the circulating immunoglobulin-gamma (IgG) antibody molecules changes in rheumatoid arthritis. The extent of the changes correlates with the disease severity and reverses in remission. We demonstrate here that the alteration in glycosylation associated with rheumatoid arthritis can create a new mode for the interaction of IgG with complement through binding to the collagenous lectin mannose-binding protein (MBP). Rheumatoid arthritis is associated with a marked increases in IgG glycoforms that lack galactose (referred to as G0 glycoforms) in the Fc region of the molecule and that terminate in N-acetyl glucosamine (GlcNAc). We show, using nuclear magnetic resonance (NMR) and X-ray data, that these terminal GlcNAc residues become accessible for MBP binding. We further demonstrate that multiple presentation of IgG-G0 glycoforms to MBP results in activation of the complement. This suggests that a contribution to the chronic inflammation of the synovial membrane could arise from the localization of the IgG-G0 glycoforms in the affected joint and from resulting activation of complement.
Many different theories have been advanced concerning the biological roles of the oligosaccharide units of individual classes of glycoconjugates. Analysis of the evidence indicates that while all of these theories are correct, exceptions to each can also be found. The biological roles of oligosaccharides appear to span the spectrum from those that are trivial, to those that are crucial for the development, growth, function or survival of an organism. Some general principles emerge. First, it is difficult to predict a priori the functions a given oligosaccharide on a given glycoconjugate might be mediating, or their relative importance to the organism. Second, the same oligosaccharide sequence may mediate different functions at different locations within the same organism, or at different times in its ontogeny or life cycle. Third, the more specific and crucial biological roles of oligosaccharides are often mediated by unusual oligosaccharide sequences, unusual presentations of common terminal sequences, or by further modifications of the sugars themselves. However, such oligosaccharide sequences are also more likely to be targets for recognition by pathogenic toxins and microorganisms. As such, they are subject to more intra- and inter-species variation because of ongoing host-pathogen interactions during evolution. In the final analysis, the only common features of the varied functions of oligosaccharides are that they either mediate 'specific recognition' events or that they provide 'modulation' of biological processes. In so doing, they generate much of the functional diversity required for the development and differentiation of complex organisms, and for their interactions with other organisms in the environment.
Acute and chronic inflammation-induced expression of sialyl LewisX has already been shown to occur on alpha1-acid glycoprotein. We now demonstrate that this phenomenon is not restricted to alpha1-acid glycoprotein but also occurs on two other acute-phase proteins. ie on alpha1-antichymotrypsin and on haptoglobin. The level of expression of sialyl LewisX on these proteins was lower than on alpha1-acid glycoprotein, in all likelihood because alpha1-acid glycoprotein is the only acute-phase protein containing tetraantennary glycans. No expression of sialyl LewisX was detectable on alpha1-protease inhibitor, a protein with a high diantennary glycan content. Non-sialylated LewisX was not detectable on these major acute-phase proteins in any of the conditions studied. This indicates that the majority of the a3-linked fucose residues are present as sialyl LewisX on alpha1-acid glycoprotein, alpha1-antichymotrypsin and haptoglobin. The absolute contribution to the total phenotype in plasma of protein containing this determinant in a multivalent form was highest for alpha1-acid glycoprotein. This leads us to propose that alpha1-acid glycoprotein is, among the acute-phase proteins studied, the one with the highest potential for interference with the extravasation of leukocytes by binding to the selectins.