An Update on the Pathogenesis of COVID-19 and the Reportedly Rare Thrombotic Events Following Vaccination.

Today the coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has become a global health problem. After more than a year with the pandemic, although our knowledge has progressed on COVID-19, there are still many unknowns in virological, pathophysiological and immunological aspects. It is obvious that the most efficient solution to end this pandemic are safe and efficient vaccines. This manuscript summarizes the pathophysiological and thrombotic features of COVID-19 and the safety and efficacy of currently approved COVID-19 vaccines with an aim to clarify the recent concerns of thromboembolic events after COVID-19 vaccination. The influx of newer information is rapid, requiring periodic updates and objective assessment of the data on the pathogenesis of COVID-19 variants and the safety and efficacy of currently available vaccines.

How glycobiology can help us treat and beat the COVID-19 pandemic.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged during the last months of 2019, spreading throughout the world as a highly transmissible infectious illness designated as COVID-19. Vaccines have now appeared, but the challenges in producing sufficient material and distributing them around the world means that effective treatments to limit infection and improve recovery are still urgently needed. This review focuses on the relevance of different glycobiological molecules that could potentially serve as or inspire therapeutic tools during SARS-CoV-2 infection. As such, we highlight the glycobiology of the SARS-CoV-2 infection process, where glycans on viral proteins and on host glycosaminoglycans have critical roles in efficient infection. We also take notice of the glycan-binding proteins involved in the infective capacity of virus and in human defense. In addition, we critically evaluate the glycobiological contribution of candidate drugs for COVID-19 therapy such as glycans for vaccines, anti-glycan antibodies, recombinant lectins, lectin inhibitors, glycosidase inhibitors, polysaccharides, and numerous glycosides, emphasizing some opportunities to repurpose FDA-approved drugs. For the next-generation drugs suggested here, biotechnological engineering of new probes to block the SARS-CoV-2 infection might be based on the essential glycobiological insight on glycosyltransferases, glycans, glycan-binding proteins, and glycosidases related to this pathology.

Drug delivery systems based on CD44-targeted glycosaminoglycans for cancer therapy.

The polysaccharide-based biomaterials hyaluronic acid (HA) and chondroitin sulfate (CS) have aroused great interest for use in drug delivery systems for tumor therapy, as they have outstanding biocompatibility and great targeting ability for cluster determinant 44 (CD44). In addition, modified HA and CS can self-assemble into micelles or micellar nanoparticles (NPs) for targeted drug delivery. This review discusses the formation of HA- and CS-based NPs, and various types of CS-based NPs including CS-drug conjugates, CS-polymer NPs, CS-small molecule NPs, polyelectrolyte nanocomplexes (PECs), CS-metal NPs, and nanogels. We then focus on the applications of HA- and CS-based NPs in tumor chemotherapy, gene therapy, photothermal therapy (PTT), photodynamic therapy (PDT), sonodynamic therapy (SDT), and immunotherapy. Finally, this review is expected to provide guidelines for the development of various HA- and CS-based NPs used in multiple cancer therapies.

A biological extract of turmeric (Curcuma longa) modulates response of cartilage explants to lipopolysaccharide.

BACKGROUND: Turmeric is commonly used as a dietary treatment for inflammation, but few studies have evaluated the direct effect of turmeric on cartilage. The purpose of this study was to characterize cartilage explants' inflammatory responses to lipopolysaccharide in the presence of a simulated biological extract of turmeric. METHODS: Turmeric was incubated in simulated gastric and intestinal fluid, followed by inclusion of liver microsomes and NADPH. The resulting extract (TURsim) was used to condition cartilage explants in the presence or absence of lipopolysaccharide. Explants were cultured for 96 h (h); the first 24 h in basal tissue culture media and the remaining 72 h in basal tissue culture media containing TURsim (0, 3, 9 or 15 µg/mL). Lipopolysaccharide (0 or 5 µg/mL) was added for the final 48 H. media samples were collected immediately prior to lipopolysaccharide exposure (0 h) and then at 24 and 48 h after, and analyzed for prostaglandin E2 (PGE2), glycosaminoglycan (GAG), and nitric oxide (NO). Explants were stained with calcein-AM for an estimate of live cells. Data were analyzed using a 2-way repeated measures (GAG, PGE2, NO) or 1-way ANOVA without repeated measures (viability). Significance accepted at p < 0.05. RESULTS: TURsim significantly reduced PGE2, NO and GAG, and calcein fluorescence was reduced. CONCLUSIONS: These data contribute to the growing body of evidence for the utility of turmeric as an intervention for cartilage inflammation.

Sticking for a Cause: The Falciparum Malaria Parasites Cytoadherence Paradigm.

After a successful invasion, malaria parasite Plasmodium falciparum extensively remodels the infected erythrocyte cellular architecture, conferring cytoadhesive properties to the infected erythrocytes. Cytoadherence plays a central role in the parasite's immune-escape mechanism, at the same time contributing to the pathogenesis of severe falciparum malaria. In this review, we discuss the cytoadhesive interactions between P. falciparum infected erythrocytes and various host cell types, and how these events are linked to malaria pathogenesis. We also highlight the limitations faced by studies attempting to correlate diversity in parasite ligands and host receptors with the development of severe malaria.

Nanotechnology and Glycosaminoglycans: Paving the Way Forward for Ovarian Cancer Intervention.

Ovarian cancer (OC) has gained a great deal of attention due to its aggressive proliferative capabilities, high death rates and poor treatment outcomes, rendering the disease the ultimate lethal gynaecological cancer. Nanotechnology provides a promising avenue to combat this malignancy by the niche fabrication of optimally-structured nanomedicines that ensure potent delivery of chemotherapeutics to OC, employing nanocarriers to act as "intelligent" drug delivery vehicles, functionalized with active targeting approaches for precision delivery of chemotherapeutics to overexpressed biomarkers on cancer cells. Recently, much focus has been implemented to optimize these active targeting mechanisms for treatment/diagnostic purposes employing nanocarriers. This two-part article aims to review the latest advances in active target-based OC interventions, where the impact of the newest antibody, aptamer and folate functionalization on OC detection and treatment is discussed in contrast to the limitations of this targeting mechanism. Furthermore, we discuss the latest advances in nanocarrier based drug delivery in OC, highlighting their commercial/clinical viability of these systems beyond the realms of research. Lastly, in the second section of this review, we comprehensively discussed a focus shift in OC targeting from the well-studied OC cells to the vastly neglected extracellular matrix and motivate the potential for glycosaminoglycans (GAGs) as a more focused extracellular molecular target.

Glycosaminoglycan Interactions Fine-Tune Chemokine-Mediated Neutrophil Trafficking: Structural Insights and Molecular Mechanisms.

Circulating neutrophils, rapidly recruited in response to microbial infection, form the first line in host defense. Humans express ~50 chemokines, of which a subset of seven chemokines, characterized by the conserved "Glu-Leu-Arg" motif, mediate neutrophil recruitment. Neutrophil-activating chemokines (NACs) share similar structures, exist as monomers and dimers, activate the CXCR2 receptor on neutrophils, and interact with tissue glycosaminoglycans (GAGs). Considering cellular assays have shown that NACs have similar CXCR2 activity, the question has been and remains, why do humans express so many NACs? In this review, we make the case that NACs are not redundant and that distinct GAG interactions determine chemokine-specific in vivo functions. Structural studies have shown that the GAG-binding interactions of NACs are distinctly different, and that conserved and specific residues in the context of structure determine geometries that could not have been predicted from sequences alone. Animal studies indicate recruitment profiles of monomers and dimers are distinctly different, monomer-dimer equilibrium regulates recruitment, and that recruitment profiles vary between chemokines and between tissues, providing evidence that GAG interactions orchestrate neutrophil recruitment. We propose in vivo GAG interactions impact several chemokine properties including gradients and lifetime, and that these interactions fine-tune and define the functional response of each chemokine that can vary between different cell and tissue types for successful resolution of inflammation.

Glycosaminoglycans are important mediators of neutrophilic inflammation in vivo.

The pro-inflammatory chemokine interleukin-8 (CXCL8) exerts its function by establishing a chemotactic gradient in infected or damaged tissues to guide neutrophil granulocytes to the site of inflammation via its G protein-coupled receptors (GPCRs) CXCR1 and CXCR2 located on neutrophils. Endothelial glycosaminoglycans (GAGs) have been proposed to support the chemotactic gradient formation and thus the inflammatory response by presenting the chemokine to approaching leukocytes. In this study, we show that neutrophil transmigration in vitro can be reduced by adding soluble GAGs and that this process is specific with respect to the nature of the glycan. To further investigate the GAG influence on neutrophil migration, we have used an engineered CXCL8 mutant protein (termed PA401) which exhibits a much higher affinity towards GAGs and an impaired GPCR activity. This dominant-negative mutant chemokine showed anti-inflammatory activity in various animal models of neutrophil-driven inflammation, i.e. in urinary tract infection, bleomycin-induced lung fibrosis, and experimental autoimmune uveitis. In all cases, treatment with PA401 resulted in a strong reduction of transmigrated inflammatory cells which became evident from histology sections and bronchoalveolar lavage. Since our CXCL8-based decoy targets GAGs and not GPCRs, our results show for the first time the crucial involvement of this glycan class in CXCL8/neutrophil-mediated inflammation and will thus pave the way to novel approaches of anti-inflammatory treatment.

Understanding the Chemistry and Biology of Glycosylation with Glycan Synthesis.

Glycoscience research has been significantly impeded by the complex compositions of the glycans present in biological molecules and the lack of convenient tools suitable for studying the glycosylation process and its function. Polysaccharides and glycoconjugates are not encoded directly by genes; instead, their biosynthesis relies on the differential expression of carbohydrate enzymes, resulting in heterogeneous mixtures of glycoforms, each with a distinct physiological activity. Access to well-defined structures is required for functional study, and this has been provided by chemical and enzymatic synthesis and by the engineering of glycosylation pathways. This review covers general methods for preparing glycans commonly found in mammalian systems and applying them to the synthesis of therapeutically significant glycoconjugates (glycosaminoglycans, glycoproteins, glycolipids, glycosylphosphatidylinositol-anchored proteins) and the development of carbohydrate-based vaccines.

Mechanistic and therapeutic overview of glycosaminoglycans: the unsung heroes of biomolecular signaling.

Immune regulation is a complex biological signaling pathway in which several classes of biomolecules and small molecules play a complacent role to mediate this process. Glycoimmunology is a rapidly evolving research area that deals with the structure, binding interactions and immunological functions of glycans. Great deal of information regarding proteins and nucleic acids in molecular recognition events have been established owing to their well-established structural features and straight forward replication, transcription and translation principles. However considering the complexities of template free synthesis and structural heterogeneity, role of carbohydrates in immune regulation are still unsung to a large extent. In the current review, we illuminate the canonical structural features, emerging and significant pathophysiological functions of glycosaminoglycans (GAGs), the negatively charged linear carbohydrate molecules that are primarily present on all types of cell surfaces and extra cellular matrix. A snap shot of their association with protein counterparts of diversified protein families has been updated exclusively to provide mechanistic insights into their cellular signaling functions. Eventually, this review throws light on the recent biomedical/biotechnological advances of GAG based biomarkers, nutraceuticals, therapeutics, and nanocomposites for inflammatory, immune disorders and their invaluable contribution in tissue engineering.

Arresting progressive atherosclerosis by immunization with an anti-glycosaminoglycan monoclonal antibody in apolipoprotein E-deficient mice.

Atherogenesis is associated with the early retention of low-density lipoproteins (LDL) in the arterial intima by interaction with glycosaminoglycan (GAG)-side chains of proteoglycans. Retained LDL undergo reactive oxygen species-mediated oxidation. Oxidized LDL trigger oxidative stress (OS) and inflammation, contributing to atherosclerosis development. Recently, we reported the preventive anti-atherogenic properties of the chimeric mouse/human monoclonal antibody (mAb) chP3R99-LALA, which were related to the induction of anti-chondroitin sulfate antibody response able to inhibit chondroitin sulfate dependent LDL-enhanced oxidation. In the present work, we aimed at further investigating the impact of chP3R99-LALA mAb vaccination on progressive atherosclerosis in apolipoprotein E-deficient mice (apoE(-/-)) fed with a high-fat high-cholesterol diet receiving 5 doses (50 µg) of the antibody subcutaneously, when ~5% of the aortic area was covered by lesions. Therapeutic immunization with chP3R99-LALA mAb halted atherosclerotic lesions progression. In addition, aortic OS was modulated, as shown by a significant (p<0.05) reduction of lipid and protein oxidation, preservation of antioxidant enzymes activity and reduced glutathione, together with a decrease of nitric oxide levels. chP3R99-LALA mAb immunization also regulated aortic NF-κB activation, diminishing the proinflammatory IL1-ß and TNF-α gene expression as well as the infiltration of macrophages into the arterial wall. The therapeutic immunization of apoE(-/-) with progressive atheromas and persistent hypercholesterolemia using chP3R99-LALA mAb arrested further development of lesions, accompanied by a decrease of aortic OS and NF-κB-regulated pro-inflammatory cytokine gene expression. These results contribute to broaden the potential use of this anti-GAG antibody-based immunotherapy as a novel approach to target atherosclerosis at different phases of progression.

Atomic description of the immune complex involved in heparin-induced thrombocytopenia.

Heparin-induced thrombocytopenia (HIT) is an autoimmune thrombotic disorder caused by immune complexes containing platelet factor 4 (PF4), antibodies to PF4 and heparin or cellular glycosaminoglycans (GAGs). Here we solve the crystal structures of the: (1) PF4 tetramer/fondaparinux complex, (2) PF4 tetramer/KKO-Fab complex (a murine monoclonal HIT-like antibody) and (3) PF4 monomer/RTO-Fab complex (a non-HIT anti-PF4 monoclonal antibody). Fondaparinux binds to the 'closed' end of the PF4 tetramer and stabilizes its conformation. This interaction in turn stabilizes the epitope for KKO on the 'open' end of the tetramer. Fondaparinux and KKO thereby collaborate to 'stabilize' the ternary pathogenic immune complex. Binding of RTO to PF4 monomers prevents PF4 tetramerization and inhibits KKO and human HIT IgG-induced platelet activation and platelet aggregation in vitro, and thrombus progression in vivo. The atomic structures provide a basis to develop new diagnostics and non-anticoagulant therapeutics for HIT.

Modulation of inflammation and angiogenesis and changes in ECM GAG-activity via dual delivery of nucleic acids.

Tissue-engineered organs and implants hold promise for the replacement of damaged and diseased organs. However, the foreign body response (FBR) is a major obstacle that compromises the function of tissue-engineered constructs, typically causing them to fail. Two components of FBR are an inflammatory response and a lack of vascularization. To overcome these limitations, a collagen system was developed to release interleukin-6 (IL-6) siRNA and endothelial nitric oxide synthase (eNOS) pDNA in a staggered manner. Hollow collagen microspheres were assembled into a collagen sphere-in-hydrogel system that displayed a staggered release profile in vitro. This system was assessed in vivo in a subcutaneous rat model. The doses of IL-6 siRNA and eNOS pDNA were first individually optimized for their ability to reduce the volume fraction of inflammatory cells (7 days) and increase the length density of blood vessels (14 days), respectively. The identified optimal doses were combined, and the ability of the system to decrease the volume fraction of inflammatory cells and increase the length density of blood vessels was confirmed at both 7 and 14 days. Analysis of the tissue using Raman microspectroscopy revealed that in addition to changes in inflammation and angiogenesis, there were also changes in the extracellular matrix (ECM) at seven days. While changes in sulfated glycosaminoglycan (sGAG) content of the ECM were not detected, changes in the binding of sGAG of the ECM to growth factors were observed. Two growth factors tested, VEGF165 and bFGF showed increased binding to sGAG extracted from eNOS pDNA-treated samples at seven days, increasing the angiogenic potential of the ECM. Thus, we observe that changes in the tissue in terms of the balance of inflammation and angiogenesis as well changes in the activity of sGAG of the ECM occurs following dual delivery of nucleic acids from the collagen sphere-in-hydrogel system.

The Positively Charged COOH-terminal Glycosaminoglycan-binding CXCL9(74-103) Peptide Inhibits CXCL8-induced Neutrophil Extravasation and Monosodium Urate Crystal-induced Gout in Mice.

The ELR(-)CXC chemokine CXCL9 is characterized by a long, highly positively charged COOH-terminal region, absent in most other chemokines. Several natural leukocyte- and fibroblast-derived COOH-terminally truncated CXCL9 forms missing up to 30 amino acids were identified. To investigate the role of the COOH-terminal region of CXCL9, several COOH-terminal peptides were chemically synthesized. These peptides display high affinity for glycosaminoglycans (GAGs) and compete with functional intact chemokines for GAG binding, the longest peptide (CXCL9(74-103)) being the most potent. The COOH-terminal peptide CXCL9(74-103) does not signal through or act as an antagonist for CXCR3, the G protein-coupled CXCL9 receptor, and does not influence neutrophil chemotactic activity of CXCL8 in vitro. Based on the GAG binding data, an anti-inflammatory role for CXCL9(74-103) was further evidenced in vivo. Simultaneous intravenous injection of CXCL9(74-103) with CXCL8 injection in the joint diminished CXCL8-induced neutrophil extravasation. Analogously, monosodium urate crystal-induced neutrophil migration to the tibiofemural articulation, a murine model of gout, is highly reduced by intravenous injection of CXCL9(74-103). These data show that chemokine-derived peptides with high affinity for GAGs may be used as anti-inflammatory peptides; by competing with active chemokines for binding and immobilization on GAGs, these peptides may lower chemokine presentation on the endothelium and disrupt the generation of a chemokine gradient, thereby preventing a chemokine from properly performing its chemotactic function. The CXCL9 peptide may serve as a lead molecule for further development of inhibitors of inflammation based on interference with chemokine-GAG interactions.

Hyaluronic acid decreases IL-6 and IL-8 secretion and permeability in an inflammatory model of interstitial cystitis.

Hyaluronic acid (HA) has received a lot of attention recently as a biomaterial with applications in wound healing, drug delivery, vascular repair and cell and/or gene delivery. Interstitial cystitis (IC) is characterised by an increase in the permeability of the bladder wall urothelium due to loss of the glycosaminoglycan (GAG) layer. The degradation of the urothelium leads to chronic pain and urinary dysfunction. The aetiology of the degradation of the GAG layer in this instance is currently unknown. At a clinical level, GAG replacement therapy using a HA solution is currently utilised as a treatment for IC. However, there is a significant lack of data on the mechanism of action of HA in IC. The current study investigates the mechanistic effect of clinically relevant HA treatment on an in vitro model of IC using urothelial cells, examining cytokine secretion, GAG secretion and trans-epithelial permeability. This study demonstrates that HA can significantly decrease induced cytokine secretion (4-5 fold increase), increase sulphated GAG production (2-fold increase) and without altering tight junction expression, decrease trans-epithelial permeability, suggesting that the HA pathway is a clinical target and potential treatment vector.

Sulfated sugars in the extracellular matrix orchestrate ovarian cancer development: 'when sweet turns sour'.

Considering the high mortality of ovarian cancer, novel approaches for diagnostics and therapy are urgently needed. Cancer initiation, progression, and invasion occur in a complex and dynamic microenvironment which depends on the interplay between host cell responses and tumor activity. Chondroitin sulfate (CS), a special highly sulfated sugar, forms an important intermediate player in this respect. Depending on the (micro)structural diversity of chondroitin sulfate chains, various ligands interact with this special group of glycosaminoglycans, making it a key molecule for many physiological and pathological processes, including cancer development. This review focuses on the various functions of chondroitin sulfate in tumor growth, angiogenesis, dissemination and immunosilencing of ovarian cancer. We also shed light on possible future diagnostic and therapeutic modalities for ovarian cancer based on the large variety in chondroitin sulfate microstructure and function. It is concluded that the class of chondroitin sulfate represents an attractive target to interfere with the process of ovarian tumorigenesis.

Immunosuppressants increase the levels of natural autoantibodies reactive with glycosaminoglycans in myasthenia gravis.

Increasing number of evidences support the role of glycosylation in the evolution of autoimmunity. We examined carbohydrate-reactive natural autoantibodies systematically for the first time in patients with autoimmune myasthenia gravis. Antibodies reactive to glycosaminoglycans were measured with CovaLink ELISA in the sera of 59 myasthenia patients as well as in 54 healthy controls. We used the GlycoChip carbohydrate array to characterize individual carbohydrate recognition patterns. Chondroitin-sulphate C and anti-α-mannose-specific IgG levels were significantly elevated in myasthenia patients. Unexpectedly, we found that immunosuppressants increased the levels of the protective IgM glycosaminoglycan-reactive natural antibodies demonstrating a new role for these agents in immunoregulation.

Meniscal tear as potential steering factor for inflammation may aggravate arthritis: two case reports.

INTRODUCTION: Meniscal tear is thought to play a crucial role in onset as well as progression of arthritis. However, role of cytokine response to meniscal injury and resulting inflammation is not clearly understood. Because synovial fluid is juxtaposed to cartilage and serves as a biological connection between chondrocytes and synoviocytes, we chose synovial fluid analysis to ascertain biochemical response associated with a meniscal tear. CASE PRESENTATION: We report the cases of two patients with clinically different inflammatory arthritis, both of whom are Indian men. Patient 1 was 30 years of age, and patient 2 was 50 years of age. They both had a history of meniscal tear, which we confirmed by magnetic resonance imaging scans. Synovial fluid samples obtained from these two patients were analyzed for proinflammatory markers, such as interleukin 1ß (IL-1ß) and nitric oxide, and also for glycosaminoglycan as a cartilage degradation indicator. Relatively high levels of IL-1ß (2000.0 ± 15.7 pg/ml) and nitric oxide (4.73 ± 0.05 µM/ml) and relatively low glycosaminoglycan (93.75 ± 6.3 µg/ml) were observed in patient 1, corroborating the diagnosis of traumatic meniscal tear. Compared to patient 1, Patient 2 had relatively low levels of IL-1ß (54.55 ± 14.5 pg/ml) and nitric oxide (20.00 ± 0.6 µM/ml) and remarkably high glycosaminoglycan levels (553.33 ± 1.7 µg/ml), coupled with significant osteophytes and profound cartilage loss, which indicated severe arthritis and a diagnosis of degenerative meniscal tear. CONCLUSION: The elevated levels of inflammatory IL-1ß aggravated the severity of arthritis attributable to meniscal tear in both patients, as found in follow-up visits. This was quite evident in patient 2, whereas patient 1, being younger, had less serious symptoms. Meniscal tear has emerged as a potential confounding factor in arthritis with different clinical backgrounds, which leads to increased levels of inflammation and results in accelerated disease progression.