alpha-Neup5Ac-(2--3)-beta-D-Galp-(1--4)-[alpha-L-Fucp-(1--3)]-D-GlcpNAc and Inflammation

Sialyl Lewis X (sLeX) antigen, Neu5Acα2,3Galβ1,4(Fucα1,3)GlcNAc-R, is expressed on the glycoproteins in sera or the surface of the cells and the expression of sLeX is enhanced in various conditions such as the inflammation and cancer. SLeX in the serum is utilized as a tumor marker. To clarify the roles of sLeX on secreted glycoproteins in vivo, we investigate the regulation of natural killer (NK) cell-dependent cytotoxicity through sLeX. NK cells express many receptors to kill the target cells such as cancerous cells and non-self, and their protein ligands have been elucidated. Of the killer lectin-like receptors (KLRs) on NK cells, several have been reported to recognize glycans. Using recombinant extracellular domains of KLRs (rKLRs: rNKG2A, C, D and rCD94), we evaluated their glycan ligand specificity and binding affinities using EIA methods. We clarified that all of these rKLRs can bind to high sLeX-expressing glycoprotein and heparin, heparan sulfate and highly sulfated polysaccharides and that glycan binding sites on NKG2D are mostly overlapped with those of protein ligands. In this review, we show the recent findings concerning the glycan ligands of these KLRs.

Topics: Animals; Biomarkers; Cytotoxicity, Immunologic; Glycoproteins; Heparin; Heparitin Sulfate; Humans; Inflammation; Killer Cells, Natural; Lewis X Antigen; Ligands; Mice; Neoplasms; Polysaccharides; Protein Binding; Receptors, NK Cell Lectin-Like; Sialyl Lewis X Antigen

Selectins form a family of Ca2+ -dependent carbohydrate binding proteins that mediate the initial step of leukocyte recruitment in the inflammatory process. Blocking of selectins is therefore considered a promising therapeutic approach to treat acute and chronic inflammatory diseases which are caused by excessive extravasation of leukocytes. This mini-review highlights the major structural differences between E- and P-selectin and summarizes the resulting strategies for the design of selectin antagonists.

Topics: Anti-Inflammatory Agents; Cell Adhesion; Drug Design; E-Selectin; Inflammation; Leukocytes; Membrane Glycoproteins; Models, Molecular; Oligosaccharides; P-Selectin; Protein Binding; Sialyl Lewis X Antigen

Asthma and COPD are chronic inflammatory conditions that affect hundreds of millions of patients worldwide. New therapeutics are desperately needed, especially those that target the underlying causes and prevent disease progression. Although asthma and COPD have distinct etiologies, both are associated with reduced airflow caused by excess infiltration of inflammatory cells into healthy lung tissues. As selectin-mediated adhesion of leukocytes to the vascular endothelium is a key early event in the initiation of the inflammatory response, selectin inhibition is thought to be a good target for therapeutic intervention. Three known selectins are expressed in distinct subsets of cells: P-selectin is presented on the surface of activated platelets and endothelial cells, L-selectin is constitutively expressed on leukocytes, and E-selectin synthesis is upregulated in activated endothelial cells. They mediate cell-cell adhesion in the shear flow of the bloodstream via specialized interactions with clusters of oligosaccharides presented on cell surface glycopeptide ligands. The role of selectin-ligand interactions in the inflammatory response has been demonstrated in various animal models, prompting considerable attention from the pharmaceutical industry.Drug discovery efforts have yielded many different classes of selectin inhibitors, including soluble protein ligands, antibodies, oligosaccharides and small molecules. Although many selectin inhibitors have shown activity in preclinical models, clinical progress of selectin-directed therapies has been slow. Early approaches employed carbohydrate-based inhibitors to mimic the natural ligand sialyl Lewis X; however, these compounds proved challenging to develop. Cytel's CY 1503, a complex oligosaccharide, progressed to phase II/III trials for reperfusion injury, but further development was halted when it failed to demonstrate clinical efficacy. Two protein-based selectin inhibitors have reached phase II development. These included Wyeth's recombinant soluble P-selectin ligand, TSI (PSGL-1), which was discontinued after disappointing results in myocardial infarction trials and Protein Design Labs' humanized anti-L-selectin monoclonal antibody, which is currently in development for trauma. Bimosiamose, discovered by Encysive Pharmaceutical and presently being developed by Revotar Biopharmaceuticals, is an 863 g/mol molecular weight dimer with minimal carbohydrate content and is, to date, the leading selectin inhibito

Topics: Animals; Asthma; Hexanes; Humans; Inflammation; Lewis X Antigen; Mannose; Oligosaccharides; P-Selectin; Pulmonary Disease, Chronic Obstructive; Selectins; Sialyl Lewis X Antigen

The discoveries of sialylated, fucosylated lacto-, and neolacto-type carbohydrate structures were accomplished with the aid of analytical methods and monoclonal antibodies such as the immunostaining of thin layer chromatograms. Based on the use of such antibodies, these structures, notably sialyl Le(a) and sialyl Le(x), were demonstrated to be highly expressed in many malignant cancers. A diagnostic assay using one of these antibodies (CA19-9) is now established as one of the more commonly used assays for pancreatic and gastrointestinal cancers worldwide. Upon further study, several laboratories have demonstrated that the level of expression of these carbohydrate tumor markers is also positively correlated with patient survival and is a prognostic indicator of metastatic disease. Concurrent with this finding, both sialyl Le(a) and sialyl Le(x) were shown to bind to a family of carbohydrate-binding proteins involved in the extravasation of cells from the bloodstream, called the selectins. Thus, sialyl Le(a) and sialyl Le(x) expressed on cell surfaces play functional roles in medical conditions that require extravasation of cells from the bloodstream which include a wide range of inflammatory diseases and cancer metastasis. Many studies have confirmed the function of sialyl Le(a) and sialyl Le(x) in animal models of these diseases and the inhibition of binding of sialyl Le(a) and sialyl Le(x) to the selectins is a validated drug target in the pharmaceutical industry. Thus, a new class of drugs, arising from the field of glycobiology, is based on the rational design of small molecule drugs that mimic the structures sialyl Le(a) and sialyl Le(x) and can potently inhibit their functional binding to the selectins.

Topics: Animals; Binding Sites; Biomarkers, Tumor; CA-19-9 Antigen; Drug Design; Gangliosides; Humans; Inflammation; Macromolecular Substances; Models, Chemical; Models, Molecular; Molecular Conformation; Neoplasms; Oligosaccharides; Selectins; Sialyl Lewis X Antigen; Stereoisomerism; Structure-Activity Relationship

Leukocyte recruitment in acute and chronic inflammation is characterized by sequential cell adhesion and activation events. E-, P- and L-selectins mediate initial leukocyte-endothelial-cell adhesion events required for this process. Each selectin recognizes related but distinct counter-receptors displayed by leukocytes and/or the endothelium. These counter-receptors correspond to specific glycoproteins whose 'activity' is enabled by carefully controlled post-translational modifications. Characterization of the glycans associated with E- and P-selectin counter-receptors, and of mice with targeted deletions of glycosyltransferase and sulfotransferase genes, disclose that neutrophil E- and/or P-selectin counter-receptor activities derive, minimally, from essential synthetic collaborations amongst polypeptide N-acetylgalactosaminyltransferase(s), a beta-N-acetylglucosaminyltransferase that assembles core-2-type O-glycans, beta-1,4-galactosyltransferase(s), protein tyrosine sulfotransferase(s), alpha-2,3-sialyltransferases, and a pair of alpha-1,3-fucosyltransferases.

Topics: Animals; CA-19-9 Antigen; Cell Adhesion; Endothelial Cells; Glycosylation; Glycosyltransferases; Golgi Apparatus; Inflammation; Leukocytes; Membrane Glycoproteins; Mice; Mice, Knockout; N-Acetylneuraminic Acid; Oligosaccharides; Selectins; Sialyl Lewis X Antigen; Sulfotransferases

Inhibition of selectin function is expected to aid in the management of diseases characterized by aberrant or chronic inflammation, as in asthma and chronic obstructive pulmonary disease (COPD). Selectin-mediated adhesion of leukocytes to the vascular endothelium is a critical early event in the initiation of the inflammatory response, making it a good target for therapeutic intervention. Many approaches to modulating selectin function have been explored, including competitive inhibition, altering cell-surface expression and inducing shedding/cleavage from the cell surface; however, clinical success has been elusive.

Topics: Animals; Anti-Asthmatic Agents; Asthma; Biphenyl Compounds; Cell Adhesion; Chemotaxis, Leukocyte; Clinical Trials as Topic; Endothelium, Vascular; Humans; Inflammation; Leukocytes; Ligands; Lung; Mannose; Mannosides; Membrane Glycoproteins; Oligosaccharides; Pulmonary Disease, Chronic Obstructive; Selectins; Sialyl Lewis X Antigen

Topics: Animals; CA-19-9 Antigen; Cell Adhesion; Cell Movement; Endothelium, Vascular; Gangliosides; Glucosyltransferases; Humans; Inflammation; Leukocytes; Ligands; Membrane Glycoproteins; Oligosaccharides; Selectins; Sialyl Lewis X Antigen; T-Lymphocytes

Topics: Animals; Carbohydrate Sequence; Endothelium, Vascular; Glycosyltransferases; Humans; Inflammation; L-Selectin; Ligands; Molecular Sequence Data; Oligosaccharides; Polysaccharides; Sialyl Lewis X Antigen

Topics: Acute-Phase Proteins; Animals; Estrogens; Female; Fucose; Fucosyltransferases; Glycosylation; Humans; Inflammation; Liver; Oligosaccharides; Orosomucoid; Pregnancy; Sialyl Lewis X Antigen

Topics: Antibodies, Monoclonal; Arteriosclerosis; Arthritis; Burns; Humans; Immunoglobulins; Inflammation; Integrins; Intercellular Adhesion Molecule-1; Leukocytes; Oligosaccharides; Reperfusion Injury; Selectins; Sialyl Lewis X Antigen

Extravasation from the blood of malignant tumour cells that form metastasis and leukocytes that go into tissues require contact between selectins and their sialyl Lewis x and sialyl Lewis a (sLe(x) and sLe(a) respectively) decorated ligands. Endothelial cells have been shown to express sLe(x) epitopes in lymph nodes and at sites of inflammation, and this is crucial for the selectin-dependent leukocyte traffic. Besides the ability to synthesize sLe(x) on sialylated N-acetyllactosamine via the action of alpha(1,3)fucosyltransferase(s), endothelial cells can also degrade sLe(x) to Lewis x through the action of alpha(2,3)sialidase(s). In addition, several epithelial tumors possess the machinery to synthesize sLe(x), which facilitates their adhesion to endothelial E- and P-selectin.

Topics: Animals; Carbohydrate Conformation; Carbohydrate Sequence; Endothelium, Vascular; Fucosyltransferases; Humans; Inflammation; Lymph Nodes; Molecular Sequence Data; Neoplasms; Neuraminidase; Oligosaccharides; Sialyl Lewis X Antigen

Topics: Animals; Blood Vessels; Carbohydrate Sequence; E-Selectin; Endothelium, Vascular; Humans; Inflammation; L-Selectin; Ligands; Lipopolysaccharides; Models, Cardiovascular; Molecular Sequence Data; Oligosaccharides; P-Selectin; Selectins; Sialyl Lewis X Antigen

The selectins are a family of carbohydrate-binding proteins, or lectins, that have stimulated tremendous interest because of their involvement in a wide array of interactions between leukocytes and endothelial cells. Highlights of recent progress include an extension of the list of instances of selectin participation in inflammatory diseases, further definition of selectin carbohydrate specificities, and identification of their carbohydrate-based ligands.

Topics: Animals; Carbohydrate Metabolism; Carbohydrate Sequence; Carbohydrates; Cell Adhesion Molecules; Cell Movement; E-Selectin; Endothelium, Vascular; Humans; Inflammation; L-Selectin; Leukocytes; Ligands; Models, Molecular; Molecular Sequence Data; Oligosaccharides; P-Selectin; Platelet Membrane Glycoproteins; Sialyl Lewis X Antigen

The specific migration of various leukocyte subsets to areas undergoing pathogenic attack is one of the cornerstones of the immune system. Assorted adhesion molecules, chemokines, non-protein signalling molecules, and their respective receptors are utilized during this process. The combinatorial matrix formed by this diversity of agents determines the cell type that migrates to a given class of inflammatory site. The selectins are a family of adhesion molecules that elicit the rolling response that initiates the inflammatory cascade. Selectins contain a type C lectin domain at their N-termini which recognizes sialylated, fucosylated carbohydrate ligands of the sialyl Lewis X (sLex) type. Recently, it has been demonstrated that these carbohydrate ligands are presented by a new class of adhesion molecules that have mucin-like structure and have been termed the sialomucin adhesion family. The function of this new family of adhesion molecules is to act as a scaffold that presents selectin carbohydrate ligands in a clustered, tissue specific manner to allow for higher avidity interactions between leukocytes and endothelial cells during the inflammatory process. In this manner, the appropriate type of leukocyte rolls adjacent to an inflammatory site in a temporally correct manner. This review will focus on the molecular and cellular biology of the sialomucin adhesion family and will discuss the possible implications of these findings on the development of specific inhibitors of selectin function.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Antigens, CD34; Cell Adhesion; Cell Adhesion Molecules; Chemotaxis, Leukocyte; Drug Design; Gangliosides; Humans; Immunoglobulins; Inflammation; Leukocytes; Ligands; Membrane Glycoproteins; Mucins; Mucoproteins; Organ Specificity; Selectins; Sialomucins; Sialyl Lewis X Antigen

The objective of this study was to isolate the impact of hydrodynamics on selectin-mediated cell rolling in branched microvessels. Significant advancements have been made in furthering the understanding of complex interactions between biochemical and physical factors in the inflammatory cascade in simplified planar geometries. However, few studies have sought to quantify the effects of branched configurations and to isolate the effects of associated fluid forces. Experimental techniques were developed to perform in vitro adhesion experiments in Y-shaped micro-slides. The micro-slides were coated with P-selectin and microspheres coated with Sialyl-Lewis

Topics: Animals; Cell Adhesion; Humans; Hydrodynamics; Inflammation; Leukocyte Rolling; Leukocytes; Lewis X Antigen; Microspheres; Models, Cardiovascular; P-Selectin; Sialyl Lewis X Antigen; Signal Transduction; Venules

Pancreatic ductal adenocarcinoma (PDAC) is characterized by an abundant stroma containing several pro-inflammatory cytokines, which are described to modulate the expression of important genes related to tumor promotion and progression. In the present work we have investigated the potential role of these cytokines in the biosynthesis of tumor-associated carbohydrate antigens such as sialyl-Lewis(x) (SLe(x)) through the regulation of specific glycosyltransferase genes.. Two human PDAC cell lines MDAPanc-3 and MDAPanc-28 were treated with pro-inflammatory cytokines IL-1β, TNFα, IL-6 or IL-8, and the content of tumor-associated carbohydrate antigens at the cell membrane was analyzed by flow cytometry. In addition, variation in the mRNA expression of sialyltransferase (ST) and fucosyltransferase (FUT) genes, which codify for the ST and FucT enzymes involved in the carbohydrate antigens' biosynthesis, was determined. The inflammatory microenvironment of PDAC tissues and the expression of Lewis-type antigens were analyzed by immunohistochemistry to find a possible correlation between inflammation status and the presence of tumor-associated carbohydrate antigens.. IL-1β stimuli increased SLe(x) and α2,6-sialic acid levels in MDAPanc-28 cells and enhanced the mRNA levels of ST3GAL3-4 and FUT5-7, which codify for ST and FucT enzymes related to SLe(x) biosynthesis, and of ST6GAL1. IL-6 and TNFα treatments increased the levels of SLe(x) and Le(y) antigens in MDPanc-3 cells and, similarly, the mRNA expression of ST3GAL3-4, FUT1-2 and FUT6, related to these Lewis-type antigens' biosynthesis, were increased. Most PDAC tissues stained for SLe(x) and SLe(a) and tended to be expressed in the tumor samples with a higher presence of inflammatory immune cells.. The inflammatory microenvironment can modulate the glycosylation pattern of PDAC cells, increasing the expression of tumor-associated sialylated antigens such as SLe(x), which contributes to pancreatic tumor malignancy.

Topics: Carcinoma, Pancreatic Ductal; Cell Line, Tumor; Cytokines; Disease Progression; Epitopes; Flow Cytometry; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Glycosyltransferases; Humans; Immunohistochemistry; Inflammation; Interleukin-1beta; Interleukin-6; Interleukin-8; Lewis X Antigen; Oligosaccharides; Pancreatic Neoplasms; Polysaccharides; Sialic Acids; Sialyl Lewis X Antigen; Tumor Necrosis Factor-alpha

Active targeting of the liposome is an attractive strategy for drug delivery and in vivo bio-imaging. We previously reported the specific accumulation of Sialyl Lewis X (SLX) liposome to inflamed tissue in arthritic model mice or tumor-bearing mice. SLX-liposome encapsulation with fluorescent substances allows for the visualization of these liposomes by the time-dependent transvascular accumulation of fluorescent signals in the histological sections. In the present study, we developed a new SLX-liposome encapsulated with colloidal gold for transmission electron microscopic observation. We herein describe the characterization of the colloidal gold-loaded SLX-liposomes and demonstrate its specific targeting to the endothelial cells of tumor blood vessels in tumor-bearing mice.

Topics: Animals; Arthritis; Drug Delivery Systems; Endothelial Cells; Female; Gold Colloid; Inflammation; Liposomes; Mice; Mice, Inbred BALB C; Microscopy, Electron, Transmission; Models, Animal; Neoplasms; Oligosaccharides; Sialyl Lewis X Antigen

Inflammation is a key component of many neurological diseases, yet our understanding of the contribution of these processes to tissue damage remains poor. For many such diseases, magnetic resonance imaging (MRI) has become the method of choice for clinical diagnosis. However, many of the MRI parameters that enable disease detection, such as passive contrast enhancement across a compromised blood-brain barrier, are weighted towards late-stage disease. Moreover, whilst these methods may report on disease severity, they are not able to provide information on either disease activity or the underlying molecular processes. There is a need, therefore, to develop methods that enable earlier disease detection, potentially long before clinical symptoms become apparent, together with identification of specific molecular processes that may guide specific therapy. This chapter describes the methodology for the synthesis and validation of two novel, functional MRI-detectable probes, based on microparticles of iron oxide (MPIO), which target endothelial adhesion molecules. These contrast agents enable the detection of acute brain inflammation in vivo, at a time when pathology is undetectable by conventional MRI. Such molecular MRI methods are opening new vistas for the acute diagnosis of CNS disease, together with the possibility for individually tailored therapy and earlier, more sensitive assessment of the efficacy of novel therapies.

Topics: Animals; Antibodies; Central Nervous System; Endothelial Cells; Ferric Compounds; Inflammation; Lewis X Antigen; Magnetic Resonance Imaging; Mice; Rats; Sialyl Lewis X Antigen; Statistics as Topic; Tumor Necrosis Factor-alpha; Vascular Cell Adhesion Molecule-1

Prior studies have shown that treatment with the peracetylated 4-fluorinated analog of glucosamine (4-F-GlcNAc) elicits anti-skin inflammatory activity by ablating N-acetyllactosamine (LacNAc), sialyl Lewis X (sLe(X)), and related lectin ligands on effector leukocytes. Based on anti-sLe(X) antibody and lectin probing experiments on 4-F-GlcNAc-treated leukocytes, it was hypothesized that 4-F-GlcNAc inhibited sLe(X) formation by incorporating into LacNAc and blocking the addition of galactose or fucose at the carbon 4-position of 4-F-GlcNAc. To test this hypothesis, we determined whether 4-F-GlcNAc is directly incorporated into N- and O-glycans released from 4-F-GlcNAc-treated human sLe(X) (+) T cells and leukemic KG1a cells. At concentrations that abrogated galectin-1 (Gal-1) ligand and E-selectin ligand expression and related LacNAc and sLe(X) structures, MALDI-TOF and MALDI-TOF/TOF mass spectrometry analyses showed that 4-F-GlcNAc 1) reduced content and structural diversity of tri- and tetra-antennary N-glycans and of O-glycans, 2) increased biantennary N-glycans, and 3) reduced LacNAc and sLe(X) on N-glycans and on core 2 O-glycans. Moreover, MALDI-TOF MS did not reveal any m/z ratios relating to the presence of fluorine atoms, indicating that 4-F-GlcNAc did not incorporate into glycans. Further analysis showed that 4-F-GlcNAc treatment had minimal effect on expression of 1200 glycome-related genes and did not alter the activity of LacNAc-synthesizing enzymes. However, 4-F-GlcNAc dramatically reduced intracellular levels of uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc), a key precursor of LacNAc synthesis. These data show that Gal-1 and E-selectin ligand reduction by 4-F-GlcNAc is not caused by direct 4-F-GlcNAc glycan incorporation and consequent chain termination but rather by interference with UDP-GlcNAc synthesis.

Topics: Acetylation; Acetylglucosamine; Amino Sugars; beta-Galactosidase; Gas Chromatography-Mass Spectrometry; Gene Expression Profiling; Gene Expression Regulation; Humans; Inflammation; Lectins; Leukocytes; Ligands; Oligosaccharides; Polysaccharides; Sialyl Lewis X Antigen

Glycosylation reactions of the ethylthio, bromo and chloro derivatives of 1-deoxy-1-ethoxysulfonyl-hept-2-ulopyranose were studied applying different acceptors under different conditions. Elimination side-reactions affording exo- and endoglycals occured in all cases, however, with different proportions. Glycosyl chloride donor was applied to glycosylate a trisaccharide acceptor obtaining a new sulfonic acid mimetic of the sialyl Lewis X tetrasaccharide in high yield.

Topics: Anti-Inflammatory Agents; Biomimetics; Glycosides; Glycosylation; Humans; Inflammation; Leukocytes; Ligands; Oligosaccharides; Protein Binding; Selectins; Sialyl Lewis X Antigen; Sulfonic Acids; Trisaccharides

The polymersome, a fully synthetic cell mimetic, is a tunable platform for drug delivery vehicles to detect and treat disease (theranostics). Here, we design a leuko-polymersome, a polymersome with the adhesive properties of leukocytes, which can effectively bind to inflammatory sites under flow. We hypothesize that optimal leukocyte adhesion can be recreated with ligands that mimic receptors of the two major leukocyte molecular adhesion pathways, the selectins and the integrins. Polymersomes functionalized with sialyl Lewis X and an antibody against ICAM-1 adhere avidly and selectively to surfaces coated with inflammatory adhesion molecules P-selectin and ICAM-1 under flow. We find that maximal adhesion occurs at intermediate densities of both sialyl Lewis X and anti-ICAM-1, owing to synergistic binding effects between the two ligands. Leuko-polymersomes bearing these two receptor mimetics adhere under physiological shear rates to inflamed endothelium in an in vitro flow chamber at a rate 7.5 times higher than those to uninflamed endothelium. This work clearly demonstrates that polymersomes bearing only a single ligand bind less avidly and with lower selectivity, thus suggesting proper mimicry of leukocyte adhesion requires contributions from both pathways. This work establishes a basis for the design of polymersomes for targeted drug delivery in inflammation.

Topics: Antibodies; Antigen-Antibody Reactions; Biomarkers; Butadienes; Cell Adhesion; Cells, Cultured; Drug Carriers; Drug Delivery Systems; Elastomers; Humans; Inflammation; Intercellular Adhesion Molecule-1; Leukocytes; Ligands; Lysine; Models, Molecular; Oligosaccharides; P-Selectin; Polyethylene Glycols; Sialyl Lewis X Antigen; Substrate Specificity

Interactions between endothelial selectins and selectin ligands expressed on tumor cells have been implicated in the binding of circulating metastatic cancer cells to the vascular endothelium during extravasation. Moreover, there is mounting evidence that inflammatory environments can accelerate the progression of metastasis by neutrophil mediated mechanisms. In this study, a physiologically relevant in vivo model of early metastasis coupled with intravital microscopy was used to visualize the trafficking of tumor cells within the liver vasculature in real time. Using GFP-labeled Lewis lung carcinoma subline H-59 cells, we show here that disrupting the interactions between endothelial selectins and tumor cell selectin ligands diminished tumor cell recruitment to the liver. Furthermore, systemic inflammation induced by intravenous injection of lipopolysaccharide significantly enhanced the metastatic potential of these lung carcinoma cells by increasing their propensity to adhere to the liver sinusoidal endothelium. Confocal microscopy revealed frequent colocalization of cancer cells with neutrophils and neutrophil depletion in vivo significantly attenuated the lipopolysaccharide-induced increase in H-59 cell adhesion. Although direct selectin-selectin ligand interactions contributed significantly to tumor cell adhesion to sinusoidal endothelial cells, we show here that in addition, interactions between adherent neutrophils within the inflamed sinusoids and circulating tumor cells may further increase tumor cell arrest in the liver.

Topics: Animals; Carcinoma, Lewis Lung; Cell Adhesion; Disease Progression; Endothelium, Vascular; Inflammation; Ligands; Lipopolysaccharides; Liver; Liver Neoplasms; Lung Neoplasms; Mice; Mice, Inbred C57BL; Neutrophils; Oligosaccharides; Selectins; Sialyl Lewis X Antigen; Tumor Cells, Cultured

The sialyl Lewis x tetrasaccharide with a propylamine aglycon was assembled by chemoselective glycosylation from a p-tolyl thioglycosyl donor obtained from an enzymatically synthesized sialodisaccharide. Combining the advantages of highly efficient enzymatic synthesis of sialoside building blocks, and diverse chemical glycosylation, this chemoenzymatic approach is practical for obtaining complex sialosides and their analogues.

Topics: Humans; Inflammation; Models, Molecular; Molecular Conformation; Neoplasm Metastasis; Oligosaccharides; Selectins; Sialic Acids; Sialyl Lewis X Antigen

We prepared the liposome binding Sialyl Lewis X (SLX) on the surface in order to specifically and efficiently deliver substances (fluorescent materials, chemical substances, proteins, genes, etc.) to inflammation or tumor regions. The liposome with SLX (SLX-Lipo-Cy5.5), in which fluorescent substance Cy5.5 was included, was administered intravenously to arthritis or Ehrlich Ascites Tumor (EAT) bearing mouse, and the accumulation of liposome was observed using two types of in vivo fluorescent imaging equipment. The result was that the accumulation of SLX-Lipo-Cy5.5 to inflammation or tumor regions was significantly higher than the control liposome without sugar chain (Lipo-Cy5.5) at 24 and 48 h after administration. In addition, it was confirmed that this accumulation showed a shift of liposome from blood vessels to the surrounding tissues. Thus, it was proven that this liposome is useful not only as an in vivo bio-imaging reagent but also as a drug delivery system (DDS).

Topics: Animals; Arthritis, Experimental; Carbocyanines; Carcinoma, Ehrlich Tumor; Female; Fluorescence; Fluorescent Dyes; Inflammation; Lewis Blood Group Antigens; Liposomes; Mice; Mice, Inbred BALB C; Oligosaccharides; Sialyl Lewis X Antigen

To analyze fucosylation of alpha(1)-acid glycoprotein (AGP) and to identify relations between AGP fucosylation and clinical and biochemical indices of disease activity in patients with rheumatoid arthritis (RA) treated with monoclonal antitumor necrosis factor (TNF) antibody infliximab, we examined 22 patients with RA who underwent a 54-week treatment with infliximab according to ATTRACT protocol. Blood samples were collected at baseline and before every infusion of infliximab. AGP fucosylation was measured using lectin-binding enzyme-linked immunosorbent assay utilizing fucose-specific lectin Aleuria aurantia (AAL). Moreover, the clinical status/activity, erythrocyte sedimentation rate, serum C-reactive protein (CRP), antitrypsin, alpha(1)-antichymotrypsin, AGP reactivity with concanavalin A, serum C3 and C4 complement components, and serum concentrations of TNF and soluble TNF type 1 and type 2 receptors were determined. In most patients, the fucosylation of AGP decreased rapidly after first infusion of infliximab and remained low during the 54-week therapy (p < 0.001). The decrease in AGP affinity to AAL closely followed changes in clinical and laboratory activity of RA and correlated with pretreatment concentrations of CRP (r = 0.4986, p < 0.05) and TNF (r = 0.5181, p < 0.05). The fucosylation of AGP can be a part of a negative feedback loop regulating migration of inflammatory cells and collagenase-3 activity in RA. The decrease in AGP fucosylation accompanied by improvement in clinical and biochemical parameters of RA could possibly reflect reduced migration of inflammatory cells to inflamed joints and AGP-mediated inhibition of collagenase-3 as a response to infliximab treatment.

Topics: Adult; Aged; Antibodies, Monoclonal; Arthritis, Rheumatoid; Enzyme-Linked Immunosorbent Assay; Erythrocytes; Female; Fucose; Humans; Inflammation; Infliximab; Lectins; Male; Matrix Metalloproteinase 13; Middle Aged; Models, Biological; Oligosaccharides; Orosomucoid; Selectins; Sialyl Lewis X Antigen

Acute inflammatory response is a complex process associated with the production of both pro- and anti-inflammatory mediators. Although it is generally considered to be a single homeostatic mechanism, there are differences associated with the nature and the site of inflammation. We examined the changes of N-linked glycans released from the serum of a patient with sepsis and a patient with acute pancreatitis during the first eight days of the disease. Sera were taken from patients at the time of reporting to hospital and then three more times. The blood from a healthy individual was drawn on one occasion only. Glycans were released using N-glycosidase F and were subjected to normal phase and weak anion exchange high-performance liquid chromatography, exoglycosidase digestions, and mass spectrometry. The levels of identified structures have been followed through the course of disease and compared to the control levels. Changes in serum glycans were found to occur very early in acute inflammation. The most prominent differences include the increase in ratio of outer arm to core fucose, increase in the amount of tetrasialylated structures, changes in the levels of mannose structures, and in the degree of branching. The relative proportions of different glycans changed daily and some differences were also observed between sepsis and pancreatitis, probably reflecting that in these two conditions, the acute phase response is triggered by a different stimulus that is associated with different patterns of production of cytokines.

Topics: Acute Disease; Anti-Inflammatory Agents; Chromatography, Ion Exchange; Cytokines; Fucose; Glycosylation; Humans; Inflammation; Lipids; Mannose; Oligosaccharides; Pancreatitis; Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; Polysaccharides; Sepsis; Sialyl Lewis X Antigen; Spectrometry, Mass, Electrospray Ionization; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

An ultrasound-based molecular imaging technique capable of detecting endothelial cell markers of inflammation may allow early, non-invasive assessment of vascular disease. Clinical application of targeted, acoustically-active microbubbles requires optimization of microbubble-endothelial adhesion strength to maximize image signal-to-noise ratio, as well as the ability to discern the degree of inflammation along a continuum of dysfunction. Accordingly, we hypothesized that adhesion of intercellular adhesion molecule-1 (ICAM-1)-targeted microbubbles is dependent on the degree of endothelial inflammation, and that microbubbles multi-targeted to both ICAM-1 (via anti-ICAM-1 antibodies) and selectins (via sialyl Lewisx) demonstrate greater adhesion strength than microbubbles targeted to either inflammatory marker alone. In a radial flow chamber, microbubbles were perfused across endothelial cells activated with interleukin-1beta to four different levels of inflammation, as assessed by quantitative ICAM-1 expression. ICAM-1-targeted microbubble adhesion strength increased with increasing degree of inflammation, with a relationship that was both positive and linear (r > 0.99). Microbubble adhesion strength was significantly higher for the multi-targeted microbubbles than either of the single-targeted microbubbles. These data thus demonstrate that multi-targeting of contrast microbubbles may offer improved adhesion characteristics, allowing for greater sensitivity to inflammation. Furthermore, the adhesion strength of targeted microbubbles is linearly dependent on the degree of inflammation, suggesting that targeted ultrasound imaging may offer differentiation between various degrees of endothelial dysfunction, and thus detect not only the presence, but also the severity of inflammatory disease processes.

Topics: Biomarkers; Cell Adhesion; Cells, Cultured; Contrast Media; Coronary Vessels; Endothelium, Vascular; Flow Cytometry; Humans; Immunoglobulin G; Inflammation; Intercellular Adhesion Molecule-1; Microbubbles; Oligosaccharides; Sialyl Lewis X Antigen; Ultrasonography, Interventional

The sialyl Lewis x (NeuAcalpha2-3Galbeta1-4(Fucalpha1-3)GlcNAc) determinants serve as ligands in the selectin-mediated adhesion of leukocytes to activated endothelium. The final step in the sialyl Lewis x synthesis is catalyzed by alpha1-3-fucosyltransferase, which transfers fucose to sialylated type 2 chain. We report the cloning of rat alpha1-3-fucosyltransferase gene (rFUT) isolated from rat lymph node and kidney allograft. The rFUT is expressed as two splice variants, but only the long one showed enzymatic activity towards sialylated lactosamine. Also flow cytometry analysis with the sLex mAbs indicated that the cloned rFuc-T was a functional enzyme and a member of the Fuc-TVII family. The rFuc-TVII mRNA expression level was strongly enhanced during acute inflammatory reaction induced by kidney allograft rejection, which could be detected by in situ hybridization and quantitative real-time PCR.

Topics: Amino Acid Sequence; Animals; Chlorocebus aethiops; Cloning, Molecular; COS Cells; Fucosyltransferases; Inflammation; Lymphoid Tissue; Molecular Sequence Data; Oligosaccharides; Rats; RNA, Messenger; Sialyl Lewis X Antigen

The de novo synthesis and expression of sulfo sLex glycan on vascular endothelial glycoproteins has a central role in the initiation of inflammatory reactions, serving as a putative ZIP code for organ-specific trafficking of leukocytes into sites of inflammation. The synthesis of sulfo sLex requires energy carrying donors, CMP-sialic acid (CMP-SA), GDP-fucose (GDP-Fuc), and adenosine 3'-phosphate 5'-phosphosulphate (PAPS) for donation of SA, Fuc, and sulfate, respectively. These donors are synthesized in the nucleus or cytosol and translocated into Golgi by specific transporters where corresponding transferase and proteins as well as enzymatic activities increase on inflammatory stimuli. Here we analyze the transcriptional coregulation of CMP-SA, GDP-Fuc, and PAPS transporters with in situ hybridization and real-time PCR in acute inflammation using kidney and heart allografts as model systems. Our results indicate that these three transporters display coordinated transcriptional regulation during the induction of the sulfo sLex glycan biosynthesis. With in silico analysis, the data generated with 230 human Affymetrix U133A gene chips indicated that the coregulated expression of CMP-SA and GDP-Fuc transporters was not common. Taken together our results suggest that inflammation-induced transcriptional regulation exists for Golgi membrane transporters required for the synthesis of the inflammation-inducible ZIP code sulfo sLex glycans.

Topics: Animals; Carrier Proteins; Cytidine Monophosphate N-Acetylneuraminic Acid; Epitopes; Golgi Apparatus; Guanosine Diphosphate Fucose; Humans; In Situ Hybridization; Inflammation; Lewis X Antigen; Oligosaccharides; Phosphoadenosine Phosphosulfate; Rats; Rats, Inbred Strains; Sialyl Lewis X Antigen; Transcription, Genetic

Excessive leukocyte infiltration causes severe tissue damage in a variety of inflammatory diseases. The initial step in leukocyte extravasation is mediated by selectins and oligosaccharides on their glycoconjugate ligands. Human milk is a rich source of lactose-derived oligosaccharides that are partly absorbed in the intestine and excreted with the urine. As these components contain binding determinants for the selectins we investigated whether human milk oligosaccharides are able to affect leukocyte rolling and adhesion to endothelial cells under dynamic conditions. Therefore, monocytes, lymphocytes, or neutrophils isolated from human peripheral blood were passed over TNF-alpha-activated HUVEC under shear stress. The influence of different oligosaccharide fractions was determined by video-microscopy and compared with the effects of various individual oligosaccharides. Within a physiological range (12.5 - 125 microg/ml) the acidic fraction significantly inhibited leukocyte rolling and adhesion (up to 24.0% and 52.8%, respectively) in a concentration-dependent manner. These effects were even more pronounced than those achieved by soluble sialyl-Lewis x, a physiological binding determinant for selectins. Several active components within the oligosaccharide fraction of human milk were identified, e.g. 3'-sialyl-lactose and 3'-sialyl-3-fucosyl-lactose. These results indicate that specific oligosaccharides in human milk may serve as anti-inflammatory components and might therefore contribute to the lower incidence of inflammatory diseases in human milk-fed infants.

Topics: Cell Adhesion; Cells, Cultured; Chromatography; Dose-Response Relationship, Drug; Endothelium, Vascular; Endotoxins; Humans; Inflammation; Lymphocytes; Mass Spectrometry; Milk, Human; Monocytes; Neutrophils; Oligosaccharides; Protein Binding; Sialyl Lewis X Antigen; Tumor Necrosis Factor-alpha; Umbilical Veins

Heparin has been used clinically as an anticoagulant and antithrombotic agent for over 60 years. Here we show that the potent anti-inflammatory property of heparin results primarily from blockade of P-selectin and L-selectin. Unfractionated heparin and chemically modified analogs were tested as inhibitors of selectin binding to immobilized sialyl Lewis(X) and of cell adhesion to immobilized selectins or thrombin-activated endothelial cells. Compared with unfractionated heparin, the modified heparinoids had inhibitory activity in this general order: over-O-sulfated heparin > heparin > 2-O,3-O-desulfated > or = N-desulfated/N-acetylated heparin > or = carboxyl-reduced heparin > or= N-,2-O,3-O-desulfated heparin >> 6-O-desulfated heparin. The heparinoids also showed similar differences in their ability to inhibit thioglycollate-induced peritonitis and oxazolone-induced delayed-type hypersensitivity. Mice deficient in P- or L-selectins showed impaired inflammation, which could be further reduced by heparin. However, heparin had no additional effect in mice deficient in both P- and L-selectins. We conclude that (a) heparin's anti-inflammatory effects are mainly mediated by blocking P- and L-selectin-initiated cell adhesion; (b) the sulfate groups at C6 on the glucosamine residues play a critical role in selectin inhibition; and (c) some non-anticoagulant forms of heparin retain anti-inflammatory activity. Such analogs may prove useful as therapeutically effective inhibitors of inflammation.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Cell Adhesion; Dermatitis, Allergic Contact; Disease Models, Animal; Glucosamine; Heparin; Humans; Inflammation; L-Selectin; Mice; Mice, Inbred C57BL; Mice, Knockout; Molecular Structure; Oligosaccharides; P-Selectin; Peritonitis; Sialyl Lewis X Antigen; Sulfates; U937 Cells

Sialyl Lewis X (sLeX) is a tetra-saccharide glycoconjugate of membrane proteins. It acts as a ligand for the selectin proteins during cell adhesion of inflammatory process. Aberrant overexpression of sLeX is also a characteristic of various cancer cells, especially for highly malignant ones. In this paper, the sLeX-specific RNA aptamer was selected using a random RNA library and its affinity and specificity were measured by Surface Plasmon Resonance technique. Affinity of the selected RNA was increased about 1000-fold as compared with the original RNA pool. RNA aptamer bound more specifically to its cognate sugar than to any other similar sugars. Inhibition of the cell adhesion was also shown by in vitro static assay of sLeX-expressing HL60 cells to the E- and P-selectins. It suggests that the high affinity carbohydrate specific RNA aptamer could be used as an alternative to the antibody.

Topics: Base Sequence; Biosensing Techniques; Cell Adhesion; Cell Membrane; Chromatography, Affinity; E-Selectin; Gene Library; HL-60 Cells; Humans; Inflammation; Kinetics; Lectins; Ligands; Models, Chemical; Molecular Sequence Data; Oligonucleotides; Oligosaccharides; P-Selectin; Protein Binding; Sensitivity and Specificity; Sialyl Lewis X Antigen; Surface Plasmon Resonance

Diabetic mellitus is attended by the development of endothelial dysfunction which is suggested to be accompanied with a chronic low-degree of inflammation. During a chronic hepatic inflammatory response, specific changes in glycosylation of the acute phase protein alpha1-acid glycoprotein (AGP) can be detected. In this report we studied the changes in glycosylation of AGP in more detail and evaluated the relation between a change in glycosylation of AGP and urinary albumin secretion in Type I diabetic patients. The glycosylation of AGP, studied by crossed affinity immunoelectrophoresis (CAIE) and high pH anion exchange chromatography with pulse amperometric detection (HPAEC-PAD), showed an increase in alpha3-fucosylation. Staining with an antibody against sialyl Lewis(x) (sLe(x)) implied that part of the alpha3-fucosylation was present in a sLe(x)-conformation. In the group of Type I diabetic patients with increased urinary albumin excretion, a significant increase in alpha3-fucosylation of AGP (p<0.0005) could be detected. Therefore, the increased alpha3-fucosylation of AGP can be used as an additional marker for the development of vascular complications in Type I diabetic patients.

Topics: Adolescent; Adult; Aged; Albuminuria; Carbohydrate Conformation; Chromatography, Ion Exchange; Concanavalin A; Diabetes Mellitus, Type 1; Diabetic Angiopathies; Female; Fucose; Fucosyltransferases; Glycosylation; Humans; Inflammation; Lectins; Male; Middle Aged; Oligosaccharides; Orosomucoid; Sialyl Lewis X Antigen; Statistics as Topic

Peptides mimicking carbohydrate structure sialyl-Lewis a (SA-Le(a)) have been selected from a diverse dodecapeptide library using monoclonal antibody (MAb) NS19-9. Families of peptides with a consensus sequence consisting of three to nine amino acids and peptides that do not show a conserved core amino acid region were identified. Peptide DLWDWVVGKPAG was selected based on the consensus sequence DXXDXXVG shared with other peptides and strong binding in Western blot. Peptide competes with antibody binding to its native carbohydrate antigen, SA-Le(a), at 50% inhibitory concentration (IC(50)), 700 microM, implying that it represents a structural mimic of the carbohydrate epitope recognized by MAb. Statistically significant reduction of neutrophil recruitment into the intraperitoneal cavity was observed upon administration of this peptide in a murine acute inflammation model in vivo. Results suggest that the peptide mimic of SA-Le(a) carbohydrate might bind to E-selectin and block its interaction with another ligand, sialyl-Lewis X (SA-LeX), expressed on neutrophils.

Topics: Amino Acid Sequence; Animals; Anti-Inflammatory Agents, Non-Steroidal; Antibodies, Monoclonal; Binding, Competitive; Carbohydrate Sequence; Disease Models, Animal; E-Selectin; Inflammation; Ligands; Mice; Molecular Sequence Data; Neutrophils; Oligopeptides; Oligosaccharides; Peptide Library; Sialyl Lewis X Antigen

The carbohydrate structure of sialyl-Lewis X (SLe(x)) can function as a ligand for E- and P-selectin, which play important roles in mediating the initial interactions of leukocytes with the endothelium in inflammatory responses. In this study we evaluated the effects of inhibiting E- and P-selectin function with the SLe(x) molecule on the inflammatory response in an experimental murine model of hypersensitivity pneumonitis (HP). Antigen exposure induced marked interstitial and especially perivascular and peribronchiolar infiltration with lymphocytes and granuloma formation, in murine lung sensitized with Saccaropolyspora rectivirgula. These pathologic changes were significantly suppressed with SLe(x) ganglioside analogues through a reduction in the numbers of lymphocytes in bronchoalveolar lavage fluid, as evidenced by the lung index and histologic scores indicating the severity of the inflammatory response. Using specific antibodies, we also evaluated the immunohistochemical localization of SLe(x) in mononuclear cells in granulomas, and of E- and P-selectin in vascular endothelium. Our findings suggest that the molecular interaction between SLe(x), and E- and P-selectin mediates lymphocyte recruitment into the lung parenchyma, which is critical for the inflammatory response in experimental murine models of HP.

Topics: Alveolitis, Extrinsic Allergic; Animals; Antigens, Fungal; Bronchoalveolar Lavage Fluid; Cell Count; E-Selectin; Endothelium, Vascular; Gangliosides; Immunohistochemistry; Inflammation; Lewis Blood Group Antigens; Lung; Male; Mice; Mice, Inbred C3H; P-Selectin; Pulmonary Circulation; Saccharopolyspora; Sialyl Lewis X Antigen; T-Lymphocytes

L-selectin mediates leukocyte rolling on vascular endothelium during inflammation. Although vascular endothelium can be activated with inflammatory cytokines to express functional L-selectin ligands, these ligands have not been well characterized. In this study, fucosyltransferase VII cDNA (Fuc-TVII) transfection of the EA.hy926 human vascular endothelial cell line (926-FtVII) induced functional L-selectin ligand expression and expression of sialyl Lewisx (sLex), as defined by HECA-452 (cutaneous lymphocyte antigen; CLA) and CSLEX-1 mAbs. Cytokine activation of human umbilical vein endothelial cells (HUVEC) also induced functional L-selectin ligand expression, with increased CLA expression and Fuc-TVII transcription. The majority of L-selectin-dependent lymphocyte attachment to activated HUVEC and 926-FtVII cells was blocked specifically by treating the endothelial cells with the HECA-452 mAb, but not the CSLEX-1 mAb. CLA-bearing ligands on vascular endothelium also required sulfation and appropriate molecular scaffolds for functional activity, but were distinct from the L-selectin ligands previously identified by the MECA-79 mAb. These findings demonstrate that the HECA-452- defined antigen, CLA, is an essential carbohydrate component of vascular L-selectin ligands.

Topics: Antibodies, Monoclonal; Antigens, Differentiation, T-Lymphocyte; Antigens, Neoplasm; Cell Adhesion; Cell Line; Endothelium, Vascular; Fluorescent Antibody Technique; Fucosyltransferases; Humans; Immunoglobulin M; Inflammation; L-Selectin; Leukocytes; Ligands; Lymphocytes; Membrane Glycoproteins; Oligosaccharides; Recombinant Fusion Proteins; Sialyl Lewis X Antigen; Transfection; Tumor Necrosis Factor-alpha

The prophylactic effects of selectin inhibitors on lipopolysaccharide-induced acute lung injury were studied in rabbits by using sialyl Lewis X-oligosaccharide and PB1.3, an anti-human P-selectin monoclonal antibody. Lipopolysaccharide-induced acute lung injury resembles that of the acute respiratory distress syndrome, in which there is a decrease in arterial blood oxygen tension (PaO2) and an increase in the difference between alveolar and arterial oxygen tension (A-aDO2). Prophylactic treatment with the selectin inhibitors, sialyl Lewis X-oligosaccharide (55 mg kg(-1) i.v. bolus injection immediately before lipopolysaccharide administration + 36 mg kg(-1) h(-1) i.v. infusion for 4 h) and PB1.3 (5 mg kg(-1) i.v. bolus injection immediately before lipopolysaccharide administration), prevented the lipopolysaccharide-induced impairments in pulmonary gas exchange. In contrast, these agents had no significant effects on lipopolysaccharide-induced systemic hypotension, the decrease in the number of circulating white blood cells and platelets, the decline in blood pH, or the increase in arterial CO2 tension (PaCO2). These results indicate that selectin inhibitors including sialyl Lewis X-oligosaccharide and the anti-P-selectin antibody, PB1.3, attenuate lipopolysaccharide-induced acute lung injury in rabbits. This is the first demonstration that P-selectin is directly involved in the development of lipopolysaccharide-induced impairments in pulmonary gas exchange.

Topics: Animals; Antibodies, Monoclonal; Blood Pressure; Dose-Response Relationship, Drug; Inflammation; Injections, Intravenous; Lipopolysaccharides; Lung Diseases; Male; Oligosaccharides; P-Selectin; Pulmonary Gas Exchange; Rabbits; Sialyl Lewis X Antigen

Acute and chronic inflammation-induced expression of sialyl LewisX has already been shown to occur on alpha1-acid glycoprotein. We now demonstrate that this phenomenon is not restricted to alpha1-acid glycoprotein but also occurs on two other acute-phase proteins. ie on alpha1-antichymotrypsin and on haptoglobin. The level of expression of sialyl LewisX on these proteins was lower than on alpha1-acid glycoprotein, in all likelihood because alpha1-acid glycoprotein is the only acute-phase protein containing tetraantennary glycans. No expression of sialyl LewisX was detectable on alpha1-protease inhibitor, a protein with a high diantennary glycan content. Non-sialylated LewisX was not detectable on these major acute-phase proteins in any of the conditions studied. This indicates that the majority of the a3-linked fucose residues are present as sialyl LewisX on alpha1-acid glycoprotein, alpha1-antichymotrypsin and haptoglobin. The absolute contribution to the total phenotype in plasma of protein containing this determinant in a multivalent form was highest for alpha1-acid glycoprotein. This leads us to propose that alpha1-acid glycoprotein is, among the acute-phase proteins studied, the one with the highest potential for interference with the extravasation of leukocytes by binding to the selectins.

Topics: Acute-Phase Proteins; alpha 1-Antichymotrypsin; alpha 1-Antitrypsin; Antibodies, Monoclonal; Arthritis, Rheumatoid; Blotting, Western; Concanavalin A; Electrophoresis; Haptoglobins; Humans; Inflammation; Lectins; Liver; Oligosaccharides; Orosomucoid; Sialyl Lewis X Antigen; Wounds and Injuries

Mice are frequently used in models for the study of immunological processes related to inflammation. Since it is known that the degree of fucosylation of human acute phase proteins (APPs) is altered as a consequence of an inflammatory response, we have undertaken this study to gain more insight into the fucosylation of acute phase proteins as it occurs in mouse liver. Mice carrying the cluster of the three genes encoding human alpha1-acid glycoprotein (AGP), one of the well known APPs, were used and the fucosylation of AGP was assessed. A complete absence of fucosylation on the transgenic human AGP was found, which is in sharp contrast to AGP in human serum, of which a major proportion is normally alpha3-fucosylated. Remarkably, a large proportion of mouse AGP did contain fucose residues. Fucosylation was also detected on another APP, mouse protease inhibitor (PI). Alpha3-fucosylation of the transgenic human AGP can be achieved in vitro, using an alpha3/4-fucosyltransferase (alpha3/4-FucT) isolated from human milk, showing that the glycoprotein is not intrinsically resistant to fucosylation. Upon subsequent measurement of the activities of the possible fucosyltransferases present in liver membranes of parent and transgenic mice, only an N-linked-core alpha6-FucT and no alpha2-, alpha3- or alpha4-FucT activity was detected. This indicates that fucose residues found on the mouse serum proteins AGP and PI, which are synthesized in the liver, are most probably in alpha6-linkage to the core chitobiosyl unit. Interestingly, both alpha6- and alpha3-FucT activity was detectable in human liver membranes. None of the above mentioned findings were influenced by the induction of an acute phase response by administration of bacterial lipopolysaccharide. This study shows that: (a) alpha6-FucT is probably a protein specific-glycosyltransferase, since mouse AGP, but not human AGP, may be used as an acceptor; (b) in contrast to human liver, mouse liver does not express any alpha3-FucT-activity, thereby making the mouse incapable of producing the Sialyl Lewis(x) epitope on APPs, which is an important part of the inflammatory reaction in humans. This last finding indicates that the mouse is not suitable as a model for the study of those phenomena related to inflammation in humans, in which glycosylation of acute phase proteins could play a significant role.

Topics: Acute-Phase Proteins; Animals; Carbohydrate Sequence; Disease Models, Animal; Epitopes; Fucose; Fucosyltransferases; Humans; In Vitro Techniques; Inflammation; Liver; Mice; Mice, Transgenic; Molecular Sequence Data; Oligosaccharides; Orosomucoid; Sialyl Lewis X Antigen; Species Specificity

Early in inflammation, adhesion occurs between leukocytes and endothelium when selectins bind to sialyl Lewis X (sLex) and related oligosaccharides. We tested novel compounds that mimic sLex for their ability to inhibit selectin-mediated adhesion of human eosinophils and neutrophils in vitro. Neutrophils and eosinophils were isolated by density gradient centrifugation, and eosinophils were further purified by immunomagnetic negative selection. Adhesion to unstimulated or interleukin-1beta-stimulated (5 ng/ml, 4-6 h) umbilical vein endothelial monolayers was tested under static or rotating conditions, where adhesion is primarily E- or L-selectin dependent, respectively. P-selectin-dependent adhesion was tested on immobilized platelets treated with or without phorbol myristate acetate (10(-7) M, 10 min). Stimulus-induced adhesion was always at least 4-fold higher than without stimulus, and selectin dependence was confirmed with specific blocking monoclonal antibodies. E-selectin-dependent adhesion of eosinophils and neutrophils was inhibited by compound GM2296 (the concentration producing 50% inhibition of adhesion [IC50] approximately 0.5-1 mM). E-selectin-dependent adhesion of neutrophils, but not eosinophils, was also inhibited by another compound, sLex with a lipid tail (30 +/- 6% inhibition at 3 mM), whereas compound GM1292 slightly inhibited adhesion of both (23 +/- 5 and 20 +/- 6% inhibition, respectively, at 1 mM). L-selectin-dependent adhesion was more effectively inhibited by GM2296 (IC50 approximately 0.2-0.5 mM), although P-selectin-dependent adhesion was also inhibited (IC50 approximately 1 mM). Inhibition was reversible without affecting viability, and no effect was seen with these compounds in assays testing neutrophil adhesion to immobilized intercellular adhesion molecule-1. Thus, compound GM2296, a carbon-fucosylated derivative of glycyrrhetinic acid, inhibits E-, L-, and P-selectin-dependent eosinophil and neutrophil adhesion. The ability of these and perhaps other related glycomimetic compounds to interfere with the function of more than one type of selectin makes them desirable candidates as anti-inflammatory agents.

Topics: Antibodies, Monoclonal; Carbohydrate Sequence; Cell Adhesion; Eosinophils; Glycyrrhizic Acid; Humans; Inflammation; Molecular Sequence Data; Molecular Structure; Neutrophils; Oligosaccharides; Selectins; Sialyl Lewis X Antigen

A novel system for active targeting of inflammatory lesions has been established. A SLeX-CMPul conjugate (2) showed accumulation that was 2.5-fold higher in inflammatory lesions in vivo than a SLN-CMPul conjugate (4) and 300-fold higher than monovalent SLeX (6).

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Carbohydrate Sequence; Drug Carriers; Edema; Inflammation; Mice; Molecular Sequence Data; Oligosaccharides; Polysaccharides; Sialyl Lewis X Antigen

Little is known about the changes in the hepatic microcirculation and the leukocyte-endothelial adhesion processes during the early reperfusion period after resuscitation in hemorrhagic shock. P-selectin and its natural ligand Sialyl Lewis(x) (SLe(x)) are involved in the early stages of reperfusion events leading to neutrophil migration. Therefore, the aim of this study was to investigate the effect of the administration of CY-1503 [corrected], a synthetic SLe(x) analog, in the liver inflammatory response and neutrophil migration after hemorrhagic shock.. Rats, each weighing 275 to 300 grams, were subjected to 60 minutes of pressure controlled hemorrhagic shock. After this period, animals were resuscitated according to the following protocol: shed blood was reinfused to equal 50% of the total volume bled, and the other 50% was replaced with 3x volume of Ringer's lactated solution. Animals were divided into sham and two study groups to receive vehicle (controls) and CY-1503 [corrected] (10 mg/kg intravenously) diluted in 1 mL of normal saline 45 minutes after initiating hemorrhagic shock. The following parameters were analyzed: 7-day survival, liver injury tests, liver tissue myeloperoxidase as an index of neutrophil infiltration, and liver histology.. Survival was significantly increased from 48% in the controls to 90% in the CY-1503 [corrected] treated group. Animals treated with the SLe(x) analog showed significantly better mean arterial blood pressure after 15 minutes after resuscitation. Also, the treated group showed a marked decrease in liver enzymes levels at 5 minutes and 4 hours after reperfusion. Neutrophil migration was significantly ameliorated as reflected by decreased myeloperoxidase levels in the SLe(x) analog treated group. Furthermore, we observed improved histologic damage scores in the treated group when compared with controls.. The SLe(x) analog, CY-1503 [corrected], had a protective effect in ischemic livers by decreasing neutrophil migration after hemorrhagic shock and resuscitation. This protective effect also resulted in improved survival and mean arterial blood pressure after resuscitation.

Topics: Animals; Disease Models, Animal; Drug Evaluation, Preclinical; Inflammation; Liver; Liver Circulation; Male; Neutrophil Activation; Oligosaccharides; P-Selectin; Rats; Rats, Sprague-Dawley; Shock, Hemorrhagic; Sialyl Lewis X Antigen; Survival Analysis

We have rationally designed a sLe(x) mimetic based on molecular modeling, synthesized type II and type II' beta-turn dipeptides (3a,b), and evaluated their biological profiles both in vitro and in vivo. Against E-selectin-sLe(x) binding, the type II beta-turn dipeptide L-Ser-D-Glu 3a (IC50, 13 microM) and the type II' beta-turn dipeptide D-Ser-L-Glu 3b (IC50, 5.5 microM) were 20-100-fold more potent blockers than sLe(x) (1; IC50, 600 microM) and a 3'-sulfated Le(x) analog (2; IC50, 280 microM). On the other hand, other stereoisomers, such as L-Ser-L-Glu 3c and D-Ser-D-Glu 3d, were very weak blockers, with IC50 > 1000 microM for both 3c,d. Against the P- and L-selectins, despite much different stereochemistry of compounds 3a-d, the dipeptides 3a-d were all more potent blockers than either sLe(x) or compound 2. Interestingly, compound 3b provided significant in vivo efficacy against an immunoglobulin E-mediated skin reaction in a mouse model. These findings indicate that sLe(x) mimetics with type II and type II' beta-turn dipeptides could be useful in the design of an active selectin blocker in vitro and/or in vivo.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Dipeptides; Drug Design; Female; Immunoglobulin E; Inflammation; Magnetic Resonance Spectroscopy; Mice; Mice, Inbred BALB C; Models, Molecular; Molecular Mimicry; Molecular Structure; Oligosaccharides; Selectins; Serine; Sialyl Lewis X Antigen; Stereoisomerism

The selectins are a family of three cell-surface-presented glycoprotein receptors. They play a key role in the initial adhesive interaction between leukocytes and endothelial cells at sites of inflammation. The presence of selectins on the cell surface is tightly regulated and inappropriate appearance is associated with a number of inflammatory disease conditions.

Topics: Amino Acid Sequence; Cell Adhesion Molecules; Cytokines; E-Selectin; Endothelium; Gene Expression; Glycoproteins; Inflammation; L-Selectin; Leukocytes; Membrane Proteins; Models, Biological; Molecular Sequence Data; Oligosaccharides; P-Selectin; Promoter Regions, Genetic; Sialyl Lewis X Antigen; Signal Transduction; Transcription Factors; Transcription, Genetic

Selectins are cell adhesion molecules known to support the initial attachment of leukocytes to inflamed vascular endothelium through their recognition of carbohydrate ligands such as the tetrasaccharide sialyl Lewisx (Neu5Ac alpha 2-3Gal beta 1-4(Fuc alpha 1-3)GlcNAc-). In the present study, we describe the inhibition of L- and P-selectin function by inositol polyanions, simple 6-carbon ring structures that have multiple ester-linked phosphate or sulfate groups. In a purified component competition assay, binding of L- and P-selectin-Ig fusion proteins to immobilized bovine serum albumin-sialyl Lewisx neoglycoprotein was inhibited by inositol hexakisphosphate (InsP6, IC50 = 2.1 +/- 1.4 microM and 160 +/- 40 microM), by inositol pentakisphosphate (InsP5, IC50 = 1.4 +/- 0.2 and 260 +/- 40 microM), and by inositol hexakissulfate (InsS6, IC50 = 210 +/- 80 microM and 2.8 +/- 0.9 mM); E-selectin-Ig binding was unaffected. Inositol polyanions diminished the adhesion of LS180 colon carcinoma cells to plates coated with L- and P-selectin-Ig but not with E-selectin-Ig. Inositol polyanions blocked polymorphonuclear leukocyte (PMN) adhesion to COS cells expressing recombinant transmembrane P-selectin but not to those expressing E-selectin. In addition, inositol polyanions diminished PMN adhesion to activated endothelial cells under rotation-induced shear stress, a process known to require L-selectin function. In vivo, the effects of inositol polyanions were studied in two murine models of acute inflammation. Intravenously administered InsP6 (two doses of 40 mumol/kg) inhibited PMN accumulation in thioglycolate-induced inflammation (55 +/- 10% inhibition) and in zymosan-induced inflammation (61 +/- 4% inhibition). InsP5 and InsS6 also inhibited inflammation in these models, although higher doses were required for InsS6. In conclusion, inositol polyanions are noncarbohydrate small molecules that inhibit L- and P-selectin function in vitro and inflammation in vivo.

Topics: Animals; Carbohydrate Sequence; Cell Adhesion; Cell Adhesion Molecules; Cells, Cultured; Haplorhini; Humans; Immunoglobulins; Inflammation; Inositol; L-Selectin; Lung; Male; Molecular Sequence Data; Neutrophils; Oligosaccharides; P-Selectin; Peritoneal Cavity; Platelet Membrane Glycoproteins; Polyelectrolytes; Polymers; Serum Albumin, Bovine; Sialyl Lewis X Antigen; Tumor Cells, Cultured

Activated platelets expressing P-selectin on their surface are known to adhere to monocytes and neutrophils. We examined the possibility that the leukocytes were activated by their adhesion to activated platelets and demonstrated that P-selectin-dependent platelet adhesion to neutrophils and monocytes induced production of extracellular superoxide anion (O2-) by these leukocytes. Leukocyte membrane glycoproteins containing Ser/Thr-linked carbohydrate chains were responsible for the signal reception leading to the leukocyte activation. Cytokines were shown to influence these processes. For example, treatments of neutrophils with interleukin-8 (IL-8) or granulocyte colony-stimulating factor (G-CSF) potentiated the P-selectin-induced O2- production. Furthermore, interleukin-1 (IL-1) and interferon-gamma (IFN-gamma) induced surface expression of P-selectin on platelets in the presence of a low concentration of thrombin and consequently enhanced their adhesion capacity to leukocytes. These results indicated that the adhesion of activated platelets to the leukocytes through the interaction between P-selectin and its carbohydrate ligand, sialyl Lewis X (LeX), was a crucial step for the activation of leukocyte function and supported the notion that activated platelets were actively involved in the inflammatory processes.

Topics: Blood Platelets; Humans; Inflammation; Leukemia, Promyelocytic, Acute; Monocytes; Neutrophils; Oligosaccharides; P-Selectin; Platelet Activation; Platelet Membrane Glycoproteins; Sialyl Lewis X Antigen; Superoxides; Tumor Cells, Cultured