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Hepatocellular carcinoma (HCC) is one of the most lethal malignancies, which is associated with a low 5-year survival rate. The importance of effective disease monitoring and prognostic evaluation is undeniable. For the present study, a systematic review was performed using extensive searches in Medline, Embase, Web of Science and Scopus up to December 29, 2023. The aim of the present study was to examine whether N-glycomics could predict the risk of developing HCC in adults with chronic liver disease and, if HCC was present, predict overall survival. As a secondary outcome, the prediction capability of HCC recurrence was assessed. After deduplication, 3,904 studies were identified, of which 30 were included. Overall, the median size of the study cohort was 144 patients, with a median follow-up time of 63.6 months. Three studies explored N-glycomics in whole serum, whereas the rest focused on individual glycoproteins, with Mac-2 binding protein glycosylation isomer (M2BPGi) being the most commonly studied. Most articles investigated baseline M2BPGi values as predictors for the development of HCC and demonstrated a median area under the curve of 0.83 with a cut-off index value of 1.8. In conclusion, it was revaled that N-glycan changes exhibit added value in determining patient prognosis in terms of survival, monitoring HCC development and recurrence., Competing Interests: The authors declare that they have no competing interests., (Copyright: © 2024 Somers et al.)

Objective: Glycans attached to the Fc region of IgG antibodies influence their pro- or anti-inflammatory effector function. We aimed to explore the interrelation of the Fc glycosylation profile and disease transition, disease activity, and outcome in patients with suspected and confirmed primary Sjögren disease (SjD)., Methods: IgG Fc sialylation and IgG Fc galactosylation serum levels were determined in 300 patients from the Belgian Sjögren's Syndrome Transition Trial. This cohort includes both suspected and confirmed patients with SjD meeting the 2016 American College of Rheumatology/EULAR criteria. Salivary gland involvement was evaluated through ultrasonography (Hocevar score 0-48) and histopathology (focus score). The relative amount of sialylated and galactosylated IgG was determined by capillary electrophoresis after using the endoglycosidase S-based assay., Results: Patients with SjD exhibited significantly lower sialylation and galactosylation levels versus asymptomatic carriers of anti-SSA and patients with sicca. Lower sialylation and galactosylation levels were significantly associated with an increase in B cell activation markers and distinct autoantibody profiles, particularly with multiple autoantibody reactivities. They were also linked to histopathological salivary gland alterations, higher Hocevar scores, and, importantly, risk factors for non-Hodgkin lymphoma (NHL) development. In contrast, patients with SjD who were mono-anti-Ro60 positive and those who were anti-SSA negative had normal IgG Fc glycosylation., Conclusion: This study points to a novel role of IgG Fc glycosylation in patients with SjD in predicting disease transition, monitoring disease activity, and stratifying risk of NHL development., (© 2024 American College of Rheumatology.)

The industrial yeast Komagataella phaffii (formerly named Pichia pastoris) is commonly used to synthesize recombinant proteins, many of which are used as human therapeutics or in food. However, the basic strain, named NRRL Y-11430, from which all commercial hosts are derived, is not available without restrictions on its use. Comparative genome sequencing leaves little doubt that NRRL Y-11430 is derived from a K. phaffii type strain deposited in the UC Davis Phaff Yeast Strain Collection in 1954. We analysed four equivalent type strains in several culture collections and identified the NCYC 2543 strain, from which we started to develop an open-access Pichia chassis strain that anyone can use to produce recombinant proteins to industry standards. NRRL Y-11430 is readily transformable, which we found to be due to a HOC1 open-reading-frame truncation that alters cell-wall mannan. We introduced the HOC1 open-reading-frame truncation into NCYC 2543, which increased the transformability and improved secretion of some but not all of our tested proteins. We provide our genome-sequenced type strain, the hoc1 tr derivative that we named OPENPichia as well as a synthetic, modular expression vector toolkit under liberal end-user distribution licences as an unencumbered OPENPichia resource for the microbial biotechnology community., (© 2024. The Author(s).)

Background: Hepatocellular carcinoma (HCC) is the most prevalent primary liver cancer with one of the highest cancer-related mortality rates worldwide. Early diagnosis is crucial for improving the therapeutic options and reducing the disease-related mortality., Aim: To investigate serum N-glycomics as diagnostic markers for HCC., Methods: We performed a comprehensive search in PubMed, EMBASE, Web of Science and Scopus through August 17, 2023. Eligible studies assessed the potential use of serum N-glycomics as diagnostic biomarkers for HCC. Study selection, data extraction and quality assessment were performed by two independent reviewers., Results: Of the 48 articles included, 11 evaluated the utility of N-glycomics for the diagnosis of HCC in whole serum while the remaining articles focused on specific protein glycoforms or protein levels. Of these specific proteins, haptoglobin, alpha-fetoprotein (AFP), kininogen (Kin), α-1-antitrypsin and Golgi protein 73 (GP73) were the most frequently studied. Increased levels of fucosylation and branching presented as the most prevalent post-translational modifications of glycoproteins in patients with HCC compared to controls. Notably, glycomics-based biomarkers may provide a clinical benefit for the diagnosis of early HCC, as several algorithms achieved AUCs between 0.92-0.97. However, these were based on single studies with limited sample sizes and should therefore be validated., Conclusions: Alterations in serum N-glycomics, characterised by increased levels of fucosylation and branching, have potential as diagnostic biomarkers for HCC. Optimisation of study design, patient selection and analysing techniques are needed before clinical implementation will be possible., (© 2023 John Wiley & Sons Ltd.)

Objectives: 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., Methods: 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., Results: 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., Conclusion: 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: 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., Competing Interests: X.V. and H.V.V. are coinventors on a patent owned by Ghent University (Belgium) for a glycomics-based biomarker for the prediction of primary nonfunction after LT. N.C. is a coinventor on a patent on GlycoCirrhoTest that is owned by VIB vzw and has been licensed to Helena Biosciences. The other authors declare no conflicts of interest. X.V. and H.V.V. participated in research design. X.V. and R.C. participated in the writing of the article. X.V., A.G., A.V., L.A.C., F.B., and X.R. participated in the performance of the research. X.V., L.M., and N.C. contributed new reagents or analytic tools. X.V., R.C., and H.V.V. participated in data analysis. X.V., A.G., A.V., H.D., L.A.C., L.M., F.B., N.C., and H.V.V. participated in reviewing the article., (Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.)

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., (© 2021. The Author(s).)

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 = 0.00001, p = 0.046, p = 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 = 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 = 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 = <0.0001 for both). The rate of seroconversion (>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 = 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 = 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., Competing Interests: Conflict of interest statement MP is an advisor of Remedus; none of the other authors has a conflict of interest related to this paper., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

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 .

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., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

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., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Influenza virus neuraminidase (NA) has been under intense study recently as a vaccine antigen, yet there remain unanswered questions regarding the immune response directed toward NA. Antibodies (Abs) that can inhibit NA activity have been shown to aid in the control of disease caused by influenza virus infection in humans and animal models, yet how and if interactions between the Fc portion of anti-NA Abs and Fcγ receptors (FcγR) contribute to protection has not yet been extensively studied. Herein, we show that poly- and monoclonal anti-NA IgG antibodies with NA inhibitory activity can control A(H1N1)pdm09 infection in the absence of FcγRs, but FcγR interaction aided in viral clearance from the lungs. In contrast, a mouse-human chimeric anti-NA IgG1 that was incapable of mediating NA inhibition (NI) solely relied on FcγR interaction to protect transgenic mice (with a humanized FcγR compartment) against A(H1N1)pdm09 infection. As such, this study suggests that NA-specific antibodies contribute to protection against influenza A virus infection even in the absence of NI activity and supports protection through multiple effector mechanisms. IMPORTANCE There is a pressing need for next-generation influenza vaccine strategies that are better able to manage antigenic drift and the cocirculation of multiple drift variants and that consistently improve vaccine effectiveness. Influenza virus NA is a key target antigen as a component of a next-generation vaccine in the influenza field, with evidence for a role in protective immunity in humans. However, mechanisms of protection provided by antibodies directed to NA remain largely unexplored. Herein, we show that antibody Fc interaction with Fcγ receptors (FcγRs) expressed on effector cells contributes to viral control in a murine model of influenza. Importantly, a chimeric mouse-human IgG1 with no direct antiviral activity was demonstrated to solely rely on FcγRs to protect mice from disease. Therefore, antibodies without NA enzymatic inhibitory activity may also play a role in controlling influenza viruses and should be of consideration when designing NA-based vaccines and assessing immunogenicity., (Copyright © 2019 Job et al.)

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-β-N-acetylglucosaminidase 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., (© 2018 Vanderschaeghe et al.)

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.

Human 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., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

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.

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.