Wheat Germ Agglutinin Binding Research Articles

Amphiphilic Low-Molecular-Weight Gelators Bearing β-S-N-Acetylglucosamine Linked to a Tartaric Acid Scaffold: Synthesis, Self-Assembly and Wheat Germ Agglutinin Binding.

The self-assembly of carbohydrate-based amphiphiles can lead to colloidal soft materials such as supramolecular gels featuring highly desirable characteristics like biodegradability and biocompatibility. The report herein presents the synthesis, characterization and supramolecular self-assembly, physical gelation and wheat lectin binding of two structurally related amphiphilic compounds having β-S-N-acetylglucosamine residues linked to a 2,3-diacyl-N,N'-dipropargylated-l-tartaric diamide. A 1-thio-β-N-acetyl-d-glucosamine precursor attached to a conveniently functionalized linker with an azido group was synthesized by means of a one-pot procedure followed by deprotection. A click reaction successfully led to the two amphiphiles, which differed in length of the fatty acid attached to the tartaric acid scaffold. Although both compounds are poorly soluble in water and organic solvents, the difference in terms of hydrophilic moieties provided them with distinct supramolecular gelation properties. While the presence of an octadecyl chain produced a hydrogelator, the dodecadecyl homologue would only form weak gels in DMSO. SEM and rheology experiments confirmed the characteristic fibrillar morphology and viscoelastic properties, in agreement with the presence of physical gels. Both amphiphiles were able to interact reversibly with wheat germ agglutinin (WGA), a lectin that specifically recognizes GlcNAc residues, indicating a potential use in the food industry, as a gluten sensitivity manager, as well as in health-related industries, for example, for drug delivery systems.

Impact of Age on Hemostasis of Patients with Immune Thrombocytopenia

Introduction Incidence of primary immune thrombocytopenia (ITP) has two peaks: one in children and young adult and one in the elderly. Management of older patients represents a challenge, because older age is associated with increased frailty, comorbidities, polypharmacy, and worse outcome. Moreover, we cannot rule out the effect of old age on hemostasis of individuals without ITP. Objectives We aimed to compare characteristics of hemostasis in healthy controls and in patients with ITP stratified according to their age in ≤65 years old (yo) and >65 yo, in order to determine if old age modifies hemostasis and if there is an increased risk of bleeding in older patients with ITP. Methods This is a prospective project that was approved by the Ethics Committee from Hospital Universitario La Paz. Informed consent was signed before sampling. ITP patients (n=100≤65 yo and n=68>65 yo) and healthy controls (n=100≤65 yo and n=28>65 yo) were recruited. Patients with ITP and participants with uncontrolled hypertension, artery disease, abnormal hepatic or renal function tests, a diagnosis of a bleeding disorder or thrombopathy, treatment with drugs that could affect hemostasis or a history of thrombotic episodes were excluded. We evaluated platelet activation markers (TRAP and ADP-induced activation of fibrinogen receptor determined through PAC1-binding, and TRAP-induced P-selectin exposure with anti-P-selectin antibody); active caspase-3, -7, -8 and -9; surface loss of sialic acid determined by the binding of Ricinus Communis Agglutinin I (RCA) to galactose residues; and presence of GlcNAc residues on platelet's surface determined by the binding of Wheat Germ Agglutinin (WGA). Neuraminidase 1 (NEU1) associated to membrane of quiescent platelets were tested with anti-NEU1-Alexa 546 antibody. All samples were analyzed by flow cytometry. Thrombin generation was measured in platelet-poor plasma by Calibrated automated thrombogram (CAT) and coagulation was triggered by proper recalcification and the addition (final concentrations) of 1 pmol/l of recombinant human tissue factor and 4 µmol/l of phospholipid mixture (PPP-Reagent LOW). The following parameters were measured: Lagtime (= time when 10 nmol/l thrombin is formed); peak height (PH = maximum thrombin concentration reached); and endogenous thrombin potential (ETP = area under the thrombin-concentration-vs-time curve) were calculated with the Thrombinoscope software package (Thrombinoscope BV). Statistical analyses were performed with GraphPrism 6.0. Comparisons between controls vs ITP patients with similar range of age; controls ≤65 vs >65 yo; and ITP patients ≤65 vs >65 yo were performed with 2 way ANOVA and p<0.05 were considered as significant. Results Table 1 shows results obtained. There was no difference in age between controls and ITP groups with the same age range. Platelets from patients with ITP had a low ability to be activated when compared to their corresponding control group, and impairment in activation of fibrinogen receptor was even more evident for the older group of ITP patients.This fact was not due to a reduction in the number of fibrinogen receptors. Caspases 3, 8 and 9 were higher in ITP patients than their age-matched controls. Moreover, caspase 3 was higher in older ITP than in patients ≤65 yo. Regarding analyses of glycoside residues, platelets from patients with ITP had less sialic acid and more GlcNAc residues on their surface when compared with the corresponding controls. Surprisingly, sialic content in platelets from controls > 65yo was higher than in the controls ≤65 yo, and this fact is in accordance to the lowest content of NEU1 associated to their membrane. Plasma from ITP patients had a higher capacity to generate thrombin when compared with their age-matched control group. Nevertheless, both groups (control and ITP)> 65 yo generated less thrombin than the groups ≤65 yo. No differences were observed in other CAT parameters. Conclusion Platelets from patients with ITP are less functional than those from control groups. This impairment seemed to be more pronounced in platelets from patients >65 yo, putting forward the possibility of an increased risk of bleeding in older patients with ITP. This study also demonstrates that old age might modify hemostasis even in individuals without ITP. Work supported by Instituto de Salud Carlos III (ISCIII) (PI19/00772, PI22/01489), co-funded by the European Union.

A Novel Preparation Technique for Human Nasal Respiratory Mucosa to Disclose Its Glycosylation Pattern for Bioadhesive Drug Delivery

To shed some light on glycotargeting as a potential strategy for nasal drug delivery, a reliable preparation method for human nasal mucosa samples and a tool to investigate the carbohydrate building blocks of the glycocalyx of the respiratory epithelium are required. Applying a simple experimental setup in a 96-well plate format together with a panel of six fluorescein-labeled lectins with different carbohydrate specificities allowed for the detection and quantification of accessible carbohydrates in the mucosa. As confirmed by binding experiments at 4 °C, both quantitatively by fluorimetry and qualitatively by microscopy, the binding of wheat germ agglutinin exceeded that of the others by 150% on average, indicating a high content of N-acetyl-D-glucosamine and sialic acid. Providing energy by raising the temperature to 37 °C revealed uptake of the carbohydrate-bound lectin into the cell. Moreover, repeated washing steps during the assay gave a slight hint as to the influence of mucus renewal on bioadhesive drug delivery. All in all, the experimental setup reported here for the first time is not only a suitable approach to estimating the basics and potential of nasal lectin-mediated drug delivery but also meets the needs for answering a broad variety of scientific questions involving the use of ex vivo tissue samples.

Microencapsulation of Probiotics with Soy Protein Isolate and Alginate for the Poultry Industry.

Many probiotic products, with properly selected microorganisms, may not be effective for the intended purpose due to the low tolerance of microorganisms to gastrointestinal digestion. The microencapsulation seems to be one of the most promising techniques to protect probiotics against adverse environmental conditions. Therefore, the aim of this work was the design of soy protein isolate-alginate microcapsules for the encapsulation of probiotics for the poultry industry by the water-in-oil emulsion technique. To this end, the strain Ligilactobacillus salivarius CRL2217, with the ability to bind wheat germ agglutinin (WGA) on its surface and protect intestinal epithelial cells from the cytotoxicity of the glycoprotein, was used as model microorganism. Several parameters were varied in order to find the better conditions for microencapsulation: oil source and nature, SPI and sodium alginate concentration, stirring equipment and time for emulsion formation, CaCl2 concentration, and absence or presence of stirring after the addition of the CaCl2 solution. The survival of entrapped cells to a simulated gastric digestion and their survival and release during simulated intestinal digestion were also investigated. The obtained particles effectively protected L. salivarius CRL2217 from the proteolytic activity and low pH present in the gastric environment. Besides, their content was released in contact with a simulated intestinal juice, as viable counts and binding of WGA after a simulated intestinal digestion revealed. This work paves the way for the design of probiotic supplements for poultry including gastrointestinal digestion-susceptible bacteria.

Surface-Templated Glycopolymer Nanopatterns Transferred to Hydrogels for Designed Multivalent Carbohydrate-Lectin Interactions across Length Scales.

Multivalent interactions between carbohydrates and proteins enable a broad range of selective chemical processes of critical biological importance. Such interactions can extend from the macromolecular scale (1-10 nm) up to much larger scales across a cell or tissue, placing substantial demands on chemically patterned materials aiming to leverage similar interactions in vitro. Here, we show that diyne amphiphiles with carbohydrate headgroups can be assembled on highly oriented pyrolytic graphite (HOPG) to generate nanometer-resolution carbohydrate patterns, with individual linear carbohydrate assemblies up to nearly 1 μm, and microscale geometric patterns. These are then photopolymerized and covalently transferred to the surfaces of hydrogels. This strategy suspends carbohydrate patterns on a relatively rigid polydiacetylene (persistence length ∼ 16 nm), exposed at the top surface of the hydrogel above the bulk pore structure. Transferred patterns of appropriate carbohydrates (e.g., N-acetyl-d-glucosamine, GlcNAc) enable selective, multivalent interactions (KD ∼ 40 nM) with wheat germ agglutinin (WGA), a model lectin that exhibits multivalent binding with appropriately spaced GlcNAc moieties. WGA binding affinity can be further improved (KD ∼ 10 nM) using diacetylenes that shift the polymer backbone closer to the displayed carbohydrate, suggesting that this strategy can be used to modulate carbohydrate presentation at interfaces. Conversely, GlcNAc-patterned surfaces do not induce specific binding of concanavalin A, and surfaces patterned with glucuronic acid, or with simple carboxylic acid or hydroxyl groups, do not induce WGA binding. More broadly, this approach may have utility in designing synthetic glycan-mimetic interfaces with features from molecular to mesoscopic scales, including soft scaffolds for cells.

Effect of Formulation Variables for the Production of WGA-Grafted, Levodopa-Loaded PLGA Nanoparticles

Levodopa is used for the treatment of Parkinson’s disease (PD) for the last few decades. However, adverse reactions such as dyskinesia, somnolence, nausea, itching, rash, as well as the need for frequent dosing and low bioavailability problems affect the success of the treatment. To prevent side effects caused by conventional therapy, a nanoparticular drug delivery system has been developed, in which receptors are constantly stimulated, and the frequency of dosing is reduced. In this study, levodopa was loaded in Poly lactic-co-glycolic acid (PLGA) nanoparticles (NP) which modified with Wheat Germ Agglutinin (WGA) To increase the effectiveness of levodopa, reduce its side effects and apply to the nasal area which is an alternative way for brain targeting with lower doses. To obtain the optimum levodopa loaded PLGA nanoparticles, the effect of some formulation variables such as polyvinyl alcohol (PVA) concentration, homogenization speed, polymer amount and molecular weight, and levodopa content on the entrapment efficiency (EE) and particle size of the nanoparticles were investigated. Besides these variables, the effect of different parameters on the WGA binding constant was also searched. In addition to in vitro release studies, Differential Scanning Calorimetry (DSC) and Fourier Transform Infrared Spectrophotometer (FT-IR), and Transmission electron microscopy (TEM) analysis were used in the characterization of nanoparticles. Among all formulations, A2 and A8a which was produced with different molcular weights of PLGA, different added levodopa amounts and with different homogenization speeds were chosen as optimum formulations due to their sustained release properties and the ability to release 80 % of their drug content.WGA binding constant was found 78.20 % for A8a-1 and 95 % for A2-1. In this study, we aimed to determine the effect of different formulation parameters on the development of levodopa loaded and WGA grafted PLGA nanoparticles and on the quality characteristics of nanoparticle formulations such as particle size, zeta potential, and EE. In this paper, our results are demonstrated for a better understanding of the effect of process parameters on the development of nanoparticle-based drug delivery systems by using the double-emulsion solvent evaporation technique and on WGA binding of drug-loaded PLGA nanoparticles.

Lectins in Cervical Screening

Cervical screening in low-resource settings remains an unmet need. Lectins are naturally occurring sugar-binding glycoproteins whose binding patterns change as cancer develops. Lectins discriminate between dysplasia and normal tissue in several precancerous conditions. We explored whether lectins could be developed for cervical screening via visual inspection. Discovery work comprised lectin histochemistry using a panel of candidate lectins on fixed-human cervix tissue (high-grade cervical intraepithelial neoplasia (CIN3, n = 20) or normal (n = 20)), followed by validation in a separate cohort (30 normal, 25 CIN1, 25 CIN3). Lectin binding was assessed visually according to staining intensity. To validate findings macroscopically, near-infra red fluorescence imaging was conducted on freshly-resected cervix (1 normal, 7 CIN3), incubated with topically applied fluorescently-labelled lectin. Fluorescence signal was compared for biopsies and whole specimens according to regions of interest, identified by the overlay of histopathology grids. Lectin histochemistry identified two lectins—wheat germ agglutinin (WGA) and Helix pomatia agglutinin (HPA)—with significantly decreased binding to CIN3 versus normal in both discovery and validation cohorts. Findings at the macroscopic level confirmed weaker WGA binding (lower signal intensity) in CIN3 vs. normal for biopsies (p = 0.0308) and within whole specimens (p = 0.0312). Our findings confirm proof-of-principle and indicate that WGA could potentially be developed further as a probe for high-grade cervical disease.

EVERYONE KNOWS AND NOBODY KNOWS: THE MYSTERY OF TER119

Background TER119 is very widely used as a marker of the erythroid lineage in mice. It is variously regarded as a marker of a protein associated with Glycophorin A or specifically reacting with Glycophorin A itself. We previously reported a TER119-negative murine pedigree, derived by ENU-mutagenesis and recessive breeding, with a T>C variant at position 4634105 on chromosome 6 and exon 14 deletion (transmembrane region) in a sialyl-O-acetyl transferase, CASD1. Methods Glycophorin A and TER119 expression were evaluated by Western blot and flow cytometry. The blood picture for the TER-119neg pedigree was evaluated using standard techniques. CASD1 expression in erythroid precursors was measured by flow cytometry. Other changes in the profile of surface carbohydrates were evaluated by lectin binding. Results While a complete loss of the TER-119 epitope was confirmed by flow cytometry and immunoblot, normal levels of Glycophorin A were noted. There was no change in haematological parameters nor in osmotic fragility. Increased levels of wheat germ agglutinin binding were found in erythroid cells in CASD1-variant homozygotes. Levels of CASD1 expression decreased during erythroid differentiation. Discussion Through studies of a CASD1-variant pedigree, we determined that TER-119 is a sialyl epitope at least partially defined by a 9-O-acetyl sialic acid. The exact nature of the epitope defined by TER119 and any human equivalent remain to be determined.

Bacterial expression, purification and biophysical characterization of wheat germ agglutinin and its four hevein-like domains.

Wheat germ agglutinin (WGA), a chitin binding lectin, has attracted increasing interest because of its unique characteristics such as conformational stability, binding specificity and transcytosis capacity. To pave the way for the study of the molecular basis of WGA's structural stability and binding capacity, as well as to facilitate its use in biomedical and biotechnological developments, we produced recombinant WGA and its 4 isolated hevein-like domains in a bacterial system. All the proteins were expressed as fusion constructs linked to a thioredoxin domain, which was enzymatically or chemically released. The structural and ligand-binding properties of recombinant WGA were similar to the wild lectin. The 4 isolated domains folded and were ligand-binding competent, indicating that each domain constitutes an independent folding unity. The biophysical characterization of the recombinant domains sheds new light on the intricate folding and binding behavior of this emblematic lectin.

Jaburetox-induced toxic effects on the hemocytes of Rhodnius prolixus (Hemiptera: Reduviidae)

Jaburetox is a recombinant peptide derived from a Canavalia ensiformis urease that presents toxic effects upon several species of insects, phytopathogenic fungi and yeasts of medical importance. So far, no toxicity of Jaburetox to mammals has been shown. Previous reports have identified biochemical targets of this toxic peptide in insect models, although its mechanism of action is not completely understood. In this work, we aimed to characterize the effects of Jaburetox in hemolymphatic insect cells. For this purpose, the model insect and Chagas' disease vector Rhodnius prolixus was used. In vivo and in vitro experiments indicated that Jaburetox interacts with a subset of hemocytes and it can be found in various subcellular compartments. In insects injected with Jaburetox there was an increase in the gene expression of the enzymes UDP-N-acetylglucosamine pyrophosphorylase (UAP), chitin synthase and nitric oxide synthase (NOS). Nevertheless, the expression of NOS protein, the enzyme activities of UAP and acid phosphatase (a possible link between UAP and NOS) as well as the phosphorylation state of proteins remained unchanged upon the in vivo Jaburetox treatment. Nitric oxide (NO) imaging using fluorescent probes showed that Jaburetox augmented NO production in the hemocyte aggregates when compared to controls. Even though Jaburetox activated the hemocytes, as demonstrated by wheat germ agglutinin binding assays, the peptide did not lead to an increase of their phagocytic behavior. Taken together, these findings contribute to our understanding of toxic effects of Jaburetox, a peptide with biotechnological applications and a prospective tool for rational insect control.

Carbohydrate Analogue Microarrays for Identification of Lectin-Selective Ligands.

Fifty-five mono- and disaccharide analogues were prepared and used for the construction of microarrays to uncover lectin-selective ligands. The microarray study showed that two disaccharide analogues, 28' and 44', selectively bind to Solanum tuberosum lectin (STL) and wheat germ agglutinin (WGA), respectively. Cell studies indicated that 28' and 44' selectively block the binding of STL and WGA to mammalian cells, unlike the natural ligand LacNAc, which suppresses binding of both STL and WGA to cells.

Increased collagen within the transverse tubules in human heart failure.

In heart failure transverse-tubule (t-tubule) remodelling disrupts calcium release, and contraction. T-tubules in human failing hearts exhibit increased labelling by wheat germ agglutinin (WGA), a lectin that binds to the dystrophin-associated glycoprotein complex. We hypothesized changes in this complex may explain the increased WGA labelling and contribute to t-tubule remodelling in the failing human heart. In this study we sought to identify the molecules responsible for this increased WGA labelling. Confocal and super-resolution fluorescence microscopy and proteomic analyses were used to quantify left ventricle samples from healthy donors and patients with idiopathic dilated cardiomyopathy (IDCM). Confocal microscopy demonstrated both WGA and dystrophin were located at t-tubules. Super-resolution microscopy revealed that WGA labelling of t-tubules is largely located within the lumen while dystrophin was restricted to near the sarcolemma. Western blots probed with WGA reveal a 5.7-fold increase in a 140 kDa band in IDCM. Mass spectrometry identified this band as type VI collagen (Col-VI) comprised of α1(VI), α2(VI), and α3(VI) chains. Pertinently, mutations in Col-VI cause muscular dystrophy. Western blotting identified a 2.4-fold increased expression and 3.2-fold increased WGA binding of Col-VI in IDCM. Confocal images showed that Col-VI is located in the t-tubules and that their diameter increased in the IDCM samples. Super-resolution imaging revealed Col-VI was restricted to the t-tubule lumen where increases were associated with displacement in the sarcolemma as identified from dystrophin labelling. Samples were also labelled for type I, III, and IV collagen. Both confocal and super-resolution imaging identified that these collagens were also present within t-tubule lumen. Increased expression and labelling of collagen in IDCM samples indicates fibrosis may contribute to t-tubule remodelling in human heart failure.

血小板活性化の初期反応部位の研究 : 血小板細胞におけるカチオン化フェリチンとwheat germ agglutininの結合部位(杏林大学学位論文要旨ならびに審査要旨)

血小板活性化の初期反応部位の研究 : 血小板細胞におけるカチオン化フェリチンとwheat germ agglutininの結合部位(杏林大学学位論文要旨ならびに審査要旨)

OC-053 Lectin-Based Near infra-red Molecular Imaging for Dysplasia Detection in Barrett’s Oesophagus: An ex-vivo Study on Human Tissue

Introduction Detection of early neoplasia in Barrett’s oesophagus (BO) by white-light endoscopy is challenging due to the inconspicuous nature of dysplasia. Molecular imaging using fluorescently labelled wheat-germ agglutinin (WGA) is a promising tool as this topically applied imaging agent shows lower binding to dysplastic versus non-dysplastic BO. 1 However in an endoscopy setting, the detection of fluorescence in the blue/green range is limited by high-levels of tissue autofluorescence. This limitation can be overcome by using near infra-red (NIR) imaging. We aimed to assess in an ex-vivo model the feasibility of WGA-based NIR imaging for detection of dysplasia in BO. Methods we recruited patients with early BO-related neoplasia undergoing endoscopic mucosal resection (EMR). Freshly collected EMR specimens were sprayed with WGA-IR800CW and then imaged with a high-sensitivity NIR camera. Fluorescence images were captured and up to two punch biopsies were collected from each EMR under fluorescence guidance. The EMRs were paraffin embedded, cut every 2 mm and processed for histopathological assessment. Each section was scored by an expert GI pathologist every 1 mm to construct a pathology grid, which was manually co-registered with the fluorescence image. The mean fluorescence intensity (MFI) of cells in dysplastic and non-dysplastic areas was compared by the Wilcoxon matched-pairs signed rank test. Only EMR specimens with at least one dysplastic gland were included in the analysis. In addition, the MFI of punch biopsies taken from dysplastic and non-dysplastic areas was also compared by Mann-Whitney test. Results Ten patients were recruited at a single centre. A total of 18 EMR specimens and 33 punch biopsies were collected, of which 10 were dysplastic. In the whole EMR analysis, we found a significantly lower MFI for dysplastic versus non-dysplastic areas (P = 0.0012), in accordance with the reported reduced binding of WGA to neoplastic BO epithelium. Similarly, we found a nearly 2 fold reduction in the MFI of punch biopsies taken from dysplastic as compared to non-dysplastic (ND) areas (P = 0.0002) (Figure 1). Conclusion WGA-based NIR imaging is an effective method for differentiating dysplastic from non-dysplastic BO mucosa ex vivo , which reduces the effects of tissue autofluorescence. In-vivo studies are now required to test the efficacy of this method for detecting dysplasia during endoscopic surveillance. Reference 1 Bird-Lieberman, et al . Nat Med 2012. Disclosure of Interest None Declared

Detection of colorectal dysplasia using fluorescently labelled lectins.

Colorectal cancer screening using conventional colonoscopy lacks molecular information and can miss dysplastic lesions. We tested here the ability of fluorescently labelled lectins to distinguish dysplasia from normal tissue when sprayed on to the luminal surface epithelium of freshly resected colon tissue from the Apcmin mouse and when applied to fixed human colorectal tissue sections. Wheat germ agglutinin (WGA) showed significantly decreased binding to adenomas in the mouse tissue and in sections of human colon from 47 patients. Changes in WGA binding to the human surface epithelium allowed regions containing normal epithelium (NE) or hyperplastic polyps (HP) to be distinguished from regions containing low-grade dysplasia (LGD), high-grade dysplasia (HGD) or carcinoma (C), with 81% sensitivity, 87% specificity and 93% positive predictive value (PPV). Helix pomatia agglutinin (HGA) distinguished epithelial regions containing NE from regions containing HP, LGD, HGD or C, with 89% sensitivity, 87% specificity and 97% PPV. The decreased binding of WGA and HPA to the luminal surface epithelium in human dysplasia suggests that these lectins may enable more sensitive detection of disease in the clinic using fluorescence colonoscopy.

Glycosylation status of serum immunoglobulin G in patients with prostate diseases.

Occurrences of high values in patients with benign prostate disease and low values in patients with highly suspicious cancer have diminished the trustworthiness of prostate‐specific antigen as an early diagnostic marker of prostate cancer. In the search for other complimentary markers, we focused on serum IgG from patients with prostate diseases as well as normal subjects. IgG purified from the sera of normal control subjects and patients with prostate diseases, was digested with peptide N‐glycanase. Released glycans were quantified using MALDI‐time of flight mass spectrometry. We report that N‐linked (N‐acetylhexosamine)2(deoxyhexose)(mannose)3(N‐acetylglucosamine)2 was significantly increased in the IgG heavy chains of patients with prostate cancer compared with that of either benign prostatic disease patients or healthy subjects, whereas (hexose)(N‐acetylhexosamine)2(deoxyhexose)(mannose)3 (N‐acetylglucosamine)2 was more abundant in the heavy chains of healthy subjects and benign prostatic disease patients. Thus, an absence of the terminal hexose of N‐linked glycans has been closely connected to the progression of prostate cancer. Furthermore, surface plasmon resonance analyses have revealed that IgG from patients with prostate cancer has a decreased binding for Sambucus nigra lectin, compared with that from the benign prostatic disease patients or from normal subjects, suggesting lower levels of (N‐acetylneuraminic acid)(α2‐6)galactose/N‐acetylgalactosamine groups in the N‐linked glycans of patient IgG. Meanwhile, wheat germ agglutinin binding to IgG of the cancer group was significantly larger than that for the benign prostatic disease group but smaller than that for normal subjects. Our study indicates that the glycosylation changes in IgG can become useful diagnostic parameters for prostate cancer.

Fluorescent imaging of endothelial glycocalyx layer with wheat germ agglutinin using intravital microscopy.

Endothelial glycocalyx (GCX) is located on the apical surface of vascular endothelial cells and is composed of a negatively-charged network of proteoglycans and glycoproteins. The GCX plays an important role in maintaining the integrity of vascular walls and preventing leakage of plasma. Therefore, degradation of the GCX is believed to lead to pathological leakage of plasma. Because the GCX is a very thin layer, its ultrastructural image has been demonstrated on electron microscope. To explore the function of the GCX, it should be visualized by a microscope in vivo. Thus, we developed in vivo visualization technique of the GCX under fluorescence microscopy using a mouse dorsal skinfold chamber (DSC) model. To label and visualize the GCX, we used fluorescein isothiocyanate (FITC)-labeled lectin, which has a high specificity for sugar moieties. We examined the affinity of the different lectins to epivascular regions under an intravital fluorescent microscope. Among seven different lectins we examined, FITC labeled Triticum vulgaris (wheat germ) agglutinin (WGA) delineated the GCX most clearly. Binding of WGA to the GCX was inhibited by chitin hydrolysate, which contained WGA-binding polysaccharide chains. Furthermore, the septic condition attenuated this structure, suggesting structural degradation of endothelial GCX layer. In conclusion, FITC-labeled WGA lectin enabled visualization of endothelial GCX under in vivo fluorescence microscopy.

Loss of the endothelial glycocalyx is associated with increased E-selectin mediated adhesion of lung tumour cells to the brain microvascular endothelium.

BackgroundArrest of metastasising lung cancer cells to the brain microvasculature maybe mediated by interactions between ligands on circulating tumour cells and endothelial E-selectin adhesion molecules; a process likely to be regulated by the endothelial glycocalyx. Using human cerebral microvascular endothelial cells and non-small cell lung cancer (NSCLC) cell lines, we describe how factors secreted by NSCLC cells i.e. cystatin C, cathepsin L, insulin-like growth factor-binding protein 7 (IGFBP7), vascular endothelial growth factor (VEGF) and tumour necrosis factor-alpha (TNF-α), damage the glycocalyx and enhance initial contacts between lung tumour and cerebral endothelial cells.MethodsEndothelial cells were treated with tumour secreted-proteins or lung tumour conditioned medium (CM). Surface levels of E-selectin were quantified by ELISA. Adhesion of A549 and SK-MES-1 cells was examined under flow conditions (1 dyne/cm2). Alterations in the endothelial glycocalyx were quantified by binding of fluorescein isothiocyanate-linked wheat germ agglutinin (WGA-FITC).ResultsA549 and SK-MES-1 CM and secreted-proteins significantly enhanced endothelial surface E-selectin levels after 30 min and 4 h and tumour cell adhesion after 30 min, 4 and 24 h. Both coincided with significant glycocalyx degradation; A549 and SK-MES-1 CM removing 55 ± 12 % and 58 ± 18.7 % of WGA-FITC binding, respectively. Inhibition of E-selectin binding by monoclonal anti-E-selectin antibody completely attenuated tumour cell adhesion.ConclusionThese data suggest that metastasising lung cancer cells facilitate their own adhesion to the brain endothelium by secreting factors that damage the endothelial glycocalyx, resulting in exposure of the previously shielded adhesion molecules and engagement of the E-selectin-mediated adhesion axis.Electronic supplementary materialThe online version of this article (doi:10.1186/s13046-015-0223-9) contains supplementary material, which is available to authorized users.

2158 The Nationwide Cancer Genome Screening Project for Gastrointestinal Cancer in Japan (GI-SCREEN): Simultaneous identification of KRAS, NRAS, BRAF, and PIK3CA mutation in advanced colorectal cancer (aCRC) (GI-SCREEN 2013-01)

resection (11.7%). Colon histological sections were stained with fluorescently labeled lectins, and the lectin binding to surface epithelium, which would be observable in colonoscopy, was compared with conventional histopathology for the presence of disease and disease stage. Results: Normal colonic epithelium (NE) occupied 40.1% of the area of all sections, hyperplastic polyps (HP) occupied 10.2%, low-grade (LGD) dysplasia 25.6%, high-grade (HGD) dysplasia 13.9%, and adenocarcinoma (C) 10.2%, as assessed by conventional histopathology based on H&E staining. Lesions were located in the ascending (10.4%), transverse (15.6%), descending (14.3%), sigmoid (42.9%) colon and rectum (7.8%), with the mean lesion size being 16.0±14.2mm (range 2−60mm). The lectin wheat germ agglutinin (WGA), when fluorescently-labeled, was capable of distinguishing epithelial regions containing NE or HP from regions containing LGD, HGD or cancer, with 81% sensitivity and 87% specificity. Conclusions: Automated analysis of fluorescently-labeled WGA binding to the surface epithelium of excised colon sections may be used for improving the assessment of early dysplasia in CRC. The same fluorescent lectin may also be useful as a topical imaging agent for improving detection of early dysplasia using fluorescence colonoscopy, increasing the early diagnosis of CRC and improving patient survival. No conflict of interest.

Abstract LB-122: molecular imaging of early colorectal dysplasia using fluorescently-labeled lectins

Abstract Objective Screening for colorectal cancer (CRC) is currently performed using bright-field colonoscopy, which has low specificity and sensitivity for the detection of early dysplasia in the colon. Fluorescently labeled lectins have been applied topically for detecting glycosylation changes in early dysplastic lesions in the esophagus, providing a route for improved endoscopic detection using fluorescence endoscopy [1]. We have identified here fluorescently labeled lectins that can detect changes in glycosylated biomarkers on the surface epithelium of early dysplasia in CRC. These probes may enable fluorescence-guided resection of dysplastic lesions at colonoscopy and subsequent automated assessment of colon histopathology. Study Design Ninety-nine colon lesions in 48 patients (34 males, 14 females; mean patient age 68.8 ± 8.3 yr, range 53-95 yr) were identified during routine colonoscopy at Addenbrooke's Hospital, Cambridge, UK. Lesions suspected of being dysplastic or neoplastic, were detected using magnification colonoscopy or surgery, and removal performed at biopsy (11.7%) and/or polypectomy (62.1%), or partial colon resection (11.7%). Colon histological sections were stained with fluorescently labeled lectins, and the lectin binding to surface epithelium, which would be observable in colonoscopy, was compared with conventional histopathology for the presence of disease and disease stage. Results Normal colonic epithelium (NE) occupied 40.1% of the area of all sections, hyperplastic polyps (HP) occupied 10.2%, low- (LGD) dysplasia 25.6%, high-grade (HGD) dysplasia 13.9%, and adenocarcinoma (C) 10.2%, as assessed by conventional histopathology based on H&E staining. Lesions were located in the ascending (10.4%), transverse (15.6%), descending (14.3%), sigmoid (42.9%) colon and rectum (7.8%), with the mean lesion size being 16.0 ± 14.2 mm (range 2-60 mm). The lectin wheat germ agglutinin (WGA), when fluorescently-labeled, was capable of distinguishing epithelial regions containing NE from regions containing LGD, HGD and cancer, with >81% sensitivity, >79% specificity and >93% positive predictive value. Conclusions Automated analysis of fluorescently-labeled WGA binding to the surface epithelium of excised colon sections may be used for improving the assessment of early dysplasia in CRC. The same fluorescent lectin may also be useful as a topical imaging agent for improving detection of early dysplasia using fluorescence colonoscopy, increasing the early diagnosis of CRC and improving patient survival.