Ulex Europaeus Agglutinin Research Articles

Endothelial progenitor cells in the peripheral blood of patients with moyamoya disease labeled with superparamagnetic iron oxide in vitro for MRI detection.

Moyamoya disease (MMD) is currently thought to involve endothelial progenitor cells (EPCs). We investigated whether superparamagnetic iron oxide (SPIO) can be used to label EPCs. Mononuclear cells from 10 moyamoya disease patients were isolated, and cluster of differentiation 133 (CD133) positive cells sorted by magnetic-activated cell sorting were cultured in vitro. The positive rates of CD133, vascular endothelial growth factor receptor (VEGFR)-2, and cluster of differentiation 34 (CD34) were detected by flow cytometry. The cells were co-cultured with fluorescence labeled Dil-acetylated-low-density lipoprotein (Dil-ac-LDL) and Ulex europaeus agglutinin-1 (UEA-1) to observe the endocytosis of Dil-ac-LDL and binding to UEA-1. Prussian blue staining and transmission electron microscopy were used to observe the endocytosis of different SPIO concentrations in EPCs, and CCK-8 was used to detect proliferation of cells transfected with different concentrations of SPIO. T2 weighted imaging (T2WI) signals from magnetic resonance imaging after SPIO endocytosis were compared. Positive rates of CD133, VEGFR-2, and CD34 on sorted mononuclear cells were 68.2±3.8, 57.5±4.2, and 36.8±6.5%, respectively. The double-positive expression rate of CD34 and VEGFR-2 was 19.6±4.7%, and 83.1±10.4% of cells, which showed the uptake of Dil-ac-LDL and binding with UEA-1. The labeling efficiencies of SPIO at concentrations of 25 and 50 μg/mL were higher than for 12.5 μg/mL. The proliferation of cells was not influenced by SPIO concentrations of 12.5 and 25 μg/mL. After labeling, the T2WI of EPCs was reduced. The concentration of 25 μg/mL SPIO had high labeling efficiency detected by magnetic resonance imaging (MRI) without decreased EPCs viability.

TSTA3 facilitates esophageal squamous cell carcinoma progression through regulating fucosylation of LAMP2 and ERBB2.

Background: TSTA3 gene encodes an enzyme responsible for synthesis of GDP-L-fucose as the only donor in fucosylation. This study was designed to explore clinical value, function and underlying mechanism of TSTA3 in the development of esophageal squamous cell carcinoma (ESCC).Methods: Whole genomic sequencing data from 663 ESCC patients and RNA sequencing data from 155 ESCC patients were used to analyze the copy number variation and mRNA expression of TSTA3 respectively. Immunohistochemistry based or not based on the tissue microarrays was used to detect its protein expression. Transwell assay and in vivo metastasis assay were used to study the effect of TSTA3 on invasion and metastasis of ESCC. Immunofluorescence was used to analyze fucosylation level. N-glycoproteomics and proteomics analysis, Lens Culinaris Agglutinin (LCA) and Ulex Europaeus Agglutinin I (UEA-I) affinity chromatography, immunoprecipitation, glycosyltransferase activity kit and rescue assay were used to explore the mechanism of TSTA3.Results: TSTA3 was frequently amplified and overexpressed in ESCC. TSTA3 amplification and protein overexpression were significantly associated with malignant progression and poor prognosis of ESCC patients. TSTA3 knockdown significantly suppressed ESCC cells invasion and tumor dissemination by decreasing fucosylation level. Conversely, exogenous overexpression of TSTA3 led to increased invasion and tumor metastasis in vitro and in vivo by increasing fucosylation level. Moreover, core fucosylated LAMP2 and terminal fucosylated ERBB2 might be mediators of TSTA3-induced pro-invasion in ESCC and had a synergistic effect on the process. Peracetylated 2-F-Fuc, a fucosyltransferase activity inhibitor, reduced TSTA3 expression and fucosylation modification of LAMP2 and ERBB2, thereby inhibiting ESCC cell invasion.Conclusion: Our results indicate that TSTA3 may be a driver of ESCC metastasis through regulating fucosylation of LAMP2 and ERBB2. Fucosylation inhibitor may have prospect to suppress ESCC metastasis by blocking aberrant fucosylation.

MyoD1 suppresses cell migration and invasion by inhibiting FUT4 transcription in human gastric cancer cells

Myogenic differentiation 1 (MyoD1) is a transcription factor that promotes expression of muscle-specific genes. MyoD1 is expressed at significantly lower levels in gastric cancer (GC) tissues and cells, and it induces apoptosis in GC cells. However, functions for MyoD1 in GC cell migration and gene expression have not been documented. We show that knockdown of MyoD1 promoted migration and invasion of GC cells, whereas MyoD1 overexpression suppressed migration and invasion. We performed chromatin immunoprecipitation (ChIP)-sequencing to identify MyoD1 target genes in MKN-45 cells. The 2-kb upstream regions (Up2k) of the transcription start sites of 57 genes were probably bound by MyoD1. Six of these genes function in signaling pathways such as synthesis of glycosphingolipid biosynthesis—lacto and neolacto series. MyoD1 inhibited transcription of fucosyltransferase IV (FUT4) by binding directly to the FUT4 F3; this finding was validated by ChIP-quantitative PCR and a luciferase reporter assay. Ulex europaeus agglutinin I, which binds Fucα1-2Galβ1-4GlcNAc, and Lewis antigens showed decreased binding to the plasma membrane of cells that overexpressed MyoD1. Knockdown of FUT4 mimicked MyoD1 overexpression by suppressing GC cell migration and invasion; this result implied that MyoD1 suppressed cell migration and invasion via inhibiting the FUT4/matrix metallopeptidase signaling pathway. In summary, this study demonstrated that MyoD1 suppresses migration and invasion of GC cells by directly binding to the F3 region in the FUT4 Up2k and inhibiting FUT4/type II Lewis antigen expression.

Impact of Fucosylation on Self-Assembly of Prostate and Breast Tumor Spheroids by Using Cyclo-RGDfK(TPP) Peptide and Image Object Detection.

IntroductionCore fucosylation of N-glycans on the integrin β1 subunit is essential for the functional activity of the integrin. The binding of α5β1 integrin with the tripeptide Arg-Gly-Asp (RGD) motif within the extracellular matrix protein fibronectin may be influenced by the α-1,6-fucose core or α-1,2-fucose and α-1,3/4-fucose peripheral N-glycan profiles. Here, we investigated whether fucosylation impacts the formation of matrix-free 3D multicellular tumor spheroids (MCTS) from human triple negative breast MDA-MB231 cell line, prostate PC3 and DU145 cell lines and DU145 gemcitabine resistant (GemR) variant by using the cyclic Arg-Gly-Asp-D-Phe-Lys peptide modified with 4-carboxybutyl-triphenylphosphonium bromide (cyclo-RGDfK(TPP)) peptide method.MethodsMicroscopic imaging, lectin histochemistry, flow cytometry, WST-1 cell viability assay and You Only Look Once version 2 (YOLOv2) training object detection using cyclic learning rates were used to evaluate the formation of MCTS, morphologic changes, and the expression levels of α-1,6-fucose and α-1,2-fucose linkages on the cell surface.ResultsDU145 prostate cancer cells expressed higher α-1,6-fucose than α-1,2-fucose linkages on their cell surface, as determined by lectin cytochemistry and flow cytometry. Blockage of the α-1,6- and α-1,2-fucose linkages with Aspergillus oryzae lectin (AOL) and Ulex Europaeus agglutinin I (UEA I) one hour before the addition of cyclic-RGDfK(TPP) peptide to the monolayer of the cancer cells resulted in a statistically significant dose-dependent reduction in spheroid volumes using threshold diameters of 40 and 60 µm. Application of a 40 µm threshold diameter measurements of spheroids resulted in fewer false-positive ones compared to the 60 µm diameter threshold previously used in our studies. A state-of-the-art, image object detection system YOLOv2 was used to automate the analysis of spheroid measurements and volumes. The results showed that YOLOv2 corroborated manual spheroid detection and volume measurements with high precision and accuracy.ConclusionFor the first time, the findings demonstrate that α-1,6- and α-1,2-fucose linkages of N-glycans on the cell surface receptors facilitate cyclo-RGDfK(TPP)-mediated self-assembly of cancer cells to form 3D multicellular tumor spheroids.

Terminal fucose mediates progression of human cholangiocarcinoma through EGF/EGFR activation and the Akt/Erk signaling pathway

Aberrant glycosylation is recognized as a cancer hallmark that is associated with cancer development and progression. In this study, the clinical relevance and significance of terminal fucose (TFG), by fucosyltransferase-1 (FUT1) in carcinogenesis and progression of cholangiocarcinoma (CCA) were demonstrated. TFG expression in human and hamster CCA tissues were determined using Ulex europaeus agglutinin-I (UEA-I) histochemistry. Normal bile ducts rarely expressed TFG while 47% of CCA human tissues had high TFG expression and was correlated with shorter survival of patients. In the CCA-hamster model, TFG was elevated in hyperproliferative bile ducts and gradually increased until CCA was developed. This evidence indicates the involvement of TFG in carcinogenesis and progression of CCA. The mechanistic insight was performed in 2 CCA cell lines. Suppression of TFG expression using siFUT1 or neutralizing the surface TFG with UEA-I significantly reduced migration, invasion and adhesion of CCA cells in correlation with the reduction of Akt/Erk signaling and epithelial-mesenchymal transition. A short pulse of EGF could stimulate Akt/Erk signaling via activation of EGF-EGFR cascade, however, decreasing TFG using siFUT1 or UEA-I treatment reduced the EGF-EGFR activation and Akt/Erk signaling. This evidence provides important insight into the relevant role and molecular mechanism of TFG in progression of CCA.

Keloid patients have higher peripheral blood endothelial progenitor cell counts and CD34+ cells with normal vasculogenic and angiogenic function that overexpress vascular endothelial growth factor and interleukin-8.

One suggested reason for aberrant wound healing in keloid scars is chronic inflammation of the dermis. We hypothesized that excessive blood vessel formation and high capillary density in keloid tissue is caused by dysfunction of endothelial progenitor cells. We compared the number of circulating endothelial progenitor cells and vasculogenic and angiogenic capacity, as well as secretory function, of circulating CD34+ cells in keloid patients and healthy individuals. Compared to mononuclear cell cultures from healthy donors, cultures of peripheral blood mononuclear cells obtained from keloid patients showed a more than twofold increase in the number of peripheral blood EPCs (fibronectin-adhering cells that phagocytized acetylated low-density lipoprotein and bound Ulex europaeus agglutinin-I lectin). However, there was no difference in colony-forming ability and participation in in vitro angiogenesis between circulating CD34+ cells isolated from keloid patients and healthy individuals. This means that circulating CD34+ /endothelial progenitor cells in keloid patients have normal vasculogenic and angiogenic function. However, CD34+ cells derived from keloid patients demonstrated a more than sevenfold expression of the interleukin-8 gene and a more than fivefold expression of the vascular endothelial growth factor gene than CD34+ cells derived from healthy individuals. These results support the role of vascular endothelial growth factor and interleukin-8 in increased recruitment of endothelial progenitor cells in keloid patients.

A Universal Photochemical Method to Prepare Carbohydrate Sensors Based on Perfluorophenylazide Modified Polydopamine for Study of Carbohydrate-Lectin Interactions by QCM Biosensor.

A universal photochemical method to prepare carbohydrate sensors based on perfluorophenylazide (PFPA) modified polydopamine (PDA) for the study of carbohydrate–lectin interactions by a quartz crystal microbalance (QCM) biosensor was developed. The PFPA was immobilized on PDA-coated gold sensors via Schiff base reactions. Upon light irradiation, the underivatized carbohydrates were inserted into the sensor surface, including mannose, galactose, fucose and N-acetylglucosamine (GlcNAc). Carbohydrate sensors were evaluated for the binding to a series of plant lectins. A kinetic study of the interactions between mannose and concanavalin A (Con A), fucose and Ulex europaeus agglutinin I (UEA-I) were performed. This method can eliminate the tedious modification of carbohydrates, improve the experimental efficiency, and reduce the experimental cost, which is of great significance for the development of QCM biosensors and the study of biomolecular interactions.

In situ molecular characterization of endoneurial microvessels that form the blood-nerve barrier in normal human adult peripheral nerves.

The blood-nerve barrier (BNB) formed by tight junction-forming endoneurial microvessels located in the innermost compartment of peripheral nerves, and the perineurium serve to maintain the internal microenvironment required for normal signal transduction. The specific molecular components that define the normal adult human BNB are not fully known. Guided by data derived from the adult human BNB transcriptome, we evaluated the in situ expression of 25 junctional complex, transporter, cell membrane, and cytoskeletal proteins in four histologically normal adult sural nerves by indirect fluorescent immunohistochemistry to determine proteins specifically expressed by restrictive endoneurial microvascular endothelium. Using Ulex Europaeus Agglutinin-1 expression to detect endothelial cells, we ascertained that the selected proteins were uniformly expressed in ≥90% of endoneurial microvessels. P-glycoprotein (also known as adenosine triphosphate-binding cassette subfamily B member 1) and solute carrier family 1 member 1 demonstrated restricted expression by endoneurial endothelium only, with classic tight junction protein claudin-5 also expressed on fenestrated epineurial macrovessels, and vascular-specific adherens junction protein cadherin-5 also expressed by the perineurium. The expression profiles of the selected proteins provide significant insight into the molecular composition of normal adult peripheral nerves. Further work is required to elucidate the human adult BNB molecular signature in order to better understand its development and devise strategies to restore function in peripheral neuropathies.

Choriocapillaris Degeneration in Geographic Atrophy

Early age-related macular degeneration (AMD) is characterized by degeneration of the choriocapillaris, the vascular supply of retinal photoreceptor cells. We assessed vascular loss during disease progression in the choriocapillaris and larger vessels in the deeper choroid. Human donor maculae from controls (n=99), early AMD (n=35), or clinically diagnosed with geographic atrophy (GA; n=9, collected from outside the zone of retinal pigment epithelium degeneration) were evaluated using Ulex europaeus agglutinin-I labeling to discriminate between vessels with intact endothelial cells and ghost vessels. Morphometric analyses of choriocapillaris density (cross-sectional area of capillary lumens divided by length) and of vascular lumen/stroma ratio in the outer choroid were performed. Choriocapillaris loss was observed in early AMD (Bonferroni-corrected P =0.024) with greater loss in GA (Bonferroni-corrected P < 10-9), even in areas of intact retinal pigment epithelium. In contrast, changes in lumen/stroma ratio in the outer choroid were not found to differ between controls and AMD or GA eyes (P>0.05), suggesting choriocapillaris changes are more prevalent in AMD than those in the outer choroid. In addition, vascular endothelial growth factor-A levels were negatively correlated with choriocapillaris vascular density. These findings support the concept that choroidal vascular degeneration, predominantly in the microvasculature, contributes to dry AMD progression. Addressing capillary loss in AMD remains an important translational target.

Adult human pancreatic acinar cells dedifferentiate into an embryonic progenitor-like state in 3D suspension culture

Human pancreatic exocrine cells were cultured in 3D suspension and formed pancreatospheres composed of acinar-derived and duct-like cells. We investigated, up to 6 days, the fate of human pancreatic acinar cells using fluorescein-conjugated Ulex Europaeus Agglutinin 1 lectin, a previously published acinar-specific non-genetic lineage tracing strategy. At day 4, fluorescence-activated cell sort for the intracellularly incorporated FITC-conjugated UEA1 lectin and the duct-specific CA19.9 surface marker, distinguished acinar-derived cells (UEA1+CA19.9−) from duct-like cells (UEA1−CA19.9+) and acinar-to-duct-like transdifferentiated cells (UEA1+CA19.9+). mRNA expression analysis of the acinar-derived (UEA1+CA19.9−) and duct-like (UEA1-CA19.9+) cell fractions with concomitant immunocytochemical analysis of the pancreatospheres revealed acquisition of an embryonic signature in the UEA1+CA19.9− acinar-derived cells characterized by de novo expression of SOX9 and CD142, robust expression of PDX1 and surface expression of GP2. The colocalisation of CD142, a multipotent pancreatic progenitor surface marker, PDX1, SOX9 and GP2 is reminiscent of a cellular state present during human embryonic development. Addition of TGF-beta signalling inhibitor Alk5iII, induced a 28-fold increased KI67-labeling in pancreatospheres, more pronounced in the CD142+GP2+ acinar-derived cells. These findings with human cells underscore the remarkable plasticity of pancreatic exocrine acinar cells, previously described in rodents, and could find applications in the field of regenerative medicine.

Vascular endothelial growth factor/polylactide-polyethyleneglycol-polylactic acid copolymer/basic fibroblast growth factor mixed microcapsules in promoting angiogenic differentiation of rat bone marrow mesenchymal stem cells in vitro

To observe the effect of vascular endothelial growth factor/polylactide-polyethyleneglycol-polylactic acid copolymer/basic fibroblast growth factor (VEGF/PELA/bFGF) mixed microcapsules in promoting the angiogenic differentiation of rat bone marrow mesenchymal stem cells (BMSCs) in vitro. The BMSCs were isolated by the method of whole bone marrow adherent, and sub-cultured. The passage 3 BMSCs were identified by Wright-Giemsa staining and flow cytometry, and used for subsequent experiments. VEGF/PELA/bFGF (group A), PELA/bFGF (group B), VEGF/PELA (group C), and PELA (group D) microcapsules were prepared. The biodegradable ability and cytotoxicity of PELA microcapsule were determined，and the slow-released ability of VEGF/PELA/bFGF mixed microcapsules was measured. The passage 3 BMSCs were co-cultured with the extracts of groups A, B, C, and D, separately. At 1, 3, 7, 14, and 20 days after being cultured, the morphological changes of induced BMSCs were recorded. At 21 days, the induced BMSCs were tested for DiI-labeled acetylated low density lipoprotein (Dil-ac-LDL) and FITC-labeled ulex europaeus agglutinin I (FITC-UEA-I) uptake ability. The tube-forming ability of the induced cells on Matrigel was also verified. The differences of the vascularize indexes in nodes, master junctions, master segments, and tot.master segments length in 4 groups were summarized and analyzed. The isolated and cultured cells were identified as BMSCs. The degradation time of PELA was more than 20 days. There was no significant effect on cell viability under co-culture conditions. At 20 days, the cumulative release of VEGF in the mixed microcapsules exceeded 95%, and the quantity of bFGF exceeded 80%. The morphology of cells in groups A, B, and C were changed. The cells in groups A and B showed the typical change of cobble-stone morphology. The numbers of double fluorescent labeled cells observed by fluorescence microscope were the most in group A, and decreases from group B and group C, with the lowest in group D. The cells in groups A and B formed a grid-like structure on Matrigel. Quantitative analysis showed that the differences in the number of nodes, master junctions, master segments, and tot.master segments length between groups A, B and groups C, D were significant ( P<0.05). The number of nodes and the tot.master segments length of group A were more than those of group B ( P<0.05). There was no significant differences in the number of master junctions and master segments between group A and group B ( P>0.05). VEGF/PELA/bFGF mixed microcapsules have significantly ability to promote the angiogenic differentiation of rat BMSCs in vitro.

Effect of Mucine 4 and Fucosyltransferase 1 genetic variants on gut homoeostasis of growing healthy pigs.

Putative genetic markers have been associated with ETEC F4 (Mucine 4 [MUC4]; MUC4GG;CG as susceptible; MUC4CC as resistant) and F18 (Fucosyltransferase 1 [FUT1]; FUT1GG;AG as susceptible; FUT1AA as resistant) resistances respectively. In this study, 71 post-weaning pigs were followed from d0 (35days old) to d42 (77days of age) to investigate the effect of MUC4 or FUT1 genotypes on the mid-jejunal microbiota composition, pigs expression of genes related to inflammation (IL8, GPX2, REG3G, TFF3, CCL20 and LBPI) and glycomic binding pattern profile (Ulex europaeus agglutinin I [UEA] fucose-binding lectin and peanut agglutinin [PNA] galactose-specific), and on blood plasma targeted metabolomics profile, faecal score and performance parameters of growing healthy pigs. The MUC4 and FUT1 resistant genotypes improved the pigs' growth performance and had firmed faecal score susceptible genotypes in d0-d21 period. Pigs with MUC4GG genotype had a higher jejunal expression of genes relate to immune function (CCL20 and REG3G) than MUC4CG and MUC4CC pigs (p<0.05). MUC4CG pigs had higher expression of TFF3 (implicated in mucosal integrity) than MUC4GG and MUC4CC (p<0.05). FUT1 influenced the alpha- and beta-jejunal microbial indices. The FUT1AA group had a higher number of operational taxonomic units (OTUs) belonging to Lactobacillus genus, while FUT1GG group had a higher number of OTUs belonging to Veillonella genus. MUC4CC pigs had lower scores for UEA on brush borders and goblet cells in villi than MUC4GG (p<0.05). FUT1AA pigs had lower UEA positivity and higher PNA positivity on brush borders and goblet cells than FUT1AG and FUT1GG (p<0.05). Both FUT1 and MUC4 influenced the metabolic profile of healthy pigs. Results highlight the role of MUC4 and FUT1 on pig intestinal homoeostasis and improved the knowledge regarding the potential interaction between host genetics, gut microbiota composition and host metabolism in a healthy status.

An Improved Method for Cell Type-Selective Glycomic Analysis of Tissue Sections Assisted by Fluorescence Laser Microdissection.

Lectin microarray (LMA) is a highly sensitive technology used to obtain the global glycomic profiles of endogenous glycoproteins in biological samples including formalin-fixed paraffin-embedded tissue sections. Here, we describe an effective method for cell type-selective glycomic profiling of tissue fragments collected by laser microdissection (LMD) under fluorescent histochemical visualization. We optimized each step of histochemical staining and confirmed the reliability and validity of glycomic profiling. Using the optimized procedure, glycomic profiles were obtained with 0.5 mm2 of stained thymic sections (5-μm-thick) from 8-week-old C57BL/6J male mice. The glycomic profiles of Ulex europaeus agglutinin-I (UEA-I)-stained medullary regions showed higher UEA-I signals than those of the morphologically determined medulla regions, indicating the utility of this method for UEA-I(+) cell-selective analysis. To further evaluate this method, tissue fragments was serially collected from stained and unstained areas of medullary epithelial cell probes (UEA-I and anti-cytokeratin 5 antibody) and a cortex-staining probe (peanut agglutinin). The medullary regions assigned by the three probes showed significantly different glycomic profiles, highlighting the difference in subpopulation recognition among the three probes, which was consistent with previous reports. In conclusion, our fluorescence LMD-LMA method enabled cell type-selective tissue glycomic analysis of pathological specimens and animal models, especially for glyco-biomarker discovery.

Neu-medullocytes, sialidase-positive B cells in the thymus, express autoimmune regulator (AIRE)

Neu-medullocytes, which were previously identified and named by our group, are sialidase (neuraminidase)-positive B cells that express immunoglobulin and Mac-1 in the mouse thymus. Recently, B cells that migrated into the thymus were reported to express autoimmune regulator (AIRE) and to contribute to self-tolerance. We sought to determine whether Neu-medullocytes also express AIRE. We obtained positive results by triple staining Neu-medullocytes for in situ sialidase activity, anti-AIRE, and either anti-IgG or anti-IgM antibodies and observing the staining with confocal microscopy. Additional molecules including CD5, IgM, major histocompatibility complex (MHC) Class II, and neuraminidase 1 (NEU1) were found in sialidase-positive cells independently. The real-time PCR results suggest that the primary sialidase in AIRE-positive cells is neuraminidase 2 (NEU2). Furthermore, some of the AIRE-positive medullary thymic epithelial cells also clearly showed sialidase activity when a triple staining of sialidase activity, anti-AIRE, and Ulex europaeus agglutinin-1 (UEA-1) was performed. Neu-medullocytes may present Aire-dependent antigens for negative selection. We discuss the negative selection steps in consideration of sialidases and sialic acids.

Lectins at Interfaces-An Atomic Force Microscopy and Multi-Parameter-Surface Plasmon Resonance Study.

Lectins are a diverse class of carbohydrate binding proteins with pivotal roles in cell communication and signaling in many (patho)physiologic processes in the human body, making them promising targets in drug development, for instance, in cancer or infectious diseases. Other applications of lectins employ their ability to recognize specific glycan epitopes in biosensors and glycan microarrays. While a lot of research has focused on lectin interaction with specific carbohydrates, the interaction potential of lectins with different types of surfaces has not been addressed extensively. Here, we screen the interaction of two specific plant lectins, Concanavalin A and Ulex Europaeus Agglutinin-I with different nanoscopic thin films. As a control, the same experiments were performed with Bovine Serum Albumin, a widely used marker for non-specific protein adsorption. In order to test the preferred type of interaction during adsorption, hydrophobic, hydrophilic and charged polymer films were explored, such as polystyrene, cellulose, N,-N,-N-trimethylchitosan chloride and gold, and characterized in terms of wettability, surface free energy, zeta potential and morphology. Atomic force microscopy images of surfaces after protein adsorption correlated very well with the observed mass of adsorbed protein. Surface plasmon resonance spectroscopy studies revealed low adsorbed amounts and slow kinetics for all of the investigated proteins for hydrophilic surfaces, making those resistant to non-specific interactions. As a consequence, they may serve as favorable supports for biosensors, since the use of blocking agents is not necessary.

Fabrication of Carbohydrate Chips Based on Polydopamine for Real-Time Determination of Carbohydrate⁻Lectin Interactions by QCM Biosensor.

A novel approach for preparing carbohydrate chips based on polydopamine (PDA) surface to study carbohydrate–lectin interactions by quartz crystal microbalance (QCM) biosensor instrument has been developed. The amino-carbohydrates were immobilized on PDA-coated quartz crystals via Schiff base reaction and/or Michael addition reaction. The resulting carbohydrate-chips were applied to QCM biosensor instrument with flow-through system for real-time detection of lectin–carbohydrate interactions. A series of plant lectins, including wheat germ agglutinin (WGA), concanavalin A (Con A), Ulex europaeus agglutinin I (UEA-I), soybean agglutinin (SBA), and peanut agglutinin (PNA), were evaluated for the binding to different kinds of carbohydrate chips. Clearly, the results show that the predicted lectin selectively binds to the carbohydrates, which demonstrates the applicability of the approach. Furthermore, the kinetics of the interactions between Con A and mannose, WGA and N-Acetylglucosamine were studied, respectively. This study provides an efficient approach to preparing carbohydrate chips based on PDA for the lectin–carbohydrate interactions study.

The content of immunomodulatory glycoepitopes in seminal plasma glycoproteins of fertile and infertile men.

According to a concept of fetoembryonic defence, protein-carbohydrate interaction may be involved in the regulation of maternal immunity that prevents rejection of allograft spermatozoa, embryo and fetus. In the present study we focussed on the evaluation of the expression of glycoepitopes that may be of crucial importance in this process: LewisY (LeY) and LewisX (LeX) as well as terminal sialylation. Polyacrylamide gel electrophoresis with sodium dodecyl sulphate was used to separate seminal plasma samples of fertile (n=10) and infertile (n=103) men; these were then probed with lectins specific to fucose (Lotus tetragonolobus agglutinin and Ulex europaeus agglutinin) and sialic acid (Sambucus nigra agglutinin and Maackia amurensis agglutinin). Differential expression of α2,3-bound sialic acid was found in six out of seven analysed bands, whereas differences in the other analysed glycoepitopes were found in fewer numbers of bands. Mass spectrometry analysis focussed on the identification of proteins carrying glycans with immunomodulatory epitopes, including fibronectin, lactoferrin, clusterin, zinc-α2-glycoprotein, prostate acid phosphatase and prostate-specific antigen; these should be submitted to further detailed analysis.

FoxN1-dependent thymic epithelial cells promote T-cell leukemia development.

T-cell acute lymphoblastic leukemia (T-ALL) and T-lymphoblastic lymphomas (T-LBL) are aggressive malignancies of thymocytes. The role of thymic microenvironmental cells and stromal factors in thymocyte malignant transformation and T-ALL development remains little explored. Here, using the TEL-JAK2 transgenic (TJ2-Tg) mouse model of T-ALL/LBL, which is driven by constitutive JAK/STAT signaling and characterized by the acquisition of Notch1 mutations, we sought to identify stromal cell alterations associated with thymic leukemogenesis. Immunofluorescence analyses showed that thymic lymphomas presented epithelial areas characterized by keratin (Krt) 5 and Krt8 expression, adjacently to epithelial-free areas negative for Krt expression. Both areas contained abundant laminin (extracellular matrix) and ER-TR7+ (fibroblasts) CD31+ (endothelial) and CD11c+ (dendritic) cells. Besides Krt5, Krt-positive areas harbored medullary thymic epithelial cells (TECs) labeled by Ulex europaeus agglutinin-1. By performing flow cytometry and RNA sequencing analyses of thymic lymphomas, we observed an enrichment in medullary TEC markers in detriment of cortical TEC markers. To assess whether TECs are important for T-ALL/LBL development, we generated TJ2-Tg mice heterozygous for the FoxN1 transcription factor nude null mutation (Foxn1+/nu). Strikingly, in TJ2-Tg;Foxn1+/nu compound mice, both emergence of malignant cells in preleukemic thymi and overt T-ALL onset were significantly delayed. Moreover, in transplantation assays, leukemic cell expansion within the thymus of recipient Foxn1+/nu mice was reduced as compared with control littermates. Since thymopoesis is largely normal in Foxn1+/nu mice, these results indicate that FoxN1 haploinsufficiency in TECs has a more profound impact in thymic leukemogenesis.

Morphological and immunohistochemical study of the rabbit vomeronasal organ.

The characterization of the rabbit mammary pheromone, which is sensed by the main olfactory system, has made this species a unique model for the study of pheromonal communication in mammals. This discovery has brought attention to the global understanding of chemosensory communication in this species. Chemocommunication is mediated by two distinct organs located in the nasal cavity, the main olfactory epithelium and the vomeronasal organ (VNO). However, there is a lack of knowledge about the vomeronasal system in rabbits. To understand the role of this system, an exhaustive anatomical and histological study of the rabbit VNO was performed. The rabbit VNO was studied macroscopically by light microscopy, and by histochemical and immunohistochemical techniques. We employed specific histological staining techniques (periodic acid-Schiff, Alcian blue, Gallego's trichrome), confocal autofluorescence, histochemical labelling with the lectin Ulex europaeus agglutinin (UEA-I), and immunohistochemical studies of the expression of the Gαi2 and Gαo proteins and olfactory marker protein. The opening of the vomeronasal duct into the nasal cavity and its indirect communication with the oral cavity through a functional nasopalatine duct was demonstrated by classical dissection and microdissection. In a series of transverse histological sections, special attention was paid to the general distribution of the various soft-tissue components of this organ (duct, glands, connective tissue, blood vessels and nerves) and to the nature of the capsule of the organ. Among the main morphological features that distinguish the rabbit VNO, the presence of a double envelope, which is bony externally and cartilaginous internally, and highly developed venous sinuses stand out. This observation indicates the crucial role played in this species by the pumping mechanism that introduces chemical signals into the vomeronasal duct. The functional properties of the organ are also confirmed by the presence of a well-developed neuroepithelium and profuse glandular tissue that is positive for neutral mucopolysaccharides. The role of glycoconjugates was assessed by the identification of the α1-2 fucose glycan system in the neuroepithelium of the VNO employing UEA-I lectin. The pattern of labelling, which was concentrated around the commissures of the sensory epithelium and more diffuse in the central segments, is different from that found in most mammals studied. According to the expression of G-proteins, two pathways have been described in the VNOs of mammals: neuroreceptor cells expressing the Gαi2 protein (associated with vomeronasal receptor type 1); and cells expressing Gαo (associated with vomeronasal receptor type 2). The latter pathway is absent in most mammals studied. The expression of both G-protein families in the rabbit VNO places Lagomorpha together with rodents and insectivores in a small group of mammals belonging to the two-path model. These findings support the notion that the rabbit possesses a highly developed VNO, with many specific morphological features, which highlights the significance of chemocommunication in this species.

Spleen immunotoxicities induced by intra-testicular injection of magnetic nanoparticles and the role of Echinacea purpurea extract: a histological and immunohistochemical study

ABSTRACTRecently, magnetic nanoparticles (MNP) have been used in several medical applications. High accumulations of MNP can be found in spleen and liver after different injection routes. Immunotoxicities produced by MNP in spleen, a lymphoid organ, need more investigations. This study investigated biodistribution, histological and immune responses to MNP in spleen after intra-testicular injection. Echinacea purpurea (EP) extract has immune stimulation and anti-oxidant properties. Investigation was done on the role of EP reduction of immunotoxicities produced by MNP in the spleen. After intra-testicular injection, MNP reached spleen parenchyma through blood circulation. Long term EP administration induced mild iron accumulation resulting from red blood cell (RBC) lysis. The opposite was observed when MNP treatment during EP administration significantly reduced lysed RBC iron in the spleen as well as MNP iron. It is possible the chelating properties of EP molecules possibly explained the presence of inert iron complexes from RBC in the spleen after EP administration only. The presence of MNP induced severe splenic cell degeneration and hemorrhage. The treatment with MNP combined with EP administration reduced these abnormalities. Additionally, anti-oxidant properties of EP and presence of MNP iron in the spleen had a role in activating macrophages to recycle all splenic iron. However, F4/80+, immunoglobulin A (IgA)+ and Ulex europaeus agglutinin I (UEAI)+ expressions significantly decreased when MNP were injected without EP. Otherwise, long term EP treatment maintained normal function of these cells.