Agglutinin Binding Research Articles

Role of Wisteria floribunda agglutinin binding glycans in carcinogenesis and metastasis of cholangiocarcinoma.

Aberrant glycosylation is an important factor in facilitating tumor progression and therapeutic resistance. In this study, using Wisteria floribunda agglutinin (WFA), we examined the expression of WFA-binding glycans (WFAG) in cholangiocarcinoma (CCA). The results showed that WFAG was highly detected in precancerous and cancerous lesions of human CCA tissues, although it was rarely detected in normal bile ducts. The positive signal of WFAG in the cancerous lesion accounted for 96.2% (50/52) of the cases. Overexpression of WFAG was significantly associated with lymph node and distant metastasis (P < 0.05). The study using the CCA hamster model showed that WFAG is elevated in preneoplastic and neoplastic bile ducts as early as 1month after being infected with liver fluke and exposed to N-nitrosodimethylamine. Functional analysis was performed to reveal the role of WFAG in CCA. The CCA cell lines KKU-213A and KKU-213B were treated with WFA, followed by migration assay. Our data suggested that WFAG facilitates the migration of CCA cells via the activation of the Akt and ERK signaling pathways. In conclusion, we have demonstrated the association of WFAG with carcinogenesis and metastasis of CCA, suggesting its potential as a target for the treatment of the disease.

Surface plasmon resonance microscopy identifies glycan heterogeneity in pancreatic cancer cells that influences mucin-4 binding interactions.

Membrane proteins are the main targets of therapeutic drugs and most of them are glycosylated. Glycans play pivotal roles in several biological processes, and glycosylation changes are a well-established hallmark of several types of cancer, including pancreatic cancer, that contribute to tumor growth. Mucin-4 (MUC-4) is a membrane glycoprotein which is associated with pancreatic cancer and metastasis, and it has been targeted as a promising vaccine candidate. In this study, Surface Plasmon Resonance Microscopy (SPRM) was implemented to study complex influences of the native N-glycan cellular environment on binding interactions to the MUC-4 receptor as this is currently the only commercially available label-free technique with high enough sensitivity and resolution to measure binding kinetics and heterogeneity on single cells. Such unique capability enables for a more accurate understanding of the "true" binding interactions on human cancer cells without disrupting the native environment of the target MUC-4 receptor. Removal of N-linked glycans in pancreatic cancer cells using PNGase F exposed heterogeneity in Concanavalin (Con A) binding by revealing three new binding populations with higher affinities than the glycosylated control cells. Anti-MUC-4 binding interactions of enzymatically N-linked deglycosylated pancreatic cancer cells produced a 25x faster association and 37x higher affinity relative to the glycosylated control cells. Lastly, four interaction modes were observed for Helix Pomatia Agglutinin (HPA) binding to the glycosylated control cells, but shifted and increased in activity upon removal of N-linked glycans. These results identified predominant interaction modes of glycan and MUC-4 in pancreatic cancer cells, the kinetics of their binding interactions were quantified, and the influence of N-linked glycans in MUC-4 binding interactions was revealed.

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 &amp;gt;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&amp;gt;65 yo) and healthy controls (n=100≤65 yo and n=28&amp;gt;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 &amp;gt;65 yo; and ITP patients ≤65 vs &amp;gt;65 yo were performed with 2 way ANOVA and p&amp;lt;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 &amp;gt; 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)&amp;gt; 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 &amp;gt;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.

Altered profile of glycosylated proteins in serum samples obtained from patients with Hashimoto's thyroiditis following depletion of highly abundant proteins.

Hashimoto's thyroiditis (HT) is one of the most common autoimmune disorders; however, its underlying pathological mechanisms remain unclear. Although aberrant glycosylation has been implicated in the N-glycome of immunoglobulin G (IgG), changes in serum proteins have not been comprehensively characterized. This study aimed to investigate glycosylation profiles in serum samples depleted of highly abundant proteins from patients with HT and propose the potential functions of glycoproteins for further studies on the pathological mechanisms of HT. A lectin microarray containing 70 lectins was used to detect and analyze glycosylation of serum proteins using serum samples (N=27 HT; N=26 healthy control [HC]) depleted of abundant proteins. Significant differences in glycosylation status between HT patients and the HC group were verified using lectin blot analysis. A lectin-based pull-down assay combined with mass spectrometry was used to investigate potential glycoproteins combined with differentially present lectins, and an enzyme-linked immunosorbent assay (ELISA) was used to identify the expression of targeted glycoproteins in 131 patients with papillary thyroid carcinoma (PTC), 131 patients with benign thyroid nodules (BTN) patients, 130 patients with HT, and 128 HCs. Compared with the HC group, the majority of the lectin binding signals in HT group were weakened, while the Vicia villosa agglutinin (VVA) binding signal was increased. The difference in VVA binding signals verified by lectin blotting was consistent with the results of the lectin microarray. A total of 113 potential VVA-binding glycoproteins were identified by mass spectrometry and classified by gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) analyses. Using ELISA, we confirmed that lactoferrin (LTF) and mannan-binding lectin-associated serine protease 1 (MASP-1) levels were elevated in the serum of patients with HT and PTC. Following depletion of abundant proteins, remaining serum proteins in HT patients exhibited lower glycosylation levels than those observed in HCs. An increased level of potential VVA-binding glycoproteins may play an important role in HT development. LTF and MASP-1 expression was significantly higher in the serum of HT and PTC patients, providing novel insight into HT and PTC.

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.

Therapeutic Fractional Doses of Ionizing Radiation Promote Epithelial-Mesenchymal Transition, Enhanced Invasiveness, and Altered Glycosylation in MCF-7 Breast Cancer Cells.

The clinical outcome of radiation therapy is restricted due to the acquired radio-resistance of a subpopulation of tumour cells that may cause tumour relapse and distant metastasis. While the effects of ionizing radiation (IR) such as DNA damage and cell stress are well-documented, the potential role of IR in inducing invasive potential in cancer cells has not been broadly studied, therefore we aimed to investigate it in this study. MCF-7 cells irradiated with 0 Gy (control) or 2 Gy X-ray therapeutic doses of IR were assessed for cell viability, percentage of apoptotic cells, and reactive oxygen species (ROS) levels, DNA fragmentation, Matrigel invasion, assessment of epithelial-mesenchymal transition (EMT) markers and Helix pomatia agglutinin (HPA) binding at 30 min, 4- or 24-h post-IR. Reduction in cell viability, increase in apoptotic cells, ROS positive cells, and DNA fragmentation were observed, while functional invasiveness and EMT were exacerbated together with altered glycosylation in MCF-7 cells irradiated with 2 Gy X-ray compared to control cells. These findings indicate that despite the detrimental effects of 2 Gy X-ray IR on MCF-7 cells, a subpopulation of cells may have gained increased invasive potential. The exacerbated invasive potential may be attributed to enhanced EMT and altered glycosylation. Moreover, deregulation of transforming growth factor-beta (TGF-β) following IR may be one of the elements responsible for these changes, as it lies in the intersection of these invasion-promoting cell processes.

Evidence that Nectin-3 is the soybean agglutinin binding protein on rat corneal endothelium cell surfaces

The means by which the lectin soybean agglutinin (SBA) binds to the corneal endothelium cell surface following explantation into organ culture was investigated using Sprague-Dawley rats. SBA binding does not occur in freshly isolated and fixed rat corneal endothelium. However, after 48 h in organ culture, SBA binding occurs in a punctate pattern that clearly outlines all endothelial cells of the tissue monolayer. To determine what cell surface component was responsible for this binding, a series of experiments were employed that focused on the possibility that SBA bound to a nectin molecule(s). To this extent we performed a series of immunocytochemical localizations using antibodies against either nectin-2, nectin-3 or nectin-4. Of these, only nectin-3 bound to the endothelium in a manner that mimicked SBA binding. To further verify that nectin-3 bound SBA, displacement experiments employing non-labeled SBA were undertaken. Following a 48 h organ culture, tissues were fixed and incubated with SBA followed by exposure to nectin-3 antibody. No subsequent immunofluorescence could be detected, indicating that anti-nectin-3 binding was prevented. Likewise, when organ-cultured tissues were fixed and incubated in anti-nectin-3 antibody, followed by SBA exposure, no SBA binding could be detected. These results suggest that stresses accompanying explantation of the tissue into organ culture promote the appearance of nectin-3 around the cell periphery. The emergence of nectin-3 along the peripheral endothelial cell membrane in organ culture may imply a necessary role for this molecule in maintaining monolayer integrity and barrier function during either a pathologic condition, wound repair, or in organ storage.

Calculating Half Maximal Inhibitory Concentration (IC50) Values from Glycomics Microarray Data Using GraphPad Prism.

Half maximal inhibitory concentration (IC50) is a measurement often used to compare the efficiency of various carbohydrates and their derivatives for inhibition oflectin binding to particular ligands. IC50 values can be calculated using experimental data from various platforms including enzyme-linked immunosorbent assay- (ELISA-)type microtiter plate assays, isothermal titration calorimetry (ITC), or glycan microarrays. In this chapter, we describe methods to fluorescently label a lectin, to carry out a lectin binding inhibition experiment on glycan microarrays, and to calculate the IC50 value of a bindinginhibitory molecule using GraphPad Prism software. In the example used to illustrate the method in this chapter, IC50 calculation is demonstrated for inhibition of Maackia amurensis agglutinin (MAA) binding to 3'sialyl-N-acetyllactosamine (3SLN) using free lactose.

Pathogenic mechanisms contributing to thrombocytopenia in patients with systemic lupus erythematosus

Summary Systemic lupus erythematosus (SLE) is an autoimmune condition developing thrombocytopenia in about 10–15% of cases, however, mechanisms leading to low platelet count were not deeply investigated in this illness. Here we studied possible causes of thrombocytopenia, including different mechanisms of platelet clearance and impairment in platelet production. Twenty-five SLE patients with and without thrombocytopenia were included. Platelet apoptosis, assessed by measurement of loss of mitochondrial membrane potential, active caspase 3 and phosphatidylserine exposure, was found to increase in thrombocytopenic patients. Plasma from 67% SLE patients (thrombocytopenic and non-thrombocytopenic) induced loss of sialic acid (Ricinus communis agglutinin I and/or Peanut agglutinin binding) from normal platelet glycoproteins. Concerning platelet production, SLE plasma increased megakaryopoiesis (evaluated using normal human cord blood CD34+ hematopoietic progenitors), but inhibited thrombopoiesis (proplatelet count). Anti-platelet autoantibody depletion from SLE plasma reverted this inhibition. Overall, abnormalities were more frequently observed in thrombocytopenic than non-thrombocytopenic SLE patients and in those with active disease (SLEDAI≥5). In conclusion, platelet clearance due to apoptosis and desialylation, and impaired platelet production mainly due to inhibition of thrombopoiesis, could be relevant mechanisms leading to thrombocytopenia in SLE. These findings could provide a rational basis for the choice of proper therapies to correct platelet counts in these patients.

Spatiotemporal regulation of galectin-1-induced T-cell death in lamina propria from Crohn's disease and ulcerative colitis patients.

Inflammatory bowel disease (IBD), including Crohn's disease (CD) and ulcerative colitis (UC), is characterized by chronic, relapsing intestinal inflammation. Galectin-1 (Gal-1) is an endogenous lectin with key pro-resolving roles, including induction of T-cell apoptosis and secretion of immunosuppressive cytokines. Despite considerable progress, the relevance of Gal-1-induced T-cell death in inflamed tissue from human IBD patients has not been ascertained. Intestinal biopsies and surgical specimens from control patients (n = 52) and patients with active or inactive IBD (n = 97) were studied. Gal-1 expression was studied by RT-qPCR, immunoblotting, ELISA and immunohistochemistry. Gal-1-specific ligands and Gal-1-induced apoptosis of lamina propria (LP) T-cells were determined by TUNEL and flow cytometry. We found a transient expression of asialo core 1-O-glycans in LP T-cells from inflamed areas (p < 0.05) as revealed by flow cytometry using peanut agglutinin (PNA) binding and assessing dysregulation of the core-2 β 1-6-N-acetylglucosaminyltransferase 1 (C2GNT1), an enzyme responsible for elongation of core 2 O-glycans. Consequently, Gal-1 binding was attenuated in CD3+CD4+ and CD3+CD8+ LP T-cells isolated from inflamed sites (p < 0.05). Incubation with recombinant Gal-1 induced apoptosis of LP CD3+ T-cells isolated from control subjects and non-inflamed areas of IBD patients (p < 0.05), but not from inflamed areas. In conclusion, our findings showed that transient regulation of the O-glycan profile during inflammation modulates Gal-1 binding and LP T-cell survival in IBD patients.

Specific lectin binding sites during in vitro capacitation and acrosome reaction in boar spermatozoa

Sperm glycocalyx and plasma membrane undergo outstanding modifications during fertilization. However, it is unclear how in vitro capacitation time and acrosome reaction affect the specific location of boar sperm glycoconjugates. This study aimed to identify lectin binding patterns and to describe the sequential changes during different in vitro capacitation times (1 and 4 h) and acrosome reaction in boar spermatozoa. With Aleuria aurantia agglutinin (AAA), most uncapacitated cells were labelled in the postacrosomal region. Nevertheless, after 1 h of in vitro capacitation and the acrosome reaction, most AAA binding sites were in the acrosomal region. With Concanavalin A (ConA), most sperm were labeled in the postacrosomal region before and after capacitation. After the acrosome reaction induction, this pattern changed to a highly stained acrosomal and postacrosomal regions. Peanut agglutinin (PNA) binding sites were in the acrosomal region in uncapacitated and capacitated sperm. In acrosome reacted sperm after 4h capacitation, the most frequent pattern showed remaining positive labeling in the central area of the head. With Pisum sativum agglutinin (PSA), most uncapacitated cells showed a postacrosomal region staining. Nevertheless, faint stained all over the head and highly acrosomal region labelling was observed in the major part of capacitated and acrosome reacted sperm respectively. With Wheat germ agglutinin (WGA), the most representative pattern in uncapacitated, capacitated and acrosome reacted sperm was labelled in the acrosomal region. Regarding capacitation time, the most significant changes in the most representative pattern were observed in acrosome reacted spermatozoa after 4 h of in vitro capacitation. Highlights Lectins allow detailed descriptions of eight different populations in boar sperm. Changes in lectin binding patterns during capacitation are time dependent. Lectin binding patterns significantly change after 4 h of in vitro capacitation.

MON-LB85 Aberrant Glycan Expression Changes With the Phenotype of Follicular Thyroid Tumours

Glycosylation is the most common post‐translational modification of proteins and plays an important role in cell communication, interaction and adhesion. Aberration of glycosylation is a hallmark of cancer cells and plays an important role in oncogenesis and cancer progression including metastasis1. One of the markers of aberrant glycosylation (O-linked) is the binding of the lectin Helix pomatia agglutinin (HPA), and this has been shown in a wide range of human cancers2, especially in tumours with a more aggressive phenotype. To study the alteration in cellular glycosylation, detected by lectin Helix pomatia agglutinin (HPA) binding in various phenotypes of follicular thyroid tumours, ranging from adenoma to carcinoma and metastatic follicular thyroid cancers. Lectin histochemistry was performed on archival paraffin wax‐embedded specimens of 6 follicular adenomas, 10 minimally invasive follicular carcinomas, 13 widely invasive follicular cancers and 4 metastatic follicular thyroid cancers. For positive controls, sections of rat kidney were used, which shows strong and characteristic HPA labelling were included in each labelling experiment. For negative controls, the lectin was omitted and the specificity of binding was confirmed by incubating the sections with HPA in the presence of 0·1‐mol/l GalNAc. Sections were scored as positive when 5% or more of the cancer cells labelled positive and scored as negative when less than 5% labelled positive for HPA binding. Assessments of HPA binding were performed by two observers who were blinded to the identity of the sample, and their results were compared. Positive labelling was seen in 20% of adenomas, 60% of minimally invasive carcinomas, 77% of widely invasive carcinomas and 100% of metastatic carcinomas (p<0.05). In the minimally invasive carcinoma group, all tumours that showed vascular invasion showed positive HPA labelling, whereas only 20% of patients with capsular invasion showed positivity. We speculate that as the phenotype of the thyroid tumours changes from benign to the malignant phenotype, the glycan expressions change. However, the underlying molecular mechanisms leading to this change needs to be further investigated.

Design of Thermoresponsive Elastin-Like Glycopolypeptides for Selective Lectin Binding and Sorting.

Selective lectin binding and sorting was achieved using thermosensitive glycoconjugates derived from recombinant elastin-like polypeptides (ELPs) in simple centrifugation-precipitation assays. A recombinant ELP, (VPGXG)40, containing periodically spaced methionine residues was used to enable chemoselective postsynthetic modification via thioether alkylation using alkyne functional epoxide derivatives. The resulting sulfonium groups were selectively demethylated to give alkyne functionalized homocysteine residues, which were then reacted with azido-functionalized monosaccharides to obtain ELP glycoconjugates with periodic saccharide functionality. These modifications were also found to allow modulation of ELP temperature dependent water solubility. The multivalent ELP glycoconjugates were evaluated for specific recognition, binding and separation of the lectin Ricinus communis agglutinin (RCA120) from a complex protein mixture. RCA120 and ELP glycoconjugate interactions were evaluated using laser scanning confocal microscopy and dynamic light scattering. Due to the thermoresponsive nature of the ELP glycoconjugates, it was found that heating a mixture of galactose-functionalized ELP and RCA120 in complex media selectively yielded a phase separated pellet of ELP-RCA120 complexes. Based on these results, ELP glycoconjugates show promise as designer biopolymers for selective protein binding and sorting.

150 α6 Integrin for evaluating sperm quality in bulls with different capacities of invitro embryo production

The α6 integrin, an adhesion molecule, is expressed on bovine sperm, but major questions about the role of integrins in sperm-oocyte fusion remain unsolved. In this work, we show the results of characterisation of sperm α6 integrin from 4 bulls with different capacities of invitro embryo production using flow cytometric analysis. The bull capacities judged by the rate of blastocyst formation after invitro embryo production with semen from 5 ejaculates per animal were 44.3, 17.1, 13.2, and 15.0% for bulls 1, 2, 3, and 4, respectively (P&amp;lt;0.05). For flow cytometric analysis, surface expression of α6 integrin was evaluated using a fluorescence-activated cell sorter (FACScan, Coulter Electronics) using a 520-nm excitation from an argon laser at 150 mW for excitation. Frozen-thawed sperm were centrifuged at 700×g for 10min and washed once in warm phosphate buffered saline (PBS). Briefly, the spermatozoa (5×105 cells per sample) were resuspended in 100μL of PBS containing 1% bovine serum albumin. After washing 3 times, the live cells were incubated at room temperature for 1h with 100μL (1:500) of α6 monoclonal antibody (Chemicon) in PBS (0.1M, pH 7.4) with 1% bovine serum albumin. After washing three times, the cells were incubated at 4°C for 1h in the dark with the fluorescein isothiocyanate-conjugated F(ab)2 fragment of affinity isolated goat anti-mouse antibody (Invitrogen). The cells were washed three times, resuspended in 100μL of PBS, and analysed. For each sample, 10 000 cells were recorded at a flow rate of 200-300 cells s−1 using forward scatter (cell size) and side angle of light scatter (cell density), the first using a logarithmic amplifier and the second using a linear amplifier. The fluorescence data were collected using the logarithmic amplifier. The percentage of positive cells and the mean fluorescence channel on a 1023-channel scale were calculated using Epics Profile II software (Epics II Software). To define the forward and side-scatter regions corresponding to sperm, binding of fluorescein isothiocyanate-conjugated Pisum sativum agglutinin to the acrosome was used for setting the bitmap on the dot plot. Initially, the percentage of spermatozoa stained with α6 antibody was calculated on a per-individual basis. Subsequently, the mean±standard deviation was calculated for each group. The Mann-Whitney U test was used to compare the differences in the expression of α6 between groups. Expression of α6 integrin was higher in bull 1 (control) than in bulls with low capacities of invitro embryo production. The spermatozoa from bull 1 was distributed in a single broad peak with a mean fluorescence intensity of 36.16±4.17%. The increased distance of the fluorescence intensity in bull 1 compared with the weak fluorescence peaks in the others bulls reflects the increased width and distance of the population distribution. In conclusion, α6 integrin may be used as a biomarker to evaluate sperm quality. This study was supported by FAPESP grants 2010/01077-9, 2011/18085-7, and 2016/00976-6.

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.

BAFF, IL-4 and IL-21 separably program germinal center-like phenotype acquisition, BCL6 expression, proliferation and survival of CD40L-activated B cells invitro.

A B cell culture system using BAFF, IL-4 and IL-21 was recently developed that generates B cells with phenotypic and functional characteristics of invivo-generated germinal center (GC) B cells. Here, we observe discrete influences of each exogenous signal on the expansion and differentiation of a CD40L-activated B cell pool. IL-4 was expressly necessary, but neither BAFF nor IL-21 was required for B cell acquisition of the GC B cell phenotypes of peanut agglutinin binding and loss of CD38 and IgD expression. Both IL-4 and IL-21 enhanced cell cycle entry upon initial activation dose-dependently, and did so additively. Importantly, while both cytokines acted in concert to increase overall BCL6 expression amounts, IL-21 exposure uniquely caused a small proportion of cells to attain a higher level of BCL6 expression, reminiscent of invivo GC B cells. In contrast, BAFF supported survival ofa fraction of memory-like B cells in extended cultures after removal of surrogate T cell-help signals. Thus, by separably programming proliferation, survival and GC phenotype acquisition, IL-4, BAFF and IL-21 drive distinct components of activated B cell fate.

Egg autofluorescence and options for detecting peanut agglutinin binding for the identification of Haemonchus contortus eggs in fecal samples

Quantifying eggs from Haemonchus and other trichostrongyle genera in sheep and goat fecal samples is important for evaluating control and treatment strategies for this family of nematodes with divergent pathologies, capabilities for anthelmintic resistance and environmental susceptibilities. Unfortunately, egg morphology among most of the genera do not differ enough to support the accurate identification of these genera with standard microscopic techniques. Several studies have identified specific lectins which bind selectively to sugars located on the egg surfaces for individual genera among the trichostrongyles. To detect lectins binding to these eggs, they must be directly or indirectly bound to fluorophores, and observed with an epi-fluorescence microscope. The binding of multiple lectins to isolated eggs from a fecal sample can be simultaneously detected if fluorophores are used whose excitation and emission spectra do not overlap, and this would enable the development of a fluorescence-based diagnostic test that identifies multiple trichostrongyle genera within each sample. The present study compared the usefulness of different, commercially available detection systems for use in detecting lectin binding to trichostrongyle eggs. Comparisons were made using the detection of PNA binding to H. contortus eggs with the goal of finding three systems with color spectra that do not overlap. These evaluations included both fluorophores directly conjugated to PNA in a one-step incubation protocol and a two-step incubation protocol involving biotinylated PNA and streptavidin conjugated to different fluorophores. Autofluorescence can affect the efficiency of any fluorescence-based detection system, and significant autofluorescence was observed among the unstained H. contortus eggs with the DAPI-type fluorescence filter, but it was significantly lower with the FITC-type filter and was virtually absent with the rhodamine-type filter. This study demonstrated that all the PNA detection methods tested with H. contortus eggs generated fluorescence intensities (FIs) that were significantly above the autofluorescence generated by the eggs among the three different fluorescence filters. Fluorescence intensities from PNA directly conjugated to either the FITC or rhodamine fluorophores were not different, but the lower autofluoresence in the rhodamine-type filter will enable this fluorophore to be detected more efficiently. Use of biotinylated PNA combined with streptavidin-conjugated to synthetic fluorophores (Alexa Fluor 405, 488 and 546) significantly increased FIs over that of the directly conjugated PNA, but there were no significant differences in FIs among these three biotin-avidin conjugation fluorophores. This biotin-avidin system required two incubation steps. Doubling the concentration of PNA also provided increased FI, at least for the biotin-avidin system. Adding an additional amplification step to the biotin-avidin system involving biotinylated anti-streptavidin followed by the streptavidin-Alexa Fluor complex also provided additional fluorescence.

Effect of fluid shear stress on in vitro cultured ureteric bud cells.

Most kidney cells are continuously exposed to fluid shear stress (FSS) from either blood flow or urine flow. Recent studies suggest that changes in FSS could contribute to the function and injury of these kidney cells. However, it is unclear whether FSS influences kidney development when urinary flow starts in the embryonic kidneys. In this study, we evaluated the influence of FSS on in vitro cultured ureteric bud (UB) cells by using a pumpless microfluidic device, which offers the convenience of conducting parallel cell culture experiments while also eliminating the need for cumbersome electronic driven equipment and intricate techniques. We first validated the function of the device by both mathematical model and experimental measurements. UB cells dissected from E15.5 mouse embryonic kidneys were cultured in the pumpless microfluidic device and subjected to FSS in the range of 0.4–0.6 dyn mm−2 for 48 h (dynamic). Control UB cells were similarly cultured in the device and maintained under a no-flow condition (static). We found from our present study that the exposure to FSS for up to 48 h led to an increase in mRNA expression levels of UB tip cell marker genes (Wnt11, Ret, Etv4) with a decrease in stalk cell marker genes (Wnt7b, Tacstd2). In further support of the enrichment of UB tip cell population in response to FSS, we also found that exposure to FSS led to a remarkable reduction in the binding of lectin Dolichos Biflorus Agglutinin. In conclusion, results of our present study show that exposure to FSS led to an enrichment in UB tip cell populations, which could contribute to the development and function of the embryonic kidney when urine flow starts at around embryonic age E15.5 in mouse. Since UB tip cells are known to be the proliferative progenitor cells that contribute to the branching morphogenesis of the collecting system in the kidney, our finding could imply an important link between the FSS from the initiation of urine flow and the development and function of the kidney.

Binding of aberrant glycoproteins recognizable by Helix pomatia agglutinin in adrenal cancers.

BackgroundAberrant glycosylation is a hallmark of cancer cells and plays an important role in oncogenesis and cancer progression including metastasis. This study aimed to assess alteration in cellular glycosylation, detected by lectin Helix pomatia agglutinin (HPA) binding, in adrenal cancers and to determine whether such altered glycosylation has prognostic significance.MethodsHPA binding lectin histochemistry was performed on archival paraffin wax‐embedded specimens of adrenocortical cancers excised from patients attending two tertiary referral centres. Benign tumours were used as controls. Demographic, histological and survival data were collected and compared between patients with HPA‐positive and HPA‐negative tumours.ResultsThirty‐two patients were treated for adrenal cancer between 2000 and 2016; their median age was 49 (range 23–79) years. Fifteen patients had functioning tumours (14 adrenal Cushing's tumours and 1 Conn's tumour). Mean(s.d.) tumour size was 127·71(49·70) mm. None of 10 control tumours expressed HPA‐binding glycoproteins. Invasion was associated with HPA‐binding glycoproteins (P = 0·018). Local recurrence or metastatic disease did not significantly differ between HPA‐positive and HPA‐negative adrenocortical cancers. Overall survival was significantly longer in patients with HPA‐negative tumours (median survival not reached versus 22 months in patients with HPA‐positive tumours; P = 0·002).ConclusionAltered cellular glycosylation detected by lectin HPA is associated with poor survival in patients with adrenocortical cancer.