Ulex Europaeus Agglutinin Research Articles

Targeting the Cryptococcus neoformans var. grubii cell wall using lectins: study of the carbohydrate-binding domain.

Cryptococcus neoformans var. grubii is considered to be the major cause of cryptococcosis in immunosuppressed patients. Understanding cell wall glycoproteins using lectins is of medical interest and can contribute to specific therapy. The aim of this study was to evaluate the carbohydrates on the cell wall of Cryptococcus neoformans var. grubii clinical isolates, using a fluorescein isothiocyanate-lectin binding protocol. Thirty yeast strains stocked in the culture collection were cultivated for 2 days at 30 °C with shaking. Cells were obtained by centrifugation, washed in phosphate-buffered saline, and a suspension of 107 cells/mL was obtained. To determine the binding profile of lectins, concanavalin A (Con A), wheat germ agglutinin (WGA), Ulex europaeus agglutinin I (UEA-I), and peanut agglutinin (PNA) conjugated to fluorescein were used. All the tested clinical isolates of Cryptococcus neoformans var. grubii were intensely stained by WGA, moderately stained by Con A, and weakly stained by PNA and UEA-I. Thus, Cryptococcus can be detected in clinical specimens such as blood and cerebrospinal fluid using the fluorescent lectin WGA, which may be considered as an option for detection in cases of suspected cryptococcosis with low laboratory sensitivity. Future applications may be developed using this basic tool.

Effects of different fixatives on staining patterns of four lectins in cultured microvascular endothelial cells

Fixatives have significant influence on lectin histochemistry of tissue sections; however, their roles in lectin fluorescent staining of cultured cells remain unclear. In this study, using cultured microvascular endothelial cells (MVECs) from rat intestinal mucosa, the effects of seven fixatives on the fluorescent staining patterns of four lectins were investigated. The results indicated that every fixative gave concanavalin A (Con A) and wheat germ agglutinin (WGA) strong positive staining in different patterns. When fixed with Zenker’s, periodate–lysine–paraformaldehyde (PLP), 2·5% glutaraldehyde (GA), and 4% paraformaldehyde (PFA) solution, the cell membranes and cytoplasms stained, and the fluorescence in the cell edges was brighter. Rossman’s solution, 95% ethanol, and acetone fixation caused cytoplasmic staining, while the fluorescence was weak and evanescent when using acetone. Dolichos biflorus agglutinin (DBA) and Ulex europaeus agglutinin I (UEA I) were stained; revealing very faint fluorescence in cytoplasms just when PLP and 2·5% GA solution were used. In conclusion, this study shows that fixatives have significant effects on the fluorescent staining of MVECs. Fixatives containing aldehydes and mercury chloride are better options for cell membrane glycans, and those containing ethanol for cytoplasmic ones. Acetone fixation is not suitable for lectin histochemistry.

Modifications of carbohydrate residues in the sheep oviductal ampulla after superovulation

Epithelium of oviductal ampulla was studied in normal and in superovulated sheep using morphologic analysis and lectin glycohistochemistry. The lining epithelium consisted of two types of cells, ciliated and nonciliated cells. Unlike superovulated samples, the nonciliated cells from control ewes showed apical protrusions indicating an apocrine secretory activity. The ciliated cells showed lectin-binding sites mainly at the level of the cilia which bound all the used lectins except Peanut agglutinin, suggesting the lack of glycans terminating with Galβ1,3GalNAc. In superovulated specimens, the ciliated cells with high mannosylated glycans Concanavalin A (Con A) and GlcNAc and GalNac termini Griffonia simplicifolia agglutinin II (GSA II) and Dolicurus biflorus agglutinin (DBA) decreased. The luminal surface of nonciliated cells showed all investigated sugar residues in controls, whereas it was lacking in high mannosylated (Con A) and terminal GalNAcα1,3(LFucα1,2)Galβ1,3/4GlcNAcβ1 sequence (DBA) in superovulated ewes. Apical protrusions from control ampullae nonciliated cells showed glycans containing mannose, GlcNac, GalNAc, galactose, and α2,3-linked sialic acid (Con A, KOH-sialidase- Wheat germ agglutnin [WGA], GSA II, SBA, Griffonia simplicifolia agglutinin-isolectin B4 [GSA I-B4], Maackia amurensis agglutinin II [MAL II]). The supranuclear cytoplasm of nonciliated cells expressed terminal GlcNAc (GSA II) in all specimens, also O-linked glycans (mucin-type glycans) with GalNAc and sialic acid termini (Helix pomatia agglutinin [HPA] and MAL II) in control animals, and also N-linked glycans with fucose, galactose, lactosamine, and α2,3-linked sialic acid termini (Ulex europaeus agglutinin I [UEA I], GSA I-B4, Ricinus communis agglutinin120 [RCA120], and Sambucus nigra agglutinin [SNA] ) in superovulated ewes. These results report for the first time that the superovulation treatment affects the secretory activity and the glycan pattern of the epithelium lining the sheep oviductal ampulla.

Interleukin-10 overexpression improves the function of endothelial progenitor cells stimulated with TNF-α through the activation of the STAT3 signaling pathway.

Lentivirus vector-interleukin-10 green fluorescent protein (LV-IL-10-GFP) was transfected into endothelial progenitor cells (EPCs) in the present study. The aim was to detect the function of IL‑10‑modified EPCs and analyze the molecular mechanism. EPCs were cultured and identified by fluorescent labeling with the von Willebrand factor antibody, vascular endothelial growth factor (VEGF) receptor, Ulex europaeus agglutinin-1 and acetylated low‑density lipoprotein. Subsequently, EPCs were transfected with LV-IL-10-GFP and lentivirus vector‑noncontain‑GFP as the control group. Enzyme‑linked immunosorbent assay (ELISA) was used to detect the concentrations of cytokines in the supernatant with or without tumor necrosis factor‑α (TNF‑α). All types of cells were assessed by a tube formation assay, adhesion assay and migration assay induced with or without TNF‑α. Cell cycle was assessed by flow cytometry. Western blot analysis was applied to detect the expression of proteins in the cells. ELISA analysis showed that the levels of TNF‑α and IL‑8 in the supernatant without TNF‑α significantly decreased in EPC‑LV‑IL‑10‑GFP (P<0.05 for all). By contrast, the levels of IL‑10 and VEGF were contrasting in association with these. The concentrations of cytokines in the supernatant with TNF‑α were consistent to the supernatant without TNF‑α. There was no statistically significant difference in the average number of EPCs undergoing migration, adhesion, total length and cell growth among the EPC, EPC‑LV‑IL‑10‑GFP and EPC‑LV‑NC‑GFP groups without TNF‑α. Further study showed that EPC‑LV‑IL‑10‑GFP with TNF‑α significantly enhanced EPC migration, adhesion and promoted tube formation (P<0.05 for all). Western blot analysis revealed that the expression of VEGF, matrix metallopeptidase‑9 and phosphorylated‑signal transducer and activator of transcription 3 (p‑STAT3) significantly increased in the EPC‑LV‑IL‑10‑GFP group. Conversely, STAT‑3 expression decreased in the EPC‑LV‑IL‑10‑GFP group. The present study suggested that overexpression of IL‑10 had no effect on migration, adhesion, tubule formation and cell growth of EPCs without TNF‑α. Furthermore, in EPCs stimulated with TNF‑α, the overexpression of IL‑10 improved EPC function, including migration, adhesion and tubule formation by activating the STAT3 signal pathway.

BIODISTRIBUTION AND FUNCTIONAL REACTIVITY OF SOME IMMUNE CELLS IN PEYER’S PATCHES, CECAL PATCHES AND SPLEEN OF ALBINO MICE AFTER TREATMENT WITH THE NANOPARTICLES

Gut-associated lymphoid tissues (GALT) including Peyer's patches (PPs) and cecal patches (CPs) have a vital role in the induction of immune response against pathogens. Spleen is considered as non-GALT; it has a variety of immune cells that support the immune response. In this study, the expression of alpha L-fucose residues and biodistribution of Lectin Ulex Europaeus Agglutinin I (UEA I+) cells, IgA+ cells, 33D1+ dendritic cells (DCs) and CD11b+ macrophage subsets in the GALT (PPs and CPs) and in non-GALT (spleen) were investigated using UEAI lectin histochemistry and immunohistochemistry. The immune response induction of alpha L-fucose residue, UEAI+ cells, IgA+ cells, 33D1+ DCs and CD11+ macrophages subsets also will be investigated after treatment with amino-conjugated fluorescent silica nanoparticles (FSNPs). After treatment with FSNPs an induction of alpha L-fucose residue was observed in the PPs M cells in all cytoplasm, rather than the apical cytoplasm as shown in the control group. Also, the number of UEAI+ cells and IgA+ cells in PPs sub-epithelial dome (SED) increased 4 times and 2 times higher, respectively, as compared with that of the control group. The CD11b+ macrophages, but not 33D1+ DCs subsets located in PPs SED, had the main role to uptake FSNPs. In the case of spleen, the number of 33D1+ DCs and CD11b+ macrophages subsets was 30 times and 25 times higher, respectively, as compared with that of control group. These data indicated that alpha L-fucose residue and IgA+ cells, 33D1+ DCs and CD11b+ macrophages subsets in PPs as GALT and spleen as non-GALT had important role, but in different ratios, in the immune system induction against FSNPs.

Histological and lectin histochemical studies on the olfactory mucosae of the Korean roe deer, Capreolus pygargus.

The morphological features of the olfactory mucosae of Korean roe deer, Capreolus pygargus, were histologically studied using the ethmoid turbinates containing the olfactory mucosae from six roe deer (male, 2-3 years old). The ethmoid turbinates were embedded in paraffin, and histochemically evaluated in terms of the mucosal characteristics. Lectin histochemistry was performed to investigate the carbohydrate-binding specificity on the olfactory mucosa. Lectins, including Triticum vulgaris wheat germ agglutinin (WGA), Ulex europaeus agglutinin I (UEA-I), and soybean agglutinin (SBA) were used for the N-acetylglucosamine, fucose and N-acetylgalactosamine carbohydrate groups, respectively. Histologically, the olfactory mucosa, positioned mainly in the caudal roof of the nasal cavity, consisted of the olfactory epithelium and the lamina propria. The olfactory epithelium consisted of protein gene product (PGP) 9.5-positive olfactory receptor cells, galectin-3-positive supporting cells and basal cells. Bowman's glands in the lamina propria were stained by both the periodic acid Schiff reagent and alcian blue (pH 2.5). Two types of lectin, WGA and SBA, were labeled in free border, receptor cells, supporting cells and Bowman's glands, with the exception of basal cells, while UEA-I was labeled in free border, supporting cells and Bowman's glands, but not in receptor cells and basal cells, suggesting that carbohydrate terminals on the olfactory mucosae of roe deer vary depending on cell type. This is the first morphological study of the olfactory mucosa of the Korean roe deer to evaluate carbohydrate terminals in the olfactory mucosae.

Conversion of human pancreatic acinar cells toward a ductal-mesenchymal phenotype and the role of transforming growth factor β and activin signaling.

Epithelial-mesenchymal transition may interfere with the differentiation of cultured pancreatic acinar cells toward endocrine cells. Therefore, it will be important to investigate into detail the reprogramming of human pancreatic acinar cells toward a mesenchymal phenotype: the association with acinoductal transdifferentiation, the influence of cell adhesion, and the regulation behind this process. Human exocrine cells, isolated from donor pancreata, were cultured in suspension or as monolayers. Non-genetic lineage tracing, using labeled ulex europaeus agglutinin 1 lectin, was performed, and the role of the transforming growth factor (TGF-β) superfamily was investigated. After 7 days in monolayer culture, the human acinar cells coexpressed the mesenchymal marker vimentin and the ductal marker Sox9. However, when the human exocrine cells were cultured in suspension, epithelial-mesenchymal transition was not observed. The spontaneous transition of the human acinar cells toward a ductal and mesenchymal phenotype was decreased by inhibition of the TGF-β and activin signaling pathways. The human acinar cells spontaneously undergo TGF-β- regulated reprogramming in the monolayer culture. These observations are helpful to develop culture methods for the in vitro reprogramming of pancreatic exocrine to endocrine cells. They are also of potential interest for studies on exocrine acinar cells in the development of pancreatic cancer.

Intermediate expression of CCRL1 reveals novel subpopulations of medullary thymic epithelial cells that emerge in the postnatal thymus.

Cortical and medullary thymic epithelial cells (cTECs and mTECs, respectively) provide inductive microenvironments for T-cell development and selection. The differentiation pathway of cTEC/mTEC lineages downstream of common bipotent progenitors at discrete stages of development remains unresolved. Using IL-7/CCRL1 dual reporter mice that identify specialized TEC subsets, we show that the stepwise acquisition of chemokine (C-C motif) receptor-like 1 (CCRL1) is a late determinant of cTEC differentiation. Although cTECs expressing high CCRL1 levels (CCRL1(hi) ) develop normally in immunocompetent and Rag2(-/-) thymi, their differentiation is partially blocked in Rag2(-/-) Il2rg(-/-) counterparts. These results unravel a novel checkpoint in cTEC maturation that is regulated by the cross-talk between TECs and immature thymocytes. Additionally, we identify new Ulex europaeus agglutinin 1 (UEA)(+) mTEC subtypes expressing intermediate CCRL1 levels (CCRL1(int) ) that conspicuously emerge in the postnatal thymus and differentially express Tnfrsf11a, Ccl21, and Aire. While rare in fetal and in Rag2(-/-) thymi, CCRL1(int) mTECs are restored in Rag2(-/-) Marilyn TCR-Tg mice, indicating that the appearance of postnatal-restricted mTECs is closely linked with T-cell selection. Our findings suggest that alternative temporally restricted routes of new mTEC differentiation contribute to the establishment of the medullary niche in the postnatal thymus.

Elevations in vascular markers and eosinophils in chronic spontaneous urticarial weals with low-level persistence in uninvolved skin.

BackgroundIn chronic spontaneous urticaria (CSU) mast cell activation together with inflammatory changes in the skin are well documented and may play an important role in mechanisms of tissue oedema.ObjectivesTo confirm and extend these observations by measuring microvascular markers, leucocytes and mast cell numbers in lesional and uninvolved skin and to compare findings with a control group.MethodsPaired biopsies (one from 4–8-h spontaneous weals and one from uninvolved skin) were taken from eight patients with CSU and nine control subjects and studied using immunohistochemistry and confocal microscopy using the lectin Ulex europaeus agglutinin 1 (UEA-1).ResultsLesional skin in CSU contained significantly more CD31+ endothelial cells; CD31+ blood vessels, neutrophils, eosinophils, basophils and macrophages; and CD3+ T cells than nonlesional skin. Increased vascularity was confirmed by confocal imaging using the lectin UEA-1. Uninvolved skin from CSU contained significantly more CD31+ endothelial cells, CD31+ blood vessels and eosinophils compared with the control subjects. There was a threefold increase in mast cell numbers when CSU was compared with controls but no difference was observed between lesional and uninvolved skin.ConclusionsIncreased vascular markers together with eosinophil and neutrophil infiltration are features of lesional skin in CSU and might contribute to tissue oedema. Eosinophils and microvascular changes persist in uninvolved skin, which, together with increased mast cells, suggests that nonlesional skin is primed for further wealing.

Prospective study on Norovirus infection among allogeneic stem cell transplant recipients: Prolonged viral excretion and viral RNA in the blood

BackgroundHuman caliciviruses (Norovirus and Sapovirus) are important acute gastroenteritis agents. The Norovirus (NoV) disease is usually self-limited; however, prolonged viral excretion and complications have been reported, mainly in immunosuppressed individuals. ObjectivesIn this prospective study, we have monitored allogeneic stem cell transplant (ASCT) patients for human calicivirus infection. Study designTen ASCT patients were monitored for NoV and sapoviruses (SaV) infection, for a period of five months to a maximum of one year. Prolonged NoV excretion and long term viral RNA in the blood were assessed by multiplex RT-PCR targeting region C of the viral capsid. Secretor status of the patients was determined by enzyme immunoassay using Ulex Europaeus agglutinin. Partial genomic sequencing and phylogenetic analysis were performed to characterize the viral genotypes. ResultsNoV was detected in six out of ten patients (60%). Prolonged viral excretion in feces (mean of 61.6 days) and long term presence of NoV RNA in the sera (mean of 33.6 days) of the patients were observed. SaV was not detected in any of the samples. All patients had diarrhea, vomiting and fever during NoV positivity. All NoV-positive samples were characterized as GI.3 NoV. Three Nov-infected patients presented with acute intestinal graft versus host disease. ConclusionsThis study brings important information on NoV course of infection in ASCT patients. It also provides evidence for long term viral RNA in the blood highlighting the importance of the inclusion of NoV screening in the routine testing performed before transplantation and during follow-up of these patients.

In Vitro Analysis of Human Periodontal Microvascular Endothelial Cells

Endothelial cells (ECs) participate in key aspects of vascular biology, such as maintenance of capillary permeability, initiation of coagulation, and regulation of inflammation. According to previous reports, ECs have revealed highly specific characteristics depending on the organs and tissues. However, some reports have described the characteristics of the capillaries formed by human periodontal ECs. Therefore, the aim of the present study is to examine the functional characteristics of the periodontal microvascular ECs in vitro. Human periodontal ligament-endothelial cells (HPDL-ECs) and human gingiva-endothelial cells (HG-ECs) were isolated by immunoprecipitation with magnetic beads conjugated to a monoclonal anti-CD31 antibody. The isolated HPDL-ECs and HG-ECs were characterized to definitively demonstrate that these cell cultures represented pure ECs. Human umbilical-vein ECs and human dermal microvascular ECs were used for comparison. These cells were compared according to the proliferation potential, the formation of capillary-like tubes, the transendothelial electric resistance (TEER), and the expression of tight junction proteins. HPDL-ECs and HG-ECs with characteristic cobblestone monolayer morphology were obtained, as determined by light microscopy at confluence. Furthermore, the HPDL-ECs and HG-ECs expressed the EC markers platelet endothelial cell adhesion molecule-1 (also known as CD31), von Willebrand factor, and Ulex europaeus agglutinin 1, and the cells stained strongly positive for CD31 and CD309. In addition, the HPDL-ECs and HG-ECs were observed to form capillary-like tubes, and they demonstrated uptake of acetylated low-density lipoprotein. Functional analyses of the HPDL-ECs and HG-ECs showed that, compared to the control cells, tube formation persisted for only a brief period of time, and TEER was substantially reduced at confluence. Furthermore, the cells exhibited delocalization of zonula occludens-1 and occludin at cell-cell contact sites. The present results provide new evidence that HPDL-ECs and HG-ECs have characteristics of fenestrated capillaries. Therefore, capillaries in human periodontal tissues have functional characteristics of fenestrated capillaries, which might be related to the onset and the progression of systemic diseases and inflammation.

Anatomy, histochemistry, and immunohistochemistry of the olfactory subsystems in mice.

The four regions of the murine nasal cavity featuring olfactory neurons were studied anatomically and by labeling with lectins and relevant antibodies with a view to establishing criteria for the identification of olfactory subsystems that are readily applicable to other mammals. In the main olfactory epithelium and the septal organ the olfactory sensory neurons (OSNs) are embedded in quasi-stratified columnar epithelium; vomeronasal OSNs are embedded in epithelium lining the medial interior wall of the vomeronasal duct and do not make contact with the mucosa of the main nasal cavity; and in Grüneberg's ganglion a small isolated population of OSNs lies adjacent to, but not within, the epithelium. With the exception of Grüneberg's ganglion, all the tissues expressing olfactory marker protein (OMP) (the above four nasal territories, the vomeronasal and main olfactory nerves, and the main and accessory olfactory bulbs) are also labeled by Lycopersicum esculentum agglutinin, while Ulex europaeus agglutinin I labels all and only tissues expressing Gαi2 (the apical sensory neurons of the vomeronasal organ, their axons, and their glomerular destinations in the anterior accessory olfactory bulb). These staining patterns of UEA-I and LEA may facilitate the characterization of olfactory anatomy in other species. A 710-section atlas of the anatomy of the murine nasal cavity has been made available on line.

M-cell targeted polymeric lipid nanoparticles containing a toll-like receptor agonist to boost oral immunity

Oral delivery of antigens is patient-friendly and efficient way of treating intestinal infections. However, the efficacy of oral vaccines is limited by degradation in the gastrointestinal (GI) tract and poor absorption by enterocytes and antigen-presenting cells (APC). Here we report ulex europaeus agglutinin-1 (UEA-1) conjugated poly (d,l-lactide-co-glycolide) (PLGA)-lipid nanoparticles (NP) containing a Toll-like receptor (TLR)-agonist monophosphoryl lipid A (MPL) as an oral vaccine delivery system. The uniform-sized PLGA-lipid NPs (simplified as lipid NPs) were produced by the premix membrane emulsification method. They can protect the entrapped model antigen ovalbumin (OVA) from exposure to the GI tract and release the OVA in a controlled manner. With UEA-1 and MPL modification, the UEA-MPL/lipid NPs can be effectively transported by M-cells and captured by mucosal dendritic cells (DCs). After in vivo vaccination, the OVA-UEA-MPL/lipid NPs stimulated the most effective mucosal IgA and serum IgG antibodies during the oral formulations. These results suggest that this MPL containing M-cell targeted lipid NP can potentially be used as a universally robust oral vaccine delivery system.

Stage-dependent prognostic impact of molecular signatures in clear cell renal cell carcinoma.

PurposeTo enhance prognostic information of protein biomarkers for clear cell renal cell carcinomas (ccRCCs), we analyzed them within prognostic groups of ccRCC harboring different tumor characteristics of this clinically and molecularly heterogeneous tumor entity.MethodsTissue microarrays from 145 patients with primary ccRCC were immunohistochemically analyzed for VHL (von Hippel-Lindau tumor suppressor), Ki67 (marker of proliferation 1), p53 (tumor protein p53), p21 (cyclin-dependent kinase inhibitor 1A), survivin (baculoviral IAP repeat containing 5), and UEA-1 (Ulex europaeus agglutinin I) to assess microvessel-density.ResultsWhen analyzing all patients, nuclear staining of Ki67 (hazard ratio [HR] 1.08, 95% confidence interval [CI] 1.04–1.12) and nuclear survivin (nS; HR 1.04, 95% CI 1.01–1.08) were significantly associated with disease-specific survival (DSS). In the cohort of patients with advanced localized or metastasized ccRCC, high staining of Ki67, p53 and nS predicted shorter DSS (Ki67: HR 1.07, 95% CI 1.02–1.11; p53: HR 1.05, 95% CI 1.01–1.09; nS: HR 1.08, 95% CI 1.02–1.14). In organ-confined ccRCC, patients with high p21-staining had a longer DSS (HR 0.96, 95% CI 0.92–0.99). In a multivariate model with stepwise backward elimination, tumor size and p21-staining showed a significant association with DSS in patients with “organ-confined” ccRCCs. The p21-staining increased the concordance index of tumor size from 0.75 to 0.78. In patients with “organ-confined” ccRCC, no disease-related deaths occurred in the group with p21-expression below the threshold of 32.5% p21-positive cells (log rank test: P=0.002).ConclusionThe prognostic information of the studied protein biomarkers depended on anatomic tumor stages, which displayed different acquired biological tumor characteristics. Analysis of prognostic factors within different clinical ccRCC groups could help to enhance their prognostic power. The p21-staining was an independent prognostic factor and increased prognostic accuracy in a predictive model in “organ-confined” ccRCC.

Identification of a novel epigenetic code, histone H1 fucosylation, and its roles in macrophage plasticity in rheumatoid arthritis (IRM7P.491)

Abstract Macrophages (MΦ) are key players in rheumatoid arthritis (RA). High plasticity renders MΦ an attractive tool for studying epigenetic reprogramming. We have observed a highly positive correlation between TNFα and fucosylation catalyzing enzymes, fucosyltransferases (FUTs) 1, 2, 3, 4, 5, 6, 7, 9, 10, 11 (p=0.0001) in human RA synovia; these FUTs are predominantly detected in M1 inflammatory MΦ but not M2 MΦ and T cells from RA synovial fluid. A fucosylation inhibitor, 2-Deoxy-D-galactose (2-D-gal) precluded collagen II-induced arthritis in DBA/1J mice (scores 9.5±1.7 vs 0.5± 0.3, p&amp;lt;0.01) with reduced M1 MΦ in draining LN, decreased TNF-α and anti-CII in the serum (p&amp;lt;0.05).Ulex Europaeus Agglutinin I (UEA 1) affinity chromatography coupled to mass spectrometry indicated that the linker Histone H1, but not core histone H 2-4, is the major protein that modified by a fucose-α-(1-2)-gal moiety in M1 MΦ. Disrupting this moiety by 2-D-gal skewed the differentiation of M1 MΦ toward M2 anti-inflammatory phenotype with a reprogrammed gene expression, including i) robustly upregulated Il10, Arg1, and Megf8 (p&amp;lt;0.01) (M2 markers); ii) downregulated Ifnγ and Il6 (p&amp;lt;0.05) (M1 markers); iii) inhibited Actin and its associated molecules including Ahnak and Cap1, and suppressed the antigen presentation of M1 MΦ phenotype (p&amp;lt;0.01). We uncovered Histone 1 fucosylation as a novel epigenetic code that orchestrates MΦ transcriptome reprogramming and renders it an attractive target for RA therapy.

Calreticulin activates β1 integrin via fucosylation by fucosyltransferase 1 in J82 human bladder cancer cells

Fucosylation regulates various pathological events in cells. We reported that different levels of CRT (calreticulin) affect the cell adhesion and metastasis of bladder cancer. However, the precise mechanism of tumour metastasis regulated by CRT remains unclear. Using a DNA array, we identified FUT1 (fucosyltransferase 1) as a gene regulated by CRT expression levels. CRT regulated cell adhesion through α1,2-linked fucosylation of β1 integrin and this modification was catalysed by FUT1. To clarify the roles for FUT1in bladder cancer, we transfected the human FUT1 gene into CRT-RNAi stable cell lines. FUT1 overexpression in CRT-RNAi cells resulted in increased levels of β1 integrin fucosylation and rescued cell adhesion to type-I collagen. Treatment with UEA-1 (Ulex europaeus agglutinin-1), a lectin that recognizes FUT1-modified glycosylation structures, did not affect cell adhesion. In contrast, a FUT1-specific fucosidase diminished the activation of β1 integrin. These results indicated that α1,2-fucosylation of β1 integrin was not involved in integrin-collagen interaction, but promoted β1 integrin activation. Moreover, we demonstrated that CRT regulated FUT1 mRNA degradation at the 3'-UTR. In conclusion, the results of the present study suggest that CRT stabilized FUT1 mRNA, thereby leading to an increase in fucosylation of β1 integrin. Furthermore, increased fucosylation levels activate β1 integrin, rather than directly modifying the integrin-binding sites.

Enteroendocrine Cells Are Specifically Marked by Cell Surface Expression of Claudin-4 in Mouse Small Intestine

Enteroendocrine cells are solitary epithelial cells scattered throughout the gastrointestinal tract and produce various types of hormones, constituting one of the largest endocrine systems in the body. The study of these rare epithelial cells has been hampered by the difficulty in isolating them because of the lack of specific cell surface markers. Here, we report that enteroendocrine cells selectively express a tight junction membrane protein, claudin-4 (Cld4), and are efficiently isolated with the use of an antibody specific for the Cld4 extracellular domain and flow cytometry. Sorted Cld4+ epithelial cells in the small intestine exclusively expressed a chromogranin A gene (Chga) and other enteroendocrine cell–related genes (Ffar1, Ffar4, Gpr119), and the population was divided into two subpopulations based on the activity of binding to Ulex europaeus agglutinin-1 (UEA-1). A Cld4+UEA-1− cell population almost exclusively expressed glucose-dependent insulinotropic polypeptide gene (Gip), thus representing K cells, whereas a Cld4+UEA-1+ cell population expressed other gut hormone genes, including glucagon-like peptide 1 (Gcg), pancreatic polypeptide–like peptide with N-terminal tyrosine amide (Pyy), cholecystokinin (Cck), secretin (Sct), and tryptophan hydroxylase 1 (Tph1). In addition, we found that orally administered luminal antigens were taken up by the solitary Cld4+ cells in the small intestinal villi, raising the possibility that enteroendocrine cells might also play a role in initiation of mucosal immunity. Our results provide a useful tool for the cellular and functional characterization of enteroendocrine cells.

Development of lectin-linked immunomagnetic separation for the detection of hepatitis a virus.

The accuracy and sensitivity of PCR-based methods for detection of hepatitis A virus (HAV) are dependent on the methods used to separate and concentrate the HAV from the infected cells. The pH and ionic strength affect the binding affinity of the virus to cells. In this study, we initially investigated the effects of pH (4.0–10.0) and metal ions (Fe2+, Co2+, Cu2+, Mg2+, K+, and Ca2+) on the binding of HAV to oyster digestive cells. The lowest relative binding (RB) of HAV to the cells was found at pH 4.0 and in FeSO4 solution (64.6% and 68.1%, respectively). To develop an alternative to antibody-dependent immunomagnetic separation prior to detection of HAV using RT-PCR, the binding of HAV to five lectins, peanut agglutinin (PNA), Dolichos biflorus agglutinin (DBA), Helix pomatia agglutinin (HPA), Ulex europaeus agglutinin (UEA-1) and soybean agglutinin (SBA), was evaluated using ELISAs. SBA showed significantly higher RB to HAV than the other lectins tested. In addition, HAV could be concentrated within 30 min using SBA-linked magnetic bead separation (SMS) prior to the RT-PCR assay. Our findings demonstrate the feasibility of using SMS combined with RT-PCR to detect HAV at dilutions ranging from 10−1–10−4 of a HAV stock (titer: 104 TCID50/mL).

Lymphangiosarcoma of the Scalp

Lymphangiosarcoma is a rare, aggressive malignancy that originates from the endothelial cells lining lymphatic vessels and carries an extremely poor prognosis. Its clinical and histologic features are often indistinguishable from angiosarcoma. We sought to better characterize the clinical and histologic features of lymphangiosarcoma. Case report and review of the literature. A number of immunohistochemical markers, including Von Willebrand factor, Ulex europaeus agglutinin 1, CD31, VEGFR-3, D2-40, Prox-1, can be used to help differentiate lymphatic from vascular tissue. Recent characterization of several new biologic markers has allowed greater differentiation between these tumors and may provide new therapeutic targets for treatment.

A collecting duct carcinoma producing carcinoembryonic antigen and Ca-125 in a 29-year-old woman

Collecting duct carcinoma is a rare renal malignant neoplasm, arising from the medullary collecting duct and accounting for less than 1% of renal cell carcinomas. It is more common in middle-aged men and is usually presented with hematuria, abdominal mass and back or flank pain. Its immunohistochemical analysis detects the expression of various markers, such as low and high molecular weight keratins, Ulex europaeus agglutinin-1, epithelial membrane antigen and peanut lectin. Here, we present a case of a 29-year-old woman with CDC presented with back pain and supraclavicular lymphadenopathy that produced carcinoembryonic antigen and CA-125.