Neuraminidase Treatment Research Articles

Ganglioside GD3 Regulates Inflammation and Epithelial-to-Mesenchymal Transition in Human Nasal Epithelial Cells.

Chronic sinusitis with nasal polyps (CRSwNP) is one of the most common chronic inflammatory diseases, and involves tissue remodeling. One of the key mechanisms of tissue remodeling is the epithelial-mesenchymal transition (EMT), which also represents one of the pathophysiological processes of CRS observed in CRSwNP tissues. To date, many transcription factors and forms of extracellular stimulation have been found to regulate the EMT process. However, it is not known whether gangliosides, which are the central molecules of plasma membranes, involved in regulating signal transmission pathways, are involved in the EMT process. Therefore, we aimed to determine the role of gangliosides in the EMT process. First, we confirmed that N-cadherin, which is a known mesenchymal marker, and ganglioside GD3 were specifically expressed in CRSwNP_NP tissues. Subsequently, we investigated whether the administration of TNF-α to human nasal epithelial cells (hNECs) resulted in the upregulation of ganglioside GD3 and its synthesizing enzyme, ST8 alpha-N-acetyl-neuraminide alpha-2,8-sialytransferase 1 (ST8Sia1), and the consequently promoted inflammatory processes. Additionally, the expression of N-cadherin, Zinc finger protein SNAI2 (SLUG), and matrix metallopeptidase 9 (MMP-9) were elevated, but that of E-cadherin, which is known to be epithelial, was reduced. Moreover, the inhibition of ganglioside GD3 expression by the siRNA or exogenous treatment of neuraminidase 3 (NEU 3) led to the suppression of inflammation and EMT. These results suggest that gangliosides may play an important role in prevention and therapy for inflammation and EMT.

"Hide and seek": Misleading transferrin variants in PMM2-CDG complicate diagnostics.

Congenital disorders of glycosylation (CDG) are one of the fastest growing groups of inborn errors of metabolism. Despite the availability of next-generation sequencing techniques and advanced methods for evaluation of glycosylation, CDG screening mainly relies on the analysis of serum transferrin (Tf) by isoelectric focusing, HPLC or capillary electrophoresis. The main pitfall of this screening method is the presence ofTf protein variants within the general population. Althoughreportsdescribe the role of Tf variants leading to falsely abnormal results, their significance in confounding diagnosis in patients with CDG has not been documented so far. Here, we describe two PMM2-CDG cases, in which Tf variants complicated the diagnostic. Glycosylation investigations included classical screening techniques (capillary electrophoresis, isoelectric focusing and HPLC of Tf) and various confirmation techniques (two-dimensional electrophoresis, western blot,N-glycome, UPLC-FLR/QTOF MS with Rapifluor). Tf variants were highlighted following neuraminidase treatment. Sequencing ofPMM2 was performed. In both patients, Tf screening pointed to CDG-II, while second-line analyses pointed to CDG-I. Tf variants were found in both patients, explaining these discrepancies.PMM2causativevariants were identified in both patients. We suggest that a neuraminidase treatment should be performed when a typical CDG Tf pattern is found upon initial screening analysis.

CAR-Ts redirected against the Thomsen-Friedenreich antigen CD176 mediate specific elimination of malignant cells from leukemia and solid tumors.

Chimeric antigen receptor-engineered T cells (CAR-Ts) are investigated in various clinical trials for the treatment of cancer entities beyond hematologic malignancies. A major hurdle is the identification of a target antigen with high expression on the tumor but no expression on healthy cells, since "on-target/off-tumor" cytotoxicity is usually intolerable. Approximately 90% of carcinomas and leukemias are positive for the Thomsen-Friedenreich carbohydrate antigen CD176, which is associated with tumor progression, metastasis and therapy resistance. In contrast, CD176 is not accessible for ligand binding on healthy cells due to prolongation by carbohydrate chains or sialylation. Thus, no "on-target/off-tumor" cytotoxicity and low probability of antigen escape is expected for corresponding CD176-CAR-Ts. Using the anti-CD176 monoclonal antibody (mAb) Nemod-TF2, the presence of CD176 was evaluated on multiple healthy or cancerous tissues and cells. To target CD176, we generated two different 2nd generation CD176-CAR constructs differing in spacer length. Their specificity for CD176 was tested in reporter cells as well as primary CD8+ T cells upon co-cultivation with CD176+ tumor cell lines as models for CD176+ blood and solid cancer entities, as well as after unmasking CD176 on healthy cells by vibrio cholerae neuraminidase (VCN) treatment. Following that, both CD176-CARs were thoroughly examined for their ability to initiate target-specific T-cell signaling and activation, cytokine release, as well as cytotoxicity. Specific expression of CD176 was detected on primary tumor tissues as well as on cell lines from corresponding blood and solid cancer entities. CD176-CARs mediated T-cell signaling (NF-κB activation) and T-cell activation (CD69, CD137 expression) upon recognition of CD176+ cancer cell lines and unmasked CD176, whereby a short spacer enabled superior target recognition. Importantly, they also released effector molecules (e.g. interferon-γ, granzyme B and perforin), mediated cytotoxicity against CD176+ cancer cells, and maintained functionality upon repetitive antigen stimulation. Here, CD176L-CAR-Ts exhibited slightly higher proliferation and mediator-release capacities. Since both CD176-CAR-Ts did not react towards CD176- control cells, their response proved to be target-specific. Genetically engineered CD176-CAR-Ts specifically recognize CD176 which is widely expressed on cancer cells. Since CD176 is masked on most healthy cells, this antigen and the corresponding CAR-Ts represent a promising approach for the treatment of various blood and solid cancers while avoiding "on-target/off-tumor" cytotoxicity.

LOSS OF HAEMAGGLUTINATION ACTIVITY OF AN INFECTIOUS BRONCHITIS VIRUS AFTER ATTENUATION

Avian infectious bronchitis virus (IBV) has been reported to acquire haemagglutination (HA) activity after treatment with neuraminidase, which depends on the IBV S1 protein. The purpose of this study was to test the IBV HA activity and its relationship to the S1 sequences. A wild strain of IBV, 2575/98 possessed HA activity after neuraminidase treatment. On the contrary, its attenuated strain through 75 chicken embryo passages did not. The nucleotide sequence differences in S1 genes before and after attenuation were C166A and G280T, resulting in amino acid changes of P56T and A94S. The S1 and S genes of the wild strain were cloned and expressed in a baculovirus (B) expression system. To test the relationship between HA activity and the S sequence changes after attenuation, the two nucleotide residues were mutated. The HA activity of those recombinant baculoviruses (rBs) was tested. The results showed that rB containing S gene from wild strain possesses the HA activity, that containing S gene with C166A mutation shows partial HA but that containing G280T mutation shows no any HA activity. Thus, a Taiwan IBV strain 2575/98 loses its HA activity after attenuation and this loss might be related to amino acid changes of P56T and A94S in the IBV S protein.

Changes in glycans on platelet microparticles released during storage of apheresis platelets are associated with phosphatidylserine externalization and phagocytosis.

Platelets shed platelet microparticles (PMP) when activated or stored. As the removal of sialic acid (desialylation) promotes platelet uptake and clearance from the circulation, similar mechanisms for PMP uptake were hypothesized. The aim of the study was to investigate the role of surface glycans in the in vitro uptake of PMP from stored platelet components. Apheresis platelet components were stored in 40% plasma/60% SSP+ and sampled on day 1, 5, and 7 post-collection. PMP were characterized by staining with annexin-V (AnV) for phosphatidylserine (PS)-exposure, CD41 antibody, and fluorescently labeled glycan-binding lectins using flow cytometry. The procoagulant function of PMP following desialylation by neuraminidase treatment was assessed by AnV binding and a procoagulant phospholipid assay. PMP were isolated and stained with Deep Red, and phagocytosis by HepG2 cells was measured. Isolated PMP were deglycosylated with neuraminidase and galactosidase to assess the involvement of glycans in mediating phagocytosis. While the overall platelet surface glycan profile was unchanged during storage, PS+ platelets were sialylated, indicating different glycoproteins were changed. In contrast, sialic acid was removed from PS+ and CD41+ PMP, which specifically lost α-2,3-linked sialic acid during platelet storage. PMP were phagocytized by HepG2 cells, and PMP from platelets stored for 7 days were phagocytized to a lesser extent than on day 1. Desialylation by neuraminidase induced PS-exposure on PMP, decreased PPL clotting time, and increased PMP phagocytosis. PMP glycans change during platelet storage. Desialylation influences the procoagulant function of PMP and phagocytosis by HepG2 cells.

DCIR and its ligand asialo-biantennary N-glycan regulate DC function and osteoclastogenesis.

Dendritic cell immunoreceptor (DCIR) is a C-type lectin receptor with a carbohydrate recognition domain and an immunoreceptor tyrosine-based inhibitory motif. Previously, we showed that Dcir-/- mice spontaneously develop autoimmune enthesitis and sialadenitis, and also develop metabolic bone abnormalities. However, the ligands for DCIR functionality remain to be elucidated. Here we showed that DCIR is expressed on osteoclasts and DCs and binds to an asialo-biantennary N-glycan(s) (NA2) on bone cells and myeloid cells. Osteoclastogenesis was enhanced in Dcir-/- cells, and NA2 inhibited osteoclastogenesis. Neuraminidase treatment, which exposes excess NA2 by removing the terminal sialic acid of N-glycans, suppressed osteoclastogenesis and DC function. Neuraminidase treatment of mice ameliorated collagen-induced arthritis and experimental autoimmune encephalomyelitis in a DCIR-dependent manner, due to suppression of antigen presentation by DCs. These results suggest that DCIR activity is regulated by the modification of the terminal sialylation of biantennary N-glycans, and this interaction is important for the control of both autoimmune and bone metabolic diseases.

Sialic acid-containing glycolipids mediate binding and viral entry of SARS-CoV-2.

Emerging evidence suggests that host glycans influence severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Here, we reveal that the receptor-binding domain (RBD) of the spike (S) protein on SARS-CoV-2 recognizes oligosaccharides containing sialic acid (Sia), with preference for monosialylated gangliosides. Gangliosides embedded within an artificial membrane also bind to the RBD. The monomeric affinities (Kd = 100-200 μM) of gangliosides for the RBD are similar to another negatively charged glycan ligand of the RBD proposed as a viral co-receptor, heparan sulfate (HS) dp2-dp6 oligosaccharides. RBD binding and infection of SARS-CoV-2 pseudotyped lentivirus to angiotensin-converting enzyme 2 (ACE2)-expressing cells is decreased following depletion of cell surface Sia levels using three approaches: sialyltransferase (ST) inhibition, genetic knockout of Sia biosynthesis, or neuraminidase treatment. These effects on RBD binding and both pseudotyped and authentic SARS-CoV-2 viral entry are recapitulated with pharmacological or genetic disruption of glycolipid biosynthesis. Together, these results suggest that sialylated glycans, specifically glycolipids, facilitate viral entry of SARS-CoV-2.

Regulation of cell adhesion to galectins by glycosylation: A new concept in lymphoma cell adhesion

To investigate the biological roles of glycosylation in human lymphoma cell adhesion to galectins, I performed the cell adhesion assay using neuraminidase or glycosylation inhibitors on B cell lymphoma cell line. I show that desialylation by neuraminidase treatment results in enhancement of cell adhesion to Phaseolus vulgaris leukoagglutinating lectin (L-PHA), suggesting that neuraminidase treatment removes sialic acids from N-glycans, which we have shown react to L-PHA in a lymphoma cell line. Neuraminidase treatment resulted in enhancement of cell adhesion to galectin-3, an effect that was especially pronounced following pre-stimulation of B cell receptors (BCR) by anti-IgM antibody treatment. This suggests that sialic acid may be a candidate regulator of interactions between glycans on BCR and galectins. From treatment with tunicamycin (TM), a potent inhibitor of N-glycosylation, cell adhesion capacity decreased. Furthermore, it is suggested that IgM and CD45 have N-glycans for binding to galectins in western blot analysis. In a glycoengineering approach, I found that treatment with the sialic acid mimetic analogue 3Fax-peracetyl Neu5Ac, which is a potent inhibitor of sialyltransferases, markedly enhanced lymphoma cell adhesion to galectin-3. In conclusion, sialic acids and N-glycosylation appeared to influence lymphoma cell adhesion to galectins.

Analysis of Schistosoma mansoni Extracellular Vesicles Surface Glycans Reveals Potential Immune Evasion Mechanism and New Insights on Their Origins of Biogenesis.

Parasitic helminths are master manipulators of host immunity. Their strategy is complex and involves the release of excreted/secreted products, including extracellular vesicles (EVs). The protein and miRNA contents of EVs have been characterised for many parasitic helminths but, despite reports suggesting the importance of EV surface carbohydrate structures (glycans) in the interactions with target cells and thus subsequent effector functions, little is known about parasite EV glycomics. Using lectin microarrays, we identified several lectins that exhibit strong adhesion to Schistosoma mansoni EVs, suggesting the presence of multiple glycan structures on these vesicles. Interestingly, SNA-I, a lectin that recognises structures with terminal sialic acid, displayed strong affinity for S. mansoni EVs, which was completely abolished by neuraminidase treatment, suggesting sialylation in the EV sample. This finding is of interest, as sialic acids play important roles in the context of infection by aiding immune evasion, affecting target recognition, cell entry, etc., but are not thought to be synthesised by helminths. These data were validated by quantitative analysis of free sialic acid released from EVs following treatment with neuraminidase. Lectin histochemistry and fluorescence in situ hybridisation analyses on whole adult worms suggest the involvement of sub-tegumental cell bodies, as well as the digestive and excretory systems, in the release of EVs. These results support previous reports of EV biogenesis diversity in trematodes and potentially highlight new means of immune modulation and evasion employed by schistosomes.

Experimental infection of ostriches with H7N1 low pathogenic and H5N8 clade 2.3.4.4B highly pathogenic influenza A viruses

Infection dynamics data for influenza A virus in a species is important for understanding host-pathogen interactions and developing effective control strategies. Seven-week-old ostriches challenged with H7N1 low pathogenic viruses (LPAIV) or clade 2.3.4.4B H5N8 high pathogenic viruses (HPAIV) were co- housed with non-challenged contacts. Clinical signs, virus shed in the trachea, cloaca, and feather pulp, and antibody responses were quantified over 14 days. H7N1 LPAIV-infected ostriches remained generally healthy with some showing signs of mild conjunctivitis and rhinitis attributed to Mycoplasma co-infection. Mean tracheal virus shedding titres in contact birds peaked 3 days (106.2 EID50 equivalents / ml) and 9 days (105.28 EID50 equivalents / ml) after introduction, lasting for at least 13 days post infection. Cloacal shedding was substantially lower and ceased within 10 days of onset, and low virus levels were detected in wing feather pulp up until day 14. H5N8 HPAIV -infected ostriches showed various degrees of morbidity, with 2/3 mortalities in the in-contact group. Mean tracheal shedding in contact birds peaked 8 days after introduction (106.32 EID50 equivalents/ ml) and lasted beyond 14 days in survivors. Cloacal shedding and virus in feather pulp was generally higher and more consistently positive compared to H7N1 LPAIV, and was also detectable at least until 14 days post infection in survivors. Antibodies against H5N8 HPAIV and H7N1 LPAIV only appeared after day 7 post exposure, with higher titres induced by the HPAIV compared to the LPAIV, and neuraminidase treatment was essential to remove non-specific inhibitors from the H5N8-positive antisera.

Detection of recombinant erythropoietin biosimilar Jimaixin™ after administration in healthy subjects.

Jimaixin™ (Jintan Ltd, China) is a biosimilar of recombinant erythropoietin (rEPO) now authorized for therapeutic application in China. With a risk of abuse by athletes, a clear evaluation of its detection using the electrophoretic methods in use in antidoping laboratories was necessary. In a previous work, we showed that Jimaixin™ electrophoretic profile presented slight changes compared with the original drug (first generation rEPO) and that a spike of Jimaixin™ in urine and serum was well identified by SDS-PAGE but with less performance by IEF-PAGE unless a neuraminidase treatment was applied first. The aims of this research were to perform an intravenous administration of Jimaixin™ on three healthy subjects (one microdose [10IU/kg] and three therapeutic doses [50 IU/kg]) and to evaluate the detection in urine and blood up to 7days post administration. Analysis of the samples showed that Jimaixin™ detection was complicated by IEF-PAGE due to the loss of the most distinctive basic isoforms. In addition, a neuraminidase treatment did not improve detection (contrary to the observations from spike experiments). On the contrary, Jimaixin™ was very efficiently detected in blood and urine by SDS-PAGE: up to 40 h after a microdose and up to 7days after the therapeutic doses. The effect of Jimaixin™ on hematological parameters was limited to a clear but transitory increase of the reticulocytes. These data give new elements to better survey a potential misuse of Jimaixin™ by athletes.

The role of cell surface sialic acids for SARS-CoV-2 infection.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is a new virus that has higher contagious capacity than any other previous human coronaviruses (HCoVs) and causes the current coronavirus disease 2019 pandemic. Sialic acids are a group of nine-carbon acidic α-keto sugars, usually located at the end of glycans of cell surface glycoconjugates and serve as attachment sites for previous HCoVs. It is therefore speculated that sialic acids on the host cell surface could serve as co-receptors or attachment factors for SARS-CoV-2 cell entry as well. Recent in silico modeling, molecular modeling predictions and microscopy studies indicate potential sialic acid binding by SARS-CoV-2 upon cell entry. In particular, a flat sialic acid-binding domain was proposed at the N-terminal domain of the spike protein, which may lead to the initial contact and interaction of the virus on the epithelium followed by higher affinity binding to angiotensin-converting enzyme 2 (ACE2) receptor, likely a two-step attachment fashion. However, recent in vitro and ex vivo studies of sialic acids on ACE2 receptor confirmed an opposite role for SARS-CoV-2 binding. In particular, neuraminidase treatment of epithelial cells and ACE2-expressing 293T cells increased SARS-CoV-2 binding. Furthermore, the ACE2 glycosylation inhibition studies indicate that sialic acids on ACE2 receptor prevent ACE2–spike protein interaction. On the other hand, a most recent study indicates that gangliosides could serve as ligands for receptor-binding domain of SARS-CoV-2 spike protein. This mini-review discusses what has been predicted and known so far about the role of sialic acid for SARS-CoV-2 infection and future research perspective.

Thrombin in the Activation of the Fluid Contact Phase in Patients with Hereditary Angioedema Carrying the F12 P.Thr309Lys Variant.

Hereditary angioedema due to pathogenic FXII variants (HAE-FXII) is a rare dominant disease caused by increased activation of the plasma contact system. The most prevalent HAE-FXII variant, c.1032C > A p.Thr309Lys (FXII309Lys), results in a smaller FXII protein with increased sensitivity to fluid-phase activation by poorly understood mechanisms. We aimed to investigate the functionality of the FXII309Lys variant in 33 HAE-FXIIpatients, 25 healthy controls and 46 patients with congenital disorders of glycosylation (CDG). Activation of the plasma contact system was assessed by western blot and amidolytic assay in basal conditions or after treatment with either artificial or physiological activators. Recombinant wild-type and FXII309Lys variants were expressed in S2 insect (Drosophila) cells. Amidolytic and fibrin generation assays were performed in fresh plasma samples. FXII309Lys samples exhibited an increased electrophoretic mobility comparable with N-glycan-deficient FXII from CDG patients and asialo-FXII generated by neuraminidase treatment. They presented increased sensitivity to activation by dextran sulphate and silica which resulted in the generation of an aberrant 37-kDa heavy chain. We did not observe increased susceptibility of FXII309Lys to proteolysis by exogenous or tPA-generated plasmin. However, both exogenous and endogenous thrombin cleaved the FXII309Lys variant, releasing a 37-kDa fragment and resulting in enhanced proteolytic activation on the fluid phase. This model supports a sequential proteolytic activation process involving thrombin priming of FXII309Lys, followed by kallikrein cleavage and generation of active βFXIIa. The present results and the observation that angioedema episodes in HAE-FXII patients occur predominantly during hypercoagulable situations suggest a key role for thrombin.

Galectin-1 and platelet factor 4 (CXCL4) induce complementary platelet responses in vitro

Galectin-1 (gal-1) is a carbohydrate-binding lectin with important functions in angiogenesis, immune response, hemostasis and inflammation. Comparable functions are exerted by platelet factor 4 (CXCL4), a chemokine stored in the α-granules of platelets. Previously, gal-1 was found to activate platelets through integrin αIIbβ3. Both gal-1 and CXCL4 have high affinities for polysaccharides, and thus may mutually influence their functions. The aim of this study was to investigate a possible synergism of gal-1 and CXCL4 in platelet activation. Platelets were treated with increasing concentrations of gal-1, CXCL4 or both, and aggregation, integrin activation, P-selectin and phosphatidyl serine (PS) exposure were determined by light transmission aggregometry and by flow cytometry. To investigate the influence of cell surface sialic acid, platelets were treated with neuraminidase prior to stimulation. Gal-1 and CXCL4 were found to colocalize on the platelet surface. Stimulation with gal-1 led to integrin αIIbβ3 activation and to robust platelet aggregation, while CXCL4 weakly triggered aggregation and primarily induced P-selectin expression. Co-incubation of gal-1 and CXCL4 potentiated platelet aggregation compared with gal-1 alone. Whereas neither gal-1 and CXCL4 induced PS-exposure on platelets, prior removal of surface sialic acid strongly potentiated PS exposure. In addition, neuraminidase treatment increased the binding of gal-1 to platelets and lowered the activation threshold for gal-1. However, CXCL4 did not affect binding of gal-1 to platelets. Taken together, stimulation of platelets with gal-1 and CXCL4 led to distinct and complementary activation profiles, with additive rather than synergistic effects.

Glycosylation Profiling of Glycoproteins Secreted from Cultured Cells Using Glycan Node Analysis and GC-MS.

Glycan "node" analysis is the process by which pooled glycans within complex biological samples are chemically deconstructed in away that facilitates the analytical quantification of uniquely linked monosaccharide units (glycan "nodes"). It is based on glycan methylation analysis (a.k.a. linkage analysis) that has historically been applied to pre-isolated glycans. Thus, when using glycan node analysis, unique glycan features within whole biospecimens such as "core fucosylation," "α2-6sialylation," "β1-6branching," "β1-4branching," and "bisecting GlcNAc," are captured as single analytical signals by GC-MS. Here we describe the use of this methodology in cell culture supernatant and in the analysis of IgG (alpha-1 antitrypsin) glycans. The effect of IL-6 and IL-1β cytokines on secreted hepatocyte protein glycan features is demonstrated; likewise, the impact of neuraminidase treatment of IgG is illustrated. For the majority of glycan nodes, the assay is consistent and reproducible on a day-to-day basis; because of this, relatively subtle shifts in the relative abundance of glycan features can be captured using this approach.

SHP-1 Regulates Antigen Cross-Presentation and Is Exploited by Leishmania to Evade Immunity

Intracellular pathogens have evolved strategies to evade detection by cytotoxic CD8+ T lymphocytes (CTLs). Here, we ask whether Leishmania parasites trigger the SHP-1-FcRγ chain inhibitory axis to dampen antigen cross-presentation in dendritic cells expressing the C-type lectin receptor Mincle. We find increased cross-priming of CTLs in Leishmania-infected mice deficient for Mincle or with a selective loss of SHP-1 in CD11c+ cells. The latter also shows improved cross-presentation of cell-associated viral antigens. CTL activation invitro reveals increased MHC class I-peptide complex expression in Mincle- or SHP-1-deficient CD11c+ cells. Neuraminidase treatment also boosts cross-presentation, suggesting that Leishmania triggers SHP-1-associated sialic-acid-binding receptors. Mechanistically, enhanced antigen processing correlates with reduced endosomal acidification in the absence of SHP-1. Finally, we demonstrate that SHP-1 inhibition improves CD11c+ cell-based vaccination against the parasite. Thus, SHP-1-mediated impairment of cross-presentation can be exploited by pathogens to evade CTLs, and SHP-1 inhibition improves CTL responses during vaccination.

Spherization of red blood cells and platelet margination in COPD patients.

Red blood cells (RBCs) in pathological situations undergo biochemical and conformational changes, leading to alterations in rheology involved in cardiovascular events. The shape of RBCs in volunteers and stable and exacerbated chronic obstructive pulmonary disease (COPD) patients was analyzed. The effects of RBC spherization on platelet transport (displacement in the flow field caused by their interaction with RBCs) were studied in vitro and by numerical simulations. RBC spherization was observed in COPD patients compared with volunteers. In in vitro experiments at a shear rate of 100 s-1 , treatment of RBCs with neuraminidase induced greater sphericity, which mainly affected platelet aggregates without changing aggregate size. At 400 s-1 , neuraminidase treatment changes both the size of the aggregates and the number of platelet aggregates. Numerical simulations indicated that RBC spherization induces an increase of the platelet mean square displacement, which is traditionally linked to the platelet diffusion coefficient. RBCs of COPD patients are more spherical than healthy volunteers. Experimentally, RBC spherization induces increased platelet transport to the wall. Additional studies are needed to understand the link between the effect of RBCs on platelet transport and the increased cardiovascular events observed in COPD patients.

Electrostatic Surface Properties of Blood and Semen Extracellular Vesicles: Implications of Sialylation and HIV-Induced Changes on EV Internalization.

Although extracellular vesicle (EV) surface electrostatic properties (measured as zeta potential, ζ-potential) have been reported by many investigators, the biophysical implications of charge and EV origin remains uncertain. Here, we compared the ζ-potential of human blood EVs (BEVs) and semen EVs (SEVs) from 26 donors that were HIV-infected (HIV+, n = 13) or HIV uninfected (HIV-, n = 13). We found that, compared to BEVs that bear neutral surface charge, SEVs were significantly more negatively charged, even when BEVs and SEVs were from the same individual. Comparison of BEVs and SEVs from HIV- and HIV+ groups revealed subtle HIV-induced alteration in the ζ-potential of EVs, with the effect being more significant in SEVs (∆ζ-potential = −8.82 mV, p-value = 0.0062) than BEVs (∆ζ-potential = −1.4 mV, p-value = 0.0462). These observations were validated by differences in the isoelectric point (IEP) of EVs, which was in the order of HIV + SEV ≤ HIV-SEV ≪ HIV + BEV ≤ HIV-BEV. Functionally, the rate and efficiency of SEV internalization by the human cervical epithelial cell line, primary peripheral blood lymphocytes, and primary blood-derived monocytes were significantly higher than those of BEVs. Mechanistically, removal of sialic acids from the surface of EVs using neuraminidase treatment significantly decreased SEV’s surface charge, concomitant with a substantial reduction in SEV’s internalization. The neuraminidase effect was independent of HIV infection and insignificant for BEVs. Finally, these results were corroborated by enrichment of glycoproteins in SEVs versus BEVs. Taken together, these findings uncover fundamental tissue-specific differences in surface electrostatic properties of EVs and highlight the critical role of surface charge in EV/target cell interactions.

P0473LONGITUDINAL CHANGES OF IGA1 O-GLYCOFORM IN IGA NEPHROPATHY

Abstract Background and Aims Elevation of circulatory IgA1 with galactose-deficient (Gd) hinge-region (HR) O-glycans (Gd-IgA1) has been detected in most IgA nephropathy (IgAN) patients based on lectin ELISA. However, new approaches are needed for molecular-level characterization of IgA1 HR glycoform(s) in IgAN. We established a high-throughput method for analysis of IgA1 HR O-glycoforms using liquid chromatography-high-resolution mass spectrometry (LC-HRMS). To identify IgAN-associated IgA1 HR O-glycoforms and to assess their changes after therapy (with or without corticosteroids (CS)), we profiled IgA1 HR glycopeptides from sera collected at two time points (before and after therapy) from Japanese IgAN patients. Method Of the 10 Japanese IgAN patients recruited, 4 received CS treatment (CS group) and 6 have not (non-CS group). Japanese healthy volunteers (HC, n=10) were recruited as controls. Serum IgA1 was purified by affinity chromatography from HC and IgAN patients before and after therapy. After neuraminidase treatment and trypsin digestion, IgA1 HR glycosylation heterogeneity was analyzed by LC-HRMS. The relative abundance (RA, %) for each glycopeptide was calculated as percentage to the total IgA1 HR glycopeptide. The amount of each glycopeptide was then calculated by multiplying serum IgA concentration (mg/dL) by RA. Results Approximately 60% of IgA1 HR O-glycoforms in IgAN patients and HC were Gd O-glycoforms; these glycoforms contained one to three Gd-glycan(s), designated as 1 Gd-glycoform, 2 Gd-glycoform and 3 Gd-glycoform, respectively. In IgAN patients, the RA of non Gd-IgA1 glycoforms was elevated (P=0.002) and correlated with proteinuria (g/gCr) at renal biopsy (P=0.039, R=0.657). The amounts of non Gd- and 1Gd-glycoforms were higher in IgAN patients compared to HC (each P<0.001). After several years of follow up (2.77 years (1.44-3.85)), the RA of non Gd-glycoforms decreased in CS group of IgAN patients (P=0.039) whereas it remained unchanged in the non-CS group (P=0.488). The amount of non Gd-glycoforms exhibited similar trends, i.e., decreased in CS group (P=0.068) whereas it remained unchanged in the non-CS group (P=0.943). Conclusion This study profiled serum IgA1 for IgAN-associated IgA1 HR O-glycoforms at the molecular level and assessed their changes in response to CS vs. non-CS therapy. IgA1 HR O-glycoforms altered by treatment may serve as a biomarker(s) for monitoring patients’ responses to therapy.

The Putative TCP-1 Chaperonin Is an Important Player Involved in Sialic Acid-Dependent Host Cell Invasion by Toxoplasma gondii.

Host cell invasion by Toxoplasma gondii is crucial for the survival and proliferation of parasite. The process of T. gondii tachyzoite invasion requires interaction between parasite proteins and receptors on the surface of host cells. Sialic acid is one of the important receptors for host cell invasion by T. gondii. However, the parasite-derived proteins interacting with sialic acid have not been well characterized. In this study, a novel protein named putative TCP-1 chaperonin (TGME49_318410) in T. gondii (TgTCP-1) was targeted and characterized. TgTCP-1 protein colocalized with MIC3 protein, which could be secreted from T. gondii tachyzoites, and this protein showed a specific binding activity to sialic acid, and DC and Vero cells in vitro. The binding of TgTCP-1 protein to DC and Vero cells were inhibited by either pre-incubation with free sialic acid or neuraminidase treatment of the cells. Moreover, a significant reduction of T. gondii invasion in Vero cells was observed after pre-incubation of the cells with recombinant TgTCP-1 protein. These results illustrated that TgTCP-1 is an important molecule involved in sialic acid-dependent host cell invasion by T. gondii.