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Patients with chronic hepatitis C virus (HCV) infection have a genuine risk of developing liver fibrosis and cirrhosis, potentially resulting in hepatocellular carcinoma (HCC), a risk that remains even after sustained viral response (SVR). Glycomics-based biomarkers are an attractive tool to closely monitor these patients during and after antiviral treatment, as alterations in the abundance of N-glycans reflect an altered state of the liver. This study assessed serum glycomics for the evaluation of inflammation-related fibrosis regression during and after treatment of HCV with DAAs.The GlycoFibroTest and GlycoCirrhoTest were analyzed in the sera 36 HCV-infected patients with advanced fibrosis (F3) or established cirrhosis (F4), before (week 0), during (week 12) and after (week 24) a twelve-week oral administration of DAAs therapy - using an optimized glycomic technology on a DNA sequencer.All patients achieved SVR after treatment and two of them developed HCC in the subsequent five years. A significant decrease of the GlycoFibroTest (p 0.0001) was seen after 12 weeks, consistent with other measured biomarkers (APRI, FIB-4, FibroTest). Statistical analysis was performed in IBM SPSS Statistics version 28.0, using the non-parametric Friedman's test with a statistical significance α level of 0.05.This study suggests that the GlycoFibroTest is a serum biomarker for viral response in HCV patients. The rapid decrease of the glycomics-based biomarker probably reflects the amelioration of liver inflammation as underlying process, rather than the improvement of liver fibrosis itself.

Background: Coronavirus disease (COVID-19) is interfering heavily with the screening, diagnosis and treatment of cancer patients. Better knowledge of the seroprevalence and immune response after Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection in this population is important to manage them safely during the pandemic. Methods: 922 cancer patients, 100 non-cancer patients and 94 health care workers (HCW) attending the Multidisciplinary Oncology Unit of Antwerp University Hospital from 24th of March 2020 till 31st of May 2020, and the Oncology Unit of AZ Maria Middelares Hospital, Ghent, from 13th of April 2020 till 31st of May 2020 participated in the study. The Alinity (A; Abbott) and Liaison (D; DiaSorin) commercially available assays were used to measure SARS-CoV-2 IgG, while total SARS-CoV-2 Ig was measured by Elecsys (R; Roche). Results: In the overall study population IgG/total SARS-CoV-2 antibodies were found in respectively 32/998 (3.2%), 68/1020 (6.7%), 37/1010 (3.7%) and of individuals using the A, D or R test. Forty-six out of 618 (7.4%) persons had a positive SARS-CoV-2 polymerase chain reaction (RT-PCR) test. Seroprevalence in cancer patients (A:2.2%, D:6.2%, R:3.0%), did not significantly differ from that in non-cancer patients (A:1.1%, D:5.6%, R:0.0%), but was lower than the HCW (A:13%, D:12%, R:12%; respectively Fisher’s exact test p Z 0.00001, p Z 0.046, p Z 0.0004). A positive SARS-CoV-2 RT-PCR was found in 6.8% of the cancer patients, 2.3% of the non-cancer patients and 28.1% of the HCW (Fisher’s exact test p Z 0.0004). Correlation between absolute values of the different Ig tests was poor in the cancer population. Dichotomising a positive versus negative test result, the A and R test correlated well (kappa 0.82 p McNemar test Z 0.344), while A and D and R and D did not (respectively kappa 0.49 and 0.57; result significantly different p McNemar test Z 75%) and median absolute antibody levels (A: 7.0 versus 4.7; D 74.0 versus 26.6, R: 16.34 versus 7.32; all >P Mann Whitney U test Z 0.28) in cancer patients and HCW with a positive RT-PCR at least 7 days earlier did not show any differences. However, none (N Z 0/4) of the patients with hematological tumours had seroconversion and absolute antibody levels remained much lower compared to patients with solid tumours (R: 0.1 versus 37.6, p 0.003; D 4.1 versus 158, p 0.008) or HCW (all p < 0.0001). Conclusion: HCW were at high risk of being infected by SARS-CoV-2 during the first wave of the pandemic. Seroprevalence in cancer patients was low in the study period. Although Ig immune response in cancer patients with solid tumours does not differ from healthy volunteers, patients with hematological tumours have a very poor humoral immune response. This has to be taken into account in future vaccination programmes in this population. SARS-CoV-2 antibody tests have divergent results and seem to have little added value in the management of cancer patients.

For a long time, glycoprotein production has been limited by the inherent properties of production hosts. Glycosylation of biopharmaceuticals has been regarded as a necessary evil, often needed for protein folding or function, but also a source of heterogeneity, complicating downstream processing and product characterization. This has strongly determined the choice of production hosts. Over the last few decades, numerous glycoengineering efforts have helped solving this problem. Moreover, insights from fundamental studies have made it possible to improve therapeutic protein functionality through careful glycoengineering. Here, we will focus on how production host and in vitro glycoengineering approaches allow to design biopharmaceuticals with glycans that impart improved functionality. An important branch of research explores how glycosylation can be tuned to improve pharmacokinetics and reduce glycan heterogeneity of therapeutics. Furthermore, antibody glycoengineering to obtain homogeneous, defined glycan structures has been a major focus. An example of this is the production of Fc glycans without core fucose, exhibiting tremendously improved Antibody-Dependent Cell Cytotoxicity (ADCC). In the last part, glycoforms that allow for improved (subcellular) targeting and cellular uptake, a field that opens possibilities for enzyme replacement therapies and vaccine development, will be highlighted.

The chytrid fungal pathogens Batrachochytrium dendrobatidis and Batrachochytrium salamandrivorans cause the skin disease chytridiomycosis in amphibians, which is driving a substantial proportion of an entire vertebrate class to extinction. Mitigation of its impact is largely unsuccessful and requires a thorough understanding of the mechanisms underpinning the disease ecology. By identifying skin factors that mediate key events during the early interaction with B. salamandrivorans zoospores, we discovered a marker for host colonization. Amphibian skin associated beta-galactose mediated fungal chemotaxis and adhesion to the skin and initiated a virulent fungal response. Fungal colonization correlated with the skin glycosylation pattern, with cutaneous galactose content effectively predicting variation in host susceptibility to fungal colonization between amphibian species. Ontogenetic galactose patterns correlated with low level and asymptomatic infections in salamander larvae that were carried over through metamorphosis, resulting in juvenile mortality. Pronounced variation of galactose content within some, but not all species, may promote the selection for more colonization resistant host lineages, opening new avenues for disease mitigation., The skin disease chytridiomycosis is linked to global amphibian declines but effective mitigation measures require improved understanding of the mechanisms underpinning the disease ecology. This study identifies key mediators of interactions between the fungal pathogen and amphibian skin, providing a marker of host colonization that can predict susceptibility between amphibian species.

Over the past 30 years, it has been firmly established that a wide spectrum of (autoimmune) diseases such as rheumatoid arthritis, Crohn's and lupus, but also other pathologies like alcoholic and non-alcoholic steatohepatitis (ASH and NASH) are driven by chronic inflammation and are hallmarked by a reduced level of serum IgG galactosylation. IgG (under)galactosylation is a promising biomarker to assess disease severity, and monitor and adjust therapy. However, this biomarker has not been implemented in routine clinical chemistry because of a complex analytical procedure that necessitates IgG purification, which is difficult to perform and validate at high throughput. We addressed this issue by using endo-beta-N-acetyl-glucosaminidase from Streptococcus pyogenes (endoS) to specifically release Fc N-glycans in whole serum. The entire assay can be completed in a few hours and only entails adding endoS and labeling the glycans with APTS. Glycans are then readily analyzed through capillary electrophoresis. We demonstrate in two independent patient cohorts that IgG undergalactosylation levels obtained with this assay correlate very well with scores calculated from PNGaseF-released glycans of purified antibodies. Our new assay allows to directly and specifically measure the degree of IgG galactosylation in serum through a fast and completely liquid phase protocol, without the requirement for antibody purification. This should help advancing this biomarker toward clinical implementation.

Background. Prediction of outcome after liver transplantation (LT) is limited by the lack of robust predictors of graft failure. In this prospective study, we aimed to define a serum glycomic signature in the first week after LT that is associated with graft loss at 3 mo after LT. Methods. Patients were included between January 1, 2011, and February 28, 2017. Glycomic analysis was performed using DNA sequencer-associated fluorophore-associated capillary electrophoresis on a serum sample 1 wk after LT. Making use of Lasso regression, an optimal glycomic signature was identified associated with 3-mo graft survival. Results. In this cohort of 131 patients, graft loss at 3 mo occurred in 14 patients (11.9%). The optimal mode, called the GlycoTransplantTest, yielded an area under the curve of 0.95 for association with graft loss at 3 mo. Using an optimized cutoff for this biomarker, sensitivity was 86% and specificity 89%. Negative predictive value was 98%. Odds ratio for graft loss at 3 mo was 70.211 (P < 0.001; 95% confidence interval, 10.876-453.231). Conclusions. A serum glycomic signature is highly associated with graft loss at 3 mo. It could support decision making in early retransplantation.

Highlights • Cancer patients with COVID-19 have a higher morbidity and mortality. • Particularly patients with ongoing or recent cancer treatment, metastatic solid tumors and hematological malignancies are at risk. • Underlying immunosuppression, elevated cytokine levels, altered expression of the angiotensin converting enzyme (ACE-2) and TMPRSS2, and a prothrombotic status in cancer patients may fuel the effects of a SARS-CoV-2 sepsis. • The gene expression level of ACE2 may be an indicator of the susceptibility to SARS-CoV-2 infection, while TMPRSS2 plays a supporting role. • Better knowledge of the mechanisms involved may be a tool to identify high risk patients and to prevent severe complications by targeting the involved pathways., The outbreak of the SARS-CoV-2 pandemic has overwhelmed health care systems in many countries. The clinical presentation of the SARS-CoV-2 varies between a subclinical or flu-like syndrome to that of severe pneumonia with multi-organ failure and death. Initial reports have suggested that cancer patients may have a higher susceptibility to get infected by the SARS-CoV-2 virus but current evidence remains poor as it is biased by important confounders. Patients with ongoing or recent cancer treatment for advanced active disease, metastatic solid tumors and hematological malignancies are at higher risk of developing severe COVID-19 respiratory disease that requires hospitalization and have a poorer disease outcome compared to individuals without cancer. However it is not clear whether these are independent risk factors, or mainly driven by male gender, age, obesity, performance status, uncontrolled diabetes, cardiovascular disease and various other medical conditions. These often have a greater influence on the probability to die due to SARS-CoV-2 then cancer. Delayed diagnosis and suboptimal cancer management due to the pandemic results in disease upstaging and has considerable impact cancer on specific death rates. Surgery during the peak of the pandemic seems to increase mortality, but there is no convincing evidence that adjuvant systemic cancer therapy and radiotherapy are contraindicated, implicating that cancer treatment can be provided safely after individual risk/benefit assessment and some adaptive measures. Underlying immunosuppression, elevated cytokine levels, altered expression of the angiotensin converting enzyme (ACE-2) and TMPRSS2, and a prothrombotic status may fuel the effects of a SARS-CoV-2 in some cancer patients, but have the potential to be used as biomarkers for severe disease and therapeutic targets. The rapidly expanding literature on COVID-19 should be interpreted with care as it is often hampered by methodological and statistical flaws.

Glycosylation is an important post-translational modification, giving rise to a diverse and abundant repertoire of glycans on the cell surface, collectively known as the glycome. When focusing on immunity, glycans are indispensable in virtually all signaling and cell-cell interactions. More specifically, glycans have been shown to regulate key pathophysiological steps within T cell biology such as T cell development, thymocyte selection, T cell activity and signaling as well as T cell differentiation and proliferation. They are of major importance in determining the interaction of human T cells with tumor cells. In this review, we will describe the role of glycosylation of human T cells in more depth, elaborate on the importance of glycosylation in the interaction of human T cells with tumor cells and discuss the potential of cancer immunotherapies that are based on manipulating the glycome functions at the tumor immune interface.1,2

Glycosylation is an important post-translational modification, giving rise to a diverse and abundant repertoire of glycans on the cell surface, collectively known as the glycome. When focusing on immunity, glycans are indispensable in virtually all signaling and cell-cell interactions. More specifically, glycans have been shown to regulate key pathophysiological steps within T cell biology such as T cell development, thymocyte selection, T cell activity and signaling as well as T cell differentiation and proliferation. They are of major importance in determining the interaction of human T cells with tumor cells. In this review, we will describe the role of glycosylation of human T cells in more depth, elaborate on the importance of glycosylation in the interaction of human T cells with tumor cells and discuss the potential of cancer immunotherapies that are based on manipulating the glycome functions at the tumor immune interface.

Background:Inflammation in spondyloarthritis (SpA) is often not reflected by elevated acute phase reactants such as C-reactive protein (CRP) or erythrocyte sedimentation rate (ESR). It has been shown that IgG glycosylation patterns are subject to specific alterations (i.e. undergalactosylation) in chronic inflammatory diseases. Since these changes only occur in persistent inflammatory processes, lasting at least one to two t1/2of IgG (24 days), it was hypothesized that IgG-glycan profiles could serve as a surrogate marker for chronic inflammation in SpA patients.Objectives:To assess the value of serum IgG-undergalactosylation in SpA patients in relation to outcome measures for disease activity, determined by patient reported outcomes, serum inflammatory markers and imaging outcomes.Methods:Serum samples were obtained from SpA patients at the baseline visit of Be-Giant: a Belgian observational cohort including SpA patients who fulfill the ASAS classification criteria for axial or peripheral SpA. IgG Fc N-glycans were released directly in whole serum by endo-β-N-acetyl-glucosaminidase fromStreptococcus pyogenes(EndoS), fluorescently labeled with ATPS and analyzed by capillary electrophoresis, rendering glycan profiles with six peaks (Figure 1). Relative peak heights were combined in the undergalactosylation score (UGS), capturing the relative upregulation of non-galactosylated glycans normalized to the total peak height (1). Baseline radiographs (X-SIJ) and magnetic resonance images (MRI) of the sacroiliac joints (SIJ) were assessed by three calibrated readers for sacroiliitis (fulfillment of the modified New York criteria; grading 0 to 4 per SIJ) and for inflammatory lesions according to the Spondyloarthritis Research Consortium of Canada (SPARCC) method (score from 0 – 72) respectively. Grades and inflammatory lesions that were seen by at least 2 readers were used for further analysis.Figure 1.Example of a serum IgG-specific glycan profile. Adapted from (1), with permission.Results:Glycan profiles were obtained from 376 SpA patients; UGS was scaled (mean = 0, SD = 1) for further analysis. UGS was independently associated with ASDAS-CRP (β1= 0.15, 95% CI 0.04 – 0.26, p = 0.006) and BASFI (β1= 0.44, 95% CI 0.16 – 0.72, p = 0.002) but not with BASDAI (β1= 0.12, 95% CI -0.13 – 0.38, p = 0.34). UGS showed a weak to moderate correlation with CRP (Rs= 0.30, p < 0.001) and ESR (Rs= 0.27, p 1= 0.44, 95% CI 0.09 – 0.80, p = 0.01, Figure 2) and SPARCC score (β1= 2.64, 95% CI 0.98 – 4.31, p = 0.002). All models were adjusted for age, gender, BMI, CRP, anti-TNF treatment and symptom duration.Conclusion:This study shows and independent association of serum IgG undergalactosylation with disease activity and functional impairment in SpA patients. Moreover, UGS was significantly higher in advanced compared to early-stage axial disease and therefore may reflect the cumulative exposure to systemic inflammation.References:[1]Vanderschaeghe D, Meuris L, Raes T, et al. Endoglycosidase S Enables a Highly Simplified Clinical Chemistry Procedure for Direct Assessment of Serum IgG Undergalactosylation in Chronic Inflammatory Disease. Mol Cell Proteomics. 2018;17(12):2508-17.Figure 2.Correlation between UGS and X-SIJ total grading of sacroiliitis. R = Spearman’s correlation coefficient.Disclosure of Interests:Ann-Sophie De Craemer: None declared, Zuzanna Lukasik: None declared, Leander Meuris: None declared, Liselotte Deroo: None declared, Thomas Renson: None declared, Manouk de Hooge: None declared, Philippe Carron: None declared, Annelies Van Hecke: None declared, Nico Callewaert: None declared, Filip van den Bosch Consultant of: AbbVie, Celgene Corporation, Eli Lilly, Galapagos, Janssen, Novartis, Pfizer, and UCB, Speakers bureau: AbbVie, Celgene Corporation, Eli Lilly, Galapagos, Janssen, Novartis, Pfizer, and UCB, Dirk Elewaut: None declared

Many pro-inflammatory pathways leading to arthritis have global effects on the immune system rather than only acting locally in joints. The reason behind the regional and patchy distribution of arthritis represents a longstanding paradox. Here we show that biomechanical loading acts as a decisive factor in the transition from systemic autoimmunity to joint inflammation. Distribution of inflammation and erosive disease is confined to mechano-sensitive regions with a unique microanatomy. Curiously, this pathway relies on stromal cells but not adaptive immunity. Mechano-stimulation of mesenchymal cells induces CXCL1 and CCL2 for the recruitment of classical monocytes, which can differentiate into bone-resorbing osteoclasts. Genetic ablation of CCL2 or pharmacologic targeting of its receptor CCR2 abates mechanically-induced exacerbation of arthritis, indicating that stress-induced chemokine release by mesenchymal cells and chemo-attraction of monocytes determines preferential homing of arthritis to certain hot spots. Thus, mechanical strain controls the site-specific localisation of inflammation and tissue damage in arthritis., Pro-inflammatory factors implicated for the onset of arthritis often have systematic effects, yet arthritis symptoms are mostly limited to the joints. Here the authors show that mechanical strain at the joints promotes the recruitment of monocyte and their differentiation into bone-eroding osteoclast to contribute this tissue specificity.

A wide spectrum of (autoimmune) diseases such as rheumatoid arthritis (RA), Crohn's disease, systemic lupus erythematosus, but also other pathologies such as alcoholic and non-alcoholic steatohepatitis (ASH and NASH) are driven by chronic inflammation and hallmarked by a reduced level of serum Immunoglobulin G (IgG) galactosylation. IgG undergalactosylation is a promising biomarker to assess the severity of disease, monitor therapy efficacy and adjust therapy accordingly. The main hurdle for clinical implementation is the necessity for purifying the antibodies in order to specifically determine IgG Fc glycan galactosylation. We addressed this issue by using endo-β-N-acetylglucosaminidase from Streptococcus pyogenes (endoS) and optimized the reaction conditions in which the enzyme specifically releases the IgG Fc N-glycans in serum. The sample preparation takes two hours and only entails the addition of endoS to serum and subsequent labeling with the fluorophore 8-aminopyrene-1,3,6-trisulphonic acid (APTS). Samples are then readily analyzed on high-throughput DNA sequencers, which are able to analyze up to 96 samples in one hour. We demonstrate in two separate patient cohorts that the degree of IgG undergalactosylation from our assay correlates very well with undergalactosylation scores calculated from N-glycan profiles derived from antibodies that have been purified from serum and deglycosylated with peptide N-glycosidase from Flavobacterium meningosepticum (PNGaseF). The presented new assay thus allows to directly and specifically measure the degree of IgG galactosylation in serum without the requirement of isolating the antibodies, and should help in advancing this biomarker towards clinical implementation.

Background Many pro-inflammatory pathways leading to arthritis act systemically on the immune system rather than locally in the joint. However, the reason behind the regional and patchy distribution of arthritis represents a longstanding paradox. Objectives To explore the relation between mechanical strain and joint inflammation and to understand the underlying basis of joint pattern involvement in inflammatory rheumatic diseases. Methods Arthritis was induced by collagen-induced arthritis (CIA) and passive collagen antibody induced arthritis (CAIA) in respectively C57BL/6 and RAG2-/- (T- and B-cell deficient) mice. Animals were subjected to different regimens of mechanical strain. Increased strain occurred in voluntary running mice whereas tail suspension (unloading) abolished mechanical strain; both were compared to control housing conditions. The impact of different loading conditions was measured on clinical disease score, histology, micro-CT images and erosion quantification, gene induction in tendon and synovial tissue, immune cell recruitment in situ, development of anti-collagen antibodies and their pattern of siaylation and galactosylation. Results Voluntary running of CIA in C57BL/6 mice markedly induced an early onset and increased progression whereas no disease onset could be observed in the hind paws from animals in unloaded conditions. CAIA in running RAG2-/- mice also induced early arthritic symptoms and severe progression. Intriguingly, running conditions were sufficient to induce arthritis without the need of LPS as an inflammatory trigger. Mechanical strain did not alter however IgG autoantibody levels nor their levels of galactosylation and sialylation. Furthermore, we demonstrate that mechanical strain on stromal cells results in recruitment of classical monocytes into specialised mechano-sensitive regions characterised by a unique microanatomy. This promotes local inflammation and differentiation into local osteoclasts which induce regional erosions. A striking similarity was observed in the pattern of joint erosions in human patients with RA and SpA which were also confined to these mechanosensitive regions. Conclusions This study provides the first evidence that mechanical strain controls the transition from systemic autoimmunity into site-specific joint inflammation. Homing of inflammation and development of erosions was confined to specific mechano-sensitive regions, characterised by a high number of attachment- and contact points for tendons. This represents a novel paradigm and explains why arthritis in mice and humans is characterised by a regional and patchy distribution. Curiously, this pathway does not rely on adaptive immunity but rather on stromal cells. Mechano-stimulation of mesenchymal cells induced CXCL1 and CCL2 permitting recruitment of classical monocytes which can differentiate into bone-resorbing osteoclasts. Thus, mechanical strain controls the site-specific direction of inflammation and tissue damage in arthritis. Acknowledgements Disclosure of Interest None declared

SummaryHuman colony-stimulating factor 1 receptor (hCSF-1R) is unique among the hematopoietic receptors because it is activated by two distinct cytokines, CSF-1 and interleukin-34 (IL-34). Despite ever-growing insights into the central role of hCSF-1R signaling in innate and adaptive immunity, inflammatory diseases, and cancer, the structural basis of the functional dichotomy of hCSF-1R has remained elusive. Here, we report crystal structures of ternary complexes between hCSF-1 and hCSF-1R, including their complete extracellular assembly, and propose a mechanism for the cooperative human CSF-1:CSF-1R complex that relies on the adoption by dimeric hCSF-1 of an active conformational state and homotypic receptor interactions. Furthermore, we trace the cytokine-binding duality of hCSF-1R to a limited set of conserved interactions mediated by functionally equivalent residues on CSF-1 and IL-34 that play into the geometric requirements of hCSF-1R activation, and map the possible mechanistic consequences of somatic mutations in hCSF-1R associated with cancer.

The HEK293 human cell lineage is widely used in cell biology and biotechnology. Here we use whole-genome resequencing of six 293 cell lines to study the dynamics of this aneuploid genome in response to the manipulations used to generate common 293 cell derivatives, such as transformation and stable clone generation (293T); suspension growth adaptation (293S); and cytotoxic lectin selection (293SG). Remarkably, we observe that copy number alteration detection could identify the genomic region that enabled cell survival under selective conditions (i.c. ricin selection). Furthermore, we present methods to detect human/vector genome breakpoints and a user-friendly visualization tool for the 293 genome data. We also establish that the genome structure composition is in steady state for most of these cell lines when standard cell culturing conditions are used. This resource enables novel and more informed studies with 293 cells, and we will distribute the sequenced cell lines to this effect., The human embryonic kidney 293 (HEK293) cell lineage is widely used in cell biology and biotechnology. Here, the authors apply whole genome resequencing methods to characterise genomic variation in six HEK293 cell lines and suggest that this variation could affect experiments using these cell lines.

Heterogeneity in the N-glycans on therapeutic proteins causes difficulties for protein purification and process reproducibility and can lead to variable therapeutic efficacy. This heterogeneity arises from the multistep process of mammalian complex-type N-glycan synthesis. Here we report a glycoengineering strategy--which we call GlycoDelete--that shortens the Golgi N-glycosylation pathway in mammalian cells. This shortening results in the expression of proteins with small, sialylated trisaccharide N-glycans and reduced complexity compared to native mammalian cell glycoproteins. GlycoDelete engineering does not interfere with the functioning of N-glycans in protein folding, and the physiology of cells modified by GlycoDelete is similar to that of wild-type cells. A therapeutic human IgG expressed in GlycoDelete cells had properties, such as reduced initial clearance, that might be beneficial when the therapeutic goal is antigen neutralization. This strategy for reducing N-glycan heterogeneity on mammalian proteins could lead to more consistent performance of therapeutic proteins and modulation of biopharmaceutical functions.