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Congenital disorders of glycosylation (CDG) include 150 genetically and clinically heterogeneous diseases, showing significant glycoprotein hypoglycosylation that leads to pathological consequences in multiple organs and systems whose underlying mechanisms are not yet understood. A few cellular and animal models have been used to study specific CDG characteristics, although they have given limited information due to the few CDG mutations tested and the still missing comprehensive molecular and cellular basic research. Here, we provide specific gene expression profiles, based on ribonucleic acid (RNA) microarray analysis, together with some biochemical and cellular characteristics of a total of nine control Epstein-Barr virus-transformed lymphoblastoid B cell lines (B-LCL) and 13 CDG B-LCL from patients carrying severe mutations in the phosphomannomutase 2 (PMM2) gene, strong serum protein hypoglycosylation and neurological symptoms. Significantly dysregulated genes in PMM2-CDG cells included those regulating stress responses, transcription factors, glycosylation, motility, cell junction and, importantly, those related to development and neuronal differentiation and synapse, such as carbonic anhydrase 2 (CA2) and ADAM23. PMM2-CDG-associated biological consequences involved the unfolded protein response, RNA metabolism and the endoplasmic reticulum, Golgi apparatus and mitochondria components. Changes in the transcriptional and CA2 protein levels are consistent with the CDG physiopathology. These results demonstrate the global transcriptional impact in phosphomannomutase 2-deficient cells, reveal CA2 as a potential cellular biomarker and confirm B-LCL as an advantageous model for CDG studies., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Vascularization is essential for the regeneration of bone tissue within composite material. We measured the effect of regioselectively modified cellulose/hemicellulose as an additive for porous scaffolds of collagen/hydroxyapatite nanocomposite on the tubule formation of human vascular endothelial cells. Using a coculture of endothelial cells and fibroblasts, endothelial cells formed a network of tubules within an incubation time of 14 to 24 days. A cellulose sulfate with irregular sulfation pattern along the polysaccharide backbone (13-TACS-01) led to an additional increase in vascular endothelial growth factor (VEGF)-induced tubule formation, as observed in an in vitro angiogenesis assays. In contrast with structurally different heparin, these cellulose sulfates have no apparent affinity to VEGF. Their impact on endothelial function may possibly be due to interactions with cell surface receptors/soluble factors not yet defined.

Angiogenesis plays an important role in both soft and hard tissue regeneration, which can be modulated by therapeutic drugs. If nanoparticles (NP) are used as vectors for drug delivery, they have to encounter endothelial cells (EC) lining the vascular lumen, if applied intravenously. Herein the interaction of unloaded polyelectrolyte complex nanoparticles (PECNP) composed of cationic poly(l-lysine) (PLL) and various anionic polysaccharides with human vascular endothelial cells (HUVEC) was analyzed. In particular PECNP were tested for their cell adhesive properties, their cellular uptake and intracellular localization considering composition and net charge. PECNP may form a platform for both cell coating and drug delivery. PECNP, composed of PLL in combination with the polysaccharides dextran sulfate (DS), cellulose sulfate (CS) or heparin (HEP), either unlabeled or labeled with fluorescein isothiocyanate (FITC) and either with positive or negative net charge were prepared. PECNP were applied to human umbilical cord vein endothelial cells (HUVEC) in both, the volume phase and immobilized phase at model substrates like tissue culture dishes. The attachment of PECNP to the cell surface, their intracellular uptake, and effects on cell proliferation and growth behavior were determined. Immobilized PECNP reduced attachment of HUVEC, most prominently the systems PLL/HEP and PLL/DS. A small percentage of immobilized PECNP was taken up by cells during adhesion. PECNP in the volume phase showed no effect of the net charge sign and only minor effects of the composition on the binding and uptake of PECNP at HUVEC. PECNP were stored in endosomal vesicles in a cumulative manner without apparent further processing. During mitosis, internalized PECNP were almost equally distributed among the dividing cells. Both, in the volume phase and immobilized at the surface, PECNP composed of PLL/HEP and PLL/DS clearly reduced cell proliferation of HUVEC, however without an apparent cytotoxic effect, while PLL/CS composition showed minor impairment. PECNP have an anti-adhesive effect on HUVEC and are taken up by endothelial cells which may negatively influence the proliferation rate of HUVEC. The negative effects were less obvious with the composition PLL/CS. Since uptake and binding for PLL/HEP was more efficient than for PLL/DS, PECNP of PLL/HEP may be used to deliver growth factors to endothelial cells during vascularization of bone reconstitution material, whereas those of PLL/CS may have an advantage for substituting biomimetic bone scaffold material.

In this study, the effect of heparin-modified collagen type I/hydroxyapatite (HA) nanocomposites on key processes of bone regeneration - osteogenesis and angiogenesis - was characterised in vitro. Two approaches were applied for heparin modification: it was either integrated during material synthesis (in situ) or added to the porous scaffolds after their fabrication (post). Cultivation of human bone marrow-derived stromal cells (hBMSC), in heparin-modified versus heparin-free scaffolds, revealed a positive effect of the heparin modification on their proliferation and osteogenic differentiation. The amount of heparin rather than the method used for modification influenced the cell response favouring proliferation at smaller amount (30 mg/g collagen) and differentiation at larger amount (150 mg/g collagen). A co-culture of human umbilical vein endothelial cells (HUVEC) and osteogenically induced hBMSC was applied for in vitro angiogenesis studies. Pre-vascular networks have formed in the porous structure of scaffolds which were not modified with heparin or modified with a low amount of heparin (30 mg/g collagen). The modification with higher heparin quantities seemed to inhibit tubule formation. Pre-loading of the scaffolds with VEGF influenced formation and stability of the pre-vascular structures depending on the presence of heparin: In heparin-free scaffolds, induction of tubule formation and sprouting was more pronounced whereas heparin-modified scaffolds seemed to promote stabilisation of the pre-vascular structures. In conclusion, the modification of mineralised collagen with heparin by using both approaches was found to modulate cellular processes essential for bone regeneration; the amount of heparin has been identified to be crucial to direct cell responses.

A small percentage of the Western population carries antibodies in the peripheral blood, which are able to kill human tumors such as neuroblastoma or melanoma. Several observations indicate that these antibodies, preferentially of IgM isotype, belong to the class of naturally occurring antibodies. Here, we describe two screening methods for the detection and quantification of such antibodies in human blood samples: a cellular ELISA technique and a flow cytometric assay, based on intercalation of fluorescent propidium iodide into the DNA of dying or dead cells.

Multiple myeloma is characterized by abnormal infiltration of malignant plasma cells into bone marrow. Testing the hypothesis that bivalent galectin-8 (Gal-8) may influence homing of myeloma cells to vascular endothelium as a key prerequisite for infiltration, we analyzed the two Gal-8 splice variants (Gal-8S, Gal-8L). They differ in the length of their linker peptide connecting the two lectin domains. Both Gal-8 isoforms bind to cells of the myeloma lines Gal-8 + MOLP-8 and Gal-8 - LP-1 in a glycan-inhibitable manner. Both Gal-8 isoforms led to enhanced adhesion of myeloma cells to vascular endothelium under dynamic shear stress conditions, Gal-8L (by more than 40-fold) even stronger than Gal-8S. Additional treatment of endothelial cells with tumour necrosis factor prior to the dynamic shear stress assay entailed an almost 100-fold enhanced adhesion of myeloma cells without addition of Gal-8 variants and a further 1.5-1.7-fold enhancement by addition of Gal-8 variants. We also found that elevated expression of Gal-8 in native multiple myeloma cells is an adverse prognostic factor for overall and event-free survival using patients' gene expression profile data of the total therapy 2 and 3 myeloma studies. Also, elevated concentrations of Gal-8 were detected (45%, 19/42 patients) in sera of multiple myeloma patients compared to those of healthy, age-matched donors. Both experimental and clinical data strongly point to the significance of Gal-8 for multiple myeloma development., (© The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

A small percentage of healthy donors identified in the Western population carry antibodies in their peripheral blood which convey cytotoxic activity against certain human melanoma and neuroblastoma cell lines. We measured the cytotoxic activity of sera and plasmas from healthy donors on the human neuroblastoma cell line Kelly and various melanoma cell lines. Antibodies of IgM isotype, presumably belonging to the class of naturally occurring antibodies, exerted cytotoxic activity in a complement-dependent fashion. Apart from complement-dependent tumor cell lysis, we observed C3 opsonization in all tumor cell lines upon treatment with cytotoxic plasmas. Cell lines tested primarily expressed membrane complement regulatory proteins (mCRP) CD46, CD55 and CD59 to various extents. Blocking of mCRPs by monoclonal antibodies enhanced cell lysis and opsonization, though some melanoma cells remained resistant to complement attack. Epitopes recognized by cytotoxic antibodies were represented by gangliosides such as GD2 and GD3, as evidenced by cellular sialidase pretreatment and enhanced expression of distinct gangliosides. It remains to be clarified why only a small fraction of healthy persons carry these antitumor cytotoxic antibodies., (© 2016 UICC.)

CD (cluster of differentiation) Ags are cell surface molecules expressed on leukocytes and other cells relevant for the immune system. CD nomenclature has been universally adopted by the scientific community and is officially approved by the International Union of Immunological Societies and sanctioned by the World Health Organization. It provides a unified designation system for mAbs, as well as for the cell surface molecules that they recognize. This nomenclature was established by the Human Leukocyte Differentiation Antigens Workshops. In addition to defining the CD nomenclature, these workshops have been instrumental in identifying and determining the expression and function of cell surface molecules. Over the past 30 y, the data generated by the 10 Human Leukocyte Differentiation Antigens Workshops have led to the characterization and formal designation of more than 400 molecules. CD molecules are commonly used as cell markers, allowing the identification and isolation of leukocyte populations, subsets, and differentiation stages. mAbs against these molecules have proven to be essential for biomedical research and diagnosis, as well as in biotechnology. More recently, they have been recognized as invaluable tools for the treatment of several malignancies and autoimmune diseases. In this article, we describe how the CD nomenclature was established, present the official updated list of CD molecules, and provide a rationale for their usefulness in the 21st century., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Sialic acid acetyl esterase (SIAE) removes acetyl moieties from the hydroxyl groups in position 9 and 4 of sialic acid. Recently, a dispute has been opened on its association to autoimmunity. In order to get new insights on human SIAE biology and to clarify its seemingly contradictory molecular properties, we combined in silico characterization, phylogenetic analysis and homology modeling with cellular studies in COS7 cells. Genomic and phylogenetic analysis revealed that in most tissues only the "long" isoform, originally referred to lysosomal sialic acid esterase, is detected. Using the homology modeling approach, we predicted a model of SIAE 3D structure, which fulfills the topological features of SGNH-hydrolase family. In addition, the model and site-directed mutagenesis experiments allowed the definition of the residues involved in catalysis. SIAE transient expression revealed that the protein is glycosylated and is active in vitro as an esterase with a pH optimum corresponding to 8.4-8.5. Moreover, glycosylation influences the biological activity of the enzyme and is essential for release of SIAE into the culture medium. According to these findings, co-localization experiments demonstrated the presence of SIAE in membranous structures corresponding to endoplasmic reticulum and Golgi complex. Thus, at least in COS7 cells, SIAE behaves as a typical secreted enzyme, subjected to glycosylation and located along the classical secretory route or in the extracellular space. In these environments, the enzyme could act on 9-O-acetylated sialic acid residues, contributing to the fine-tuning of the various functions played by this acidic sugar., (© The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Sialic acids have a pivotal functional impact in many biological interactions such as virus attachment, cellular adhesion, regulation of proliferation, and apoptosis. A common modification of sialic acids is O-acetylation. O-Acetylated sialic acids occur in bacteria and parasites and are also receptor determinants for a number of viruses. Moreover, they have important functions in embryogenesis, development, and immunological processes. O-Acetylated sialic acids represent cancer markers, as shown for acute lymphoblastic leukemia, and they are known to play significant roles in the regulation of ganglioside-mediated apoptosis. Expression of O-acetylated sialoglycans is regulated by sialic acid-specific O-acetyltransferases and O-acetylesterases. Recent developments in the identification of the enigmatic sialic acid-specific O-acetyltransferase are discussed.

Gangliosides play important roles in the development, differentiation and proliferation of mammalian cells. They bind to other cell membrane components through their terminal sialic acids. Different gangliosides influence cellular functions based on the positions and linkages of sialic acids. Expression of gangliosides mainly depends on the status of sialic acid-modulatory enzymes, such as different types of sialyltransferases and sialidases. One such sialyltransferase, disialoganglioside GD3 synthase, is specifically responsible for the production of GD3. Pancreatic ductal adenocarcinoma, making up more than 90% of pancreatic cancers, is a fatal malignancy with poor prognosis. Despite higher sialylation status, the disialoganglioside GD3 level is very low in this cancer. However, the exact status and function of this disialoganglioside is still unknown. Here, we intended to study the intracellular mechanism of disialoganglioside GD3-induced apoptosis and its correlation with the adhesion and angiogenic pathways in pancreatic cancer. We demonstrated that disialoganglioside GD3 synthase-transfected cells showed enhanced apoptosis and it caused the arrest of these cells in the S-phase of the cell cycle. Integrins, a family of transmembrane proteins play important role in cell-cell recognition, invasion, adhesion and migration. disialoganglioside GD3 co-localised with integrin-β1 and thereby inhibited it's downstream signalling in transfected cells. Transfected cells exhibited inhibition of cell adhesion with extracellular matrix proteins. Enhanced GD3 expression down regulated angiogenesis-regulatory proteins and inhibited epidermal growth factor/vascular endothelial growth factor-driven angiogenic cell growth in these cells. Taken together, our study provides support for the GD3-induced cell cycle arrest, disruption of integrin-β1-mediated anchorage, inhibition of angiogenesis and thereby induced apoptosis in pancreatic cancer cells., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Aims: Recent reports suggest a possible role for extracellular (MUC1N) and transmembrane (MUC1C) subunits of Mucin 1 (MUC1) in the pathogenesis of multiple myeloma (MM). Nuclear translocation of MUC1C is involved in activation of various oncogenic signalling pathways and both MUC1 subunits are potential therapeutic targets. We aimed at performing a comprehensive expression analysis of the MUC1 subunits in plasma cell dyscrasias., Methods and Results: Immunohistochemistry with monoclonal antibodies against the MUC1N subunit (EMA and 5E10) tumour-associated glycoforms of MUC1N (5E5) and the MUC1C subunit were applied to a series of biopsies from normal controls (n = 10) and plasma cell dyscrasias (n = 121). Clonal plasma cells showed reduced MUC1N expression, and the 5E5 MUC1N epitope was expressed only in neoplastic plasma cells. Nuclear localization of MUC1C was equally frequent in all disease stages and did not differ from the control cases. Loss of both MUC1 subunits in MM (n = 12) was associated with significantly shorter overall survival and was more frequent in pretreated MM samples., Conclusions: Our findings indicate that aberrant glycosylation of MUC1 is an early event in the pathogenesis of MM. In contrast, MUC1C nuclear localization is not likely to be a driver of tumour progression., (© 2013 John Wiley & Sons Ltd.)

Phosphorylation is known to have a strong impact on protein functions. We analyzed members of the lectin family of multifunctional galectins as targets of the protein kinases CK1, CK2, and PKA. Galectins are potent growth regulators able to bind both glycan and peptide motifs at intra- and extracellular sites. Performing in vitro kinase assays, galectin phosphorylation was detected by phosphoprotein staining and autoradiography. The insertion of phosphoryl groups varied to a large extent depending on the type of kinase applied and the respective galectin substrate. Sites of phosphorylation observed in the recombinant galectins were determined by a strategic combination of phosphopeptide enrichment and nano-ultra-performance liquid chromatography tandem mass spectrometry (nanoUPLC-MS/MS). By in silico modeling, phosphorylation sites were visualized three-dimensionally. Our results reveal galectin-type-specific Ser-/Thr-dependent phosphorylation beyond the known example of galectin-3. These data are the basis for functional studies and also illustrate the analytical sensitivity of the applied methods for further work on human lectins., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The glycome, i.e. the cellular repertoire of glycan structures, contributes to important functions such as adhesion and intercellular communication. Enzymes regulating cellular glycosylation processes are related to the pathogenesis of cancer including multiple myeloma. Here we analyze the transcriptional differences in the glycome of normal (n = 10) and two cohorts of 332 and 345 malignant plasma-cell samples, association with known multiple myeloma subentities as defined by presence of chromosomal aberrations, potential therapeutic targets, and its prognostic impact. We found i) malignant vs. normal plasma cells to show a characteristic glycome-signature. They can ii) be delineated by a lasso-based predictor from normal plasma cells based on this signature. iii) Cytogenetic aberrations lead to distinct glycan-gene expression patterns for t(11;14), t(4;14), hyperdiploidy, 1q21-gain and deletion of 13q14. iv) A 38-gene glycome-signature significantly delineates patients with adverse survival in two independent cohorts of 545 patients treated with high-dose melphalan and autologous stem cell transplantation. v) As single gene, expression of the phosphatidyl-inositol-glycan protein M as part of the targetable glycosyl-phosphatidyl-inositol-anchor-biosynthesis pathway is associated with adverse survival. The prognostically relevant glycome deviation in malignant cells invites novel strategies of therapy for multiple myeloma.

CD176 (Thomsen-Friedenreich antigen) is a tumor-associated carbohydrate structure. CD176 is expressed at the surface of human leukemic cells but is almost absent in normal and benign adult human tissues. Therefore, CD176 could be a promising target for antitumor immunotherapy. In the present study, pre-immunization with asialoglycophorin A (containing the CD176 oligosaccharide chains) was able to significantly improve the survival time of mice carrying CD176+ leukemia as compared to the control mice without the immunization. Furthermore, the passive transfer of CD176 antiserum which reacted only with the tumor-associated CD176 in cancer cells, was able to effectively prolong the survival time of CD176+ leukemia mice. In particular, the CD176 antiserum treatment could inhibit the growth and spreading of CD176+ leukemic cells in bone marrow, spleen, liver, and lung as evidenced by histopathological examination. CD176 antiserum could induce the apoptosis of CD176+ leukemic cells in vivo in a manner as previously observed in vitro. The data provided strong evidence that both CD176 antigen-based active immunotherapy and CD176 antibody-based passive immunotherapy lead to a therapeutic response in CD176+ leukemia mice. Therefore, both CD176 vaccine and CD176 antibody drug may be beneficial for the treatment of CD176+ leukemia patients.

Aberrant activation of Hedgehog (HH) signaling has been identified as a key etiologic factor in many human malignancies. Signal strength, target gene specificity, and oncogenic activity of HH signaling depend profoundly on interactions with other pathways, such as epidermal growth factor receptor-mediated signaling, which has been shown to cooperate with HH/GLI in basal cell carcinoma and pancreatic cancer. Our experimental data demonstrated that the Daoy human medulloblastoma cell line possesses a fully inducible endogenous HH pathway. Treatment of Daoy cells with Sonic HH or Smoothened agonist induced expression of GLI1 protein and simultaneously prevented the processing of GLI3 to its repressor form. To study interactions between HH- and EGF-induced signaling in greater detail, time-resolved measurements were carried out and analyzed at the transcriptomic and proteomic levels. The Daoy cells responded to the HH/EGF co-treatment by downregulating GLI1, PTCH, and HHIP at the transcript level; this was also observed when Amphiregulin (AREG) was used instead of EGF. We identified a novel crosstalk mechanism whereby EGFR signaling silences proteins acting as negative regulators of HH signaling, as AKT- and ERK-signaling independent process. EGFR/HH signaling maintained high GLI1 protein levels which contrasted the GLI1 downregulation on the transcript level. Conversely, a high-level synergism was also observed, due to a strong and significant upregulation of numerous canonical EGF-targets with putative tumor-promoting properties such as MMP7, VEGFA, and IL-8. In conclusion, synergistic effects between EGFR and HH signaling can selectively induce a switch from a canonical HH/GLI profile to a modulated specific target gene profile. This suggests that there are more wide-spread, yet context-dependent interactions, between HH/GLI and growth factor receptor signaling in human malignancies.

Human Galectin-8 (Gal-8) is a member of the galectin family which shares an affinity for β-galactosides. The tandem-repeat Gal-8 consists of a N- and a C-terminal carbohydrate recognition domain (N- and C-CRD) joined by a linker peptide of various length. Despite their structural similarity both CRDs recognize different oligosaccharides. While the molecular requirements of the N-CRD for high binding affinity to sulfated and sialylated glycans have recently been elucidated by crystallographic studies of complexes with several oligosaccharides, the binding specificities of the C-CRD for a different set of oligosaccharides, as derived from experimental data, has only been explained in terms of the three-dimensional structure for the complex C-CRD with lactose. In this study we performed molecular dynamics (MD) simulations using the recently released crystal structure of the Gal-8C-CRD to analyse the three-dimensional conditions for its specific binding to a variety of oligosaccharides as previously defined by glycan-microarray analysis. The terminal β-galactose of disaccharides (LacNAc, lacto-N-biose and lactose) and the internal β-galactose moiety of blood group antigens A and B (BGA, BGB) as well as of longer linear oligosaccharide chains (di-LacNAc and lacto-N-neotetraose) are interacting favorably with conserved amino acids (H53, R57, N66, W73, E76). Lacto-N-neotetraose and di-LacNAc as well as BGA and BGB are well accommodated. BGA and BGB showed higher affinity than LacNAc and lactose due to generally stronger hydrogen bond interactions and water mediated hydrogen bonds with α1-2 fucose respectively. Our results derived from molecular dynamics simulations are able to explain the glycan binding specificities of the Gal-8C-CRD in comparison to those of the Gal-8N -CRD.

Summary: The open access comprehensive GlycoCD database application is for representation and retrieval of carbohydrate-related clusters of differentiation (CDs). The main objective of this database platform is to provide information about interactions of carbohydrate moieties with proteins that are important for identification of specific cell surface molecule with a focus on the integration of data from carbohydrate microarray databases. GlycoCD database comprises two sections: the carbohydrate recognition CD and glycan CD. It allows easy access through a user-friendly web interface to all carbohydrate-defined CDs and those that interact with carbohydrates along with other relevant information., Availability: The database is freely available at http://glycosciences.de/glycocd/index.php, Contact: r.s-albiez@dkfz.de.

Healthy persons carry within their pool of circulating antibodies immunoglobulins preferentially of IgM isotype, which are directed against a variety of tumor-associated antigens. In closer scrutiny of their nature, some of these antibodies could be defined as naturally occurring antibodies due to the germline configuration of the variable immunoglobulin region. The majority of these immunoglobulins recognize carbohydrate antigens which can be classified as oncofetal antigens. Many of these IgM antibodies present in the peripheral blood circulation can bind to tumor cells and of these a minor portion are also able to destroy tumor cells by several mechanisms, as for instance complement-mediated cytolysis or apoptosis. It was postulated that anti-carbohydrate antibodies are part of an anti-tumor immune response, while their presence in the peripheral blood of healthy donors is still waiting for a plausible explanation. It may be that recognition of defined epitopes, including carbohydrate sequences, by naturally occurring antibodies constitutes the humoral arm of an anti-tumor immune response as part of the often postulated tumor surveillance. The cytotoxic capacity of these antibodies inspired several research groups and pharmaceutical companies to design novel strategies of immunoglobulin-based anti-tumor immunotherapy.

Oligosaccharides aberrantly expressed on tumor cells influence processes such as cell adhesion and modulation of the cell's microenvironment resulting in an increased malignancy. Schmidt's imidate strategy offers an effective method to synthesize libraries of various oligosaccharide mimetics. With the aim to perturb interactions of tumor cells with extracellular matrix proteins and host cells, molecules with 3,4-bis(hydroxymethyl)furan as core structure were synthesized and screened in biological assays for their abilities to interfere in cell adhesion and other steps of the metastatic cascade, such as tumor-induced angiogenesis.The most active compound, (4-{[(β-D-galactopyranosyl)oxy]methyl}furan-3-yl)methyl hydrogen sulfate (GSF), inhibited the activation of matrix-metalloproteinase-2 (MMP-2) as well as migration of the human melanoma cells of the lines WM-115 and WM-266-4 in a two-dimensional migration assay. GSF inhibited completely the adhesion of WM-115 cells to the extracellular matrix (ECM) proteins, fibrinogen and fibronectin.In an in vitro angiogenesis assay with human endothelial cells, GSF very effectively inhibited endothelial tubule formation and sprouting of blood vessels, as well as the adhesion of endothelial cells to ECM proteins. GSF was not cytotoxic at biologically active concentrations; neither were 3,4-bis{[(β-D-galactopyranosyl)oxy]methyl}furan (BGF) nor methyl β-D-galactopyranoside nor 3,4-bis(hydroxymethyl)furan, which were used as controls, eliciting comparable biological activity. In silico modeling experiments, in which binding of GSF to the extracellular domain of the integrin α(v)β(3) was determined, revealed specific docking of GSF to the same binding site as the natural peptidic ligands of this integrin. The sulfate in the molecule coordinated with one manganese ion in the binding site.These studies show that this chemically easily accessible molecule GSF, synthesized in three steps from 3,4-bis(hydroxymethyl)furan and benzoylated galactose imidate, is nontoxic and antagonizes cell physiological processes in vitro that are important for the dissemination and growth of tumor cells in vivo.

GD3 (CD60a) and its 9-O-acetylated variant (CD60b) are intracellular regulators of apoptosis in T lymphocytes. Surface expressed 9-O-acetyl- and 7-O-acetyl-GD3 (CD60b and CD60c) may have a functional impact on activated T and B cells. In order to investigate the balance between surface and intracellular expression and synthesis and degradation of these glycosphingolipids in human lymphocytes of various differentiation stages, we analyzed (i) expression of GD3 molecules on native T and B cells and thymocytes by flow cytometry and (ii) activity and regulation of possible key enzymes for CD60a,b,c synthesis and degradation at the transcriptional level. Both, surface and cytoplasmic expression of CD60a and CD60c was highest in tonsillar T cells. In thymocytes, CD60c outweighs the other CD60 variants and was mainly found in the cytoplasm. All lymphocyte preparations contained sialate O-acetyltransferase activity producing 7-O-acetyl-GD3. Sialidase activity was highest in peripheral blood lymphocytes followed by thymocytes and tonsillar T and B cells. Transcription of GD3 synthase (ST8SiaI), the key enzyme for GD3 synthesis, was highest in tonsillar T cells, whereas transcriptional levels of sialidase NEU3 and O-acetylesterase H-Lse were lowest in activated T cells. This balance between enzymes of sialic acid metabolism may explain the strong overall staining intensity for all GD3 forms in T cells. Both CASD1, presumably encoding a sialic acid-specific O-acetyltransferase, and H-Lse showed highest transcription in peripheral B lymphocytes corresponding to the low expression of CD60b and c in these cells. Our data point to regulatory functions of these anabolic and catabolic key enzymes for the expression of GD3 and its O-acetylated variants in lymphocytes at a given differentiation stage.

CD176 (Thomsen-Friedenreich antigen) is a tumor-associated carbohydrate structure. In a previous study, we observed that the anti-CD176 antibody can induce the apoptosis of CD176-positive leukemic cells. In this study, we investigated the mechanisms of apoptosis induced by the CD176 antibody. We found that CD95 (FAS, APO-1) and the death receptor 4 (DR4) (TRAIL-R1) are co-expressed with CD176 on the surface of defined leukemic cells as observed by confocal microscopy and flow cytometry analyses. Further-more, CD95, CD45, CD43 and DR4 are carrier proteins of CD176 in hematopoietic cells recognized by means of sandwich solid-phase enzyme linked immunosorbent assay and co-immunoprecipitation. As shown by microarray analysis, 20 genes which are directly related to the execution, induction or positive regulation of apoptosis, were up-regulated after CD176 antibody treatment of the KG1 cell line. Nine differentially expressed genes observed in the microarray analysis were verified by quantitative real-time polymerase chain reaction in the KG1 and MT4 cell lines. Six genes (DAXX, CASP3, CHUK, RIPK2, NFKBIA and DFFA) out of these nine are involved in five apoptotic pathways: The CD95 signaling pathway, the DR3 and DR4/5 death receptor pathway, caspase cascade in apoptosis, the mitochondrial signaling pathway, and apoptotic DNA fragmentation and tissue homeo-stasis. Thus, we hypothesized that the CD176 antibody binds to the CD176 carbohydrate structure present on apoptosis-associated glycoproteins, such as CD95 and DR4 at the cellular surface, which activates apoptotic pathways, and consequently results in the apoptosis of CD176-positive cells. CD176 is expressed at the surface of human leukemic cells, but is almost absent in normal and benign adult human tissues. Thus, CD176 could be a promising target for anti-tumor therapy based on the induction of tumor-specific apoptosis.

Sialic acids are important sugars at the reducing end of glycoproteins and glycolipids. They are among many other functions involved in cell-cell interactions, host-pathogen recognition and the regulation of serum half-life of glycoproteins. An important modification of sialic acids is O-acetylation, which can alter or mask the biological properties of the parent sialic acid molecule. The nature of mammalian sialate-O-acetyltransferases (EC 2.3.1.45) involved in their biosynthesis is still unknown. We have identified the human CasD1 (capsule structure1 domain containing 1) gene as a candidate to encode the elusive enzyme. The human CasD1 gene encodes a protein with a serine-glycine-asparagine-histidine hydrolase domain and a hydrophobic transmembrane domain. Expression of the Cas1 protein tagged with enhanced green fluorescent protein in mammalian and insect cells directed the protein to the medial and trans-cisternae of the Golgi. Overexpression of the Cas1 protein in combination with α-N-acetyl-neuraminide α-2,8-sialyltransferase 1 (GD3 synthase) resulted in an up to 40% increased biosynthesis of 7-O-acetylated ganglioside GD3. By quantitative real-time polymerase chain reaction, we found up to 5-fold increase in CasD1 mRNA in tumor cells overexpressing O-Ac-GD3. CasD1-specific small interfering RNA reduced O-acetylation in tumor cells. These results suggest that the human Cas1 protein is directly involved in O-acetylation of α2-8-linked sialic acids.

Protein modifications of death receptor pathways play a central role in the regulation of apoptosis. It has been demonstrated that O-glycosylation of TRAIL-receptor (R) is essential for sensitivity and resistance towards TRAIL-mediated apoptosis. In this study we ask whether and how glycosylation of CD95 (Fas/APO-1), another death receptor, influences DISC formation and procaspase-8 activation at the CD95 DISC and thereby the onset of apoptosis. We concentrated on N-glycostructure since O-glycosylation of CD95 was not found. We applied different approaches to analyze the role of CD95 N-glycosylation on the signal transduction: in silico modeling of CD95 DISC, generation of CD95 glycosylation mutants (at N136 and N118), modulation of N-glycosylation by deoxymannojirimycin (DMM) and sialidase from Vibrio cholerae (VCN). We demonstrate that N-deglycosylation of CD95 does not block DISC formation and results only in the reduction of the procaspase-8 activation at the DISC. These findings are important for the better understanding of CD95 apoptosis regulation and reveal differences between apoptotic signaling pathways of the TRAIL and CD95 systems.

The emerging insights into glycan functionality direct increasing attention to monitor core modifications of N-glycans and branch-end structures. To address this issue in histochemistry, a panel of lectins with respective specificities was devised. The selection of probes with overlapping specificities facilitated to relate staining profiles to likely target structures. The experiments on fixed sections of adult murine testis and epididymis were carried out at non-saturating lectin concentrations to visualize high-affinity sites with optimal signal-to-background ratio. They revealed selectivity in lectin reactivity for distinct cell types and segment-dependent staining in the epididymis. Leydig cells, for instance, were reactive with the Sambucus nigra agglutinin and human siglec-2 (CD22), two lectins also separating principal from basal and apical cells in the caput segments I-III of the epididymis. Apical cells were reactive with the Maackia amurensis agglutinin-I, and basal cells with the erythroagglutinin of Phaseolus vulgaris. The reported differences support the concept of lectin staining as cell marker. They thus intimate to study glycogene (genes for glycosyltransferases and lectins) expression and cellular reactivity with tissue lectins. These investigations will be instrumental to assign a role as biochemical signals to the detected staining properties., (© 2010 Blackwell Verlag GmbH.)

Vascular endothelial cells undergo many molecular changes during pathological processes such as inflammation and tumour development. Tumours such as malignant lymphomas affecting bone marrow are dependent on interactions with endothelial cells for (1) site-specific homing and (2) tumour-induced angiogenesis. Modifications in glycosylation are responsible for fine-tuning of distinct endothelial surface receptors. In order to gain a comprehensive insight into the regulation of the endothelial glycome, comprising genes encoding for sugar transporters (sugar s/t), glycosyltransferases (GT), glycan-degrading enzymes (GD) and lectins (GBP), we performed gene profiling analysis of the human bone marrow-derived microvascular endothelial cell line HBMEC-60 that resembles closely in its biological behaviour primary bone marrow endothelial cells. HBMEC were activated by either angiogenic VEGF or the inflammatory cytokine TNF. Approximately 48% (207 genes) of the 432 glycome genes tested were found to be expressed in HBMEC-60 cells. Inflammatory and angiogenic signals produce different profiles of up- or down-regulated glycome genes, most prominent changes were seen under TNF stimulation in terms of signal intensity and number of alterations. Stimulation by VEGF and TNF affected primarily genes encoding for glycosyltransferases and in particular those important for terminal modulation. For instance, an enhanced alpha2,6 sialylation was observed after TNF stimulation at the transcriptional and glycan expression level whereas transcription of ST3Gal1 sialylating in alpha2,3 position was enhanced after VEGF stimulation. Transcriptional analysis of the glycome gives insights into the differential regulation of glycosylation pathways and may help to understand the functional impact of endothelial glycosylation.

Natural antibodies are produced by B lymphocytes in the absence of external antigen stimulation. With their ability to recognize self, altered self and foreign antigens, they comprise an important first-line defence against invading pathogens, but are also important for tissue homeostasis. By recognizing oligosaccharides expressed on tumour cells and modified cell surface structures accompanying necrosis, natural antibodies have an important anti-tumorigenic function. IVIg contains a wide spectrum of specificities presented in normal plasma including natural antibodies and has been shown to exert inhibitory effects on tumour cells through a subfraction of anti-vascular endothelial growth factor immunoglobulin (Ig)G antibodies with anti-angiogenic properties. IgA antibodies also have potent immunomodulatory properties, being able to both induce and suppress immune responses. IgA-mediated inhibitory function is able to inhibit several inflammatory diseases including asthma and glomerulonephritis. Autoantibodies of the IgM type, on the other hand, have shown promising results in the treatment of multiple sclerosis. These autoantibodies promote remyelination rather than modulating inflammation. Oxidation-specific epitopes, as found in atherosclerotic lesions and on apoptotic cells, comprise one important target of natural antibodies. By recognizing these epitopes, natural antibodies neutralize proinflammatory responses and mediate atheroprotection.

The expression of the histo-blood group carbohydrate structures T-nouvelle (Tn, CD175), sialylated Tn (CD175s) and the Thomsen-Friedenreich disaccharide (TF, CD176) on human leukemia cell lines was analyzed by their reactivity with specific monoclonal antibodies in flow cytometry, immunohistology and immunoprecipitation. Expression of sialylated CD176 was evaluated by comparative immunostaining with anti-CD176 antibodies before and after sialidase treatment. While only few cell lines expressed unmasked CD176, sialylated CD176 was present on all hematopoietic cell lines and native lymphocytes examined. CD175 and CD175s are preferentially expressed on erythroblastic leukemia cell lines. CD175s expression in these cells is consistent with the transcription of the gene encoding the key enzyme alpha2,6-sialyltransferase (hST6GalNAc1). The staining intensity was reduced after methanol pretreatment of cells, indicating that these glycans are partially expressed as constituents of glycosphingolipids. Immunoprecipitation and subsequent Western blotting revealed a series of distinct high molecular glycoproteins as carriers for these carbohydrate antigens. CD34 was identified as major carrier of CD176 by immunoprecipitation and microsequencing on a KG-1 subline enriched for CD176 expression. Incubation of several CD176-positive cell lines with anti-CD176 antibodies induced apoptosis of these cells, an effect not observed with anti-CD175/CD175s antibodies. Since the presence of naturally occurring anti-CD176 antibodies may represent a mechanism of immunosurveillance against CD176-positive tumor cells, we propose that sialylation of surface-expressed CD176--among other functions--protects against apoptosis., ((c) 2008 Wiley-Liss, Inc.)

Healthy individuals may contain in their peripheral blood antibodies which are able to destroy human tumour cells mediated either by complement-dependent cytotoxicity or by apoptosis. The largest proportion of these antibodies is of IgM isotype and directed against distinct tumour associated carbohydrate epitopes. Although the origin of these antibodies is not clear they seem to belong to the class of natural antibodies because they are not affinity matured and are encoded by distinct germ-line restricted gene families. It is most likely that this class of natural antibodies has in vivo an anti-tumour protective effect which may contribute to so-called tumour surveillance. On the other hand malignant tumour cells exert mechanisms to counteract such an antibody attack. These comprise soluble factors as well as cell surface expressed membrane complement regulatory proteins (mCRP). Further studies are needed to elucidate molecular mechanisms leading to either tumour destruction induced by natural antibodies or to overcome the protective strategies of the tumour against antibody attack.

During the initiation of tumor associated angiogenesis endothelial cells migrate, adhere to extracellular matrix and form cell-cell contacts. Humoral factors of malignant cells conduct this process. We investigated whether cell surface expression of the carbohydrate blood group determinant Lewis(y) (CD174) and its precursor structure H2 (CD173) on endothelial cells is influenced by soluble factors of tumor cells. Using a bone marrow derived endothelial cell line we observed an enhanced expression of CD173/CD174 and transcription of FUT1, the key enzyme for their synthesis, after treatment with tumor necrosis factor-alpha (TNF-alpha) or conditioned supernatants of the leukemia cell line KG1a. CD173/CD174 are concentrated on pseudopodial extensions responsible for initial contacts between endothelial cells. Endothelial migration induced by TNF-alpha could be diminished by antibodies to CD174 as well as by siRNA induced downmodulation of FUT1 transcription. Endothelial FUT1-siRNA-transfectants displayed impaired in vitro angiogenesis when cultivated on extracellular matrix and in spheroid assays. In vivo upregulation of CD174 expression was observed immunocytologically in capillaries of tumor-infiltrated tissue. Together, our observations point to a tumor induced transcription of endothelial FUT1 and consequently an enhanced expression of CD174 which is involved in migration and early cell-cell contacts during tumor associated angiogenesis., ((c) 2008 Wiley-Liss, Inc.)

BeWo human choriocarcinoma cells have recently been identified as an in vitro model of adhesion of Plasmodium falciparum-infected erythrocytes to the major placental receptor chondroitn-4-sulphate (CSA). In this study, we show that treatment of BeWo cells with tumour necrosis factor-alpha and/or interferon-gamma, cytokines linked with pregnancy-associated malaria and poor pregnancy outcome, does not alter the expression of cell surface CSA. BeWo cells do not express the common P. falciparum adhesion receptor cluster of differentiation 36 (CD36) on the cell surface, and this was unchanged after treatment with cytokines. These data demonstrate that in vitro cultured BeWo cells mimic the P. falciparum adhesion receptor expression profile of ex vivo placental cytotrophoblast cells.

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to placental chondroitin-4-sulfate (CSA) has been linked to the severe disease outcome of pregnancy-associated malaria. Consequently, sulfated polysaccharides with inhibitory capacity may be considered for therapeutic strategies as anti-adhesive drugs. During in vitro screening a regioselectively modified cellulose sulfate (CS10) was selected as prime candidate for further investigations because it was able to inhibit adhesion to CSA expressed on CHO cells and placental tissue, to de-adhere already bound infected erythrocytes, and to bind to infected erythrocytes. Similar to the undersulfated placental CSA preferred by placental-binding infected erythrocytes, CS10 is characterized by a clustered sulfate pattern along the polymer chain. In further evaluation of its effects on P. falciparum interactions with host erythrocytes, we now show that CS10 inhibits the in vitro asexual growth of parasites in erythrocytes. Furthermore, we show that CS10 interferes with C1 of the classical complement pathway but not with MBL of the lectin pathway. In order to gain insights into the possible interactions of CS10 with known parasite receptors at the molecular level, we designed 3D-structures of characteristic stretches of CS10. CS10 fragments with clustered sulfate groups showed complex patterns of hydrophobic and hydrophilic patches most likely suitable for interactions with protein binding partners. The significance of CS10 interactions with the complement system as well as its anti-malarial effect for prospective drug application are discussed.

The disialoganglioside GD3 (CD60 a) and its O-acetylated variants have previously been described as surface molecules of human T lymphocytes of the peripheral blood system. Here we report the expression of the 9-O-, and 7-O-acetylated disialoglycans of GD3 (CD60 b and CD60 c respectively) on human tonsillar lymphocytes. CD60 b and c are surface-expressed on activated germinal centre B cells and colocalize in raft-like structures on the cell surface together with the cytoplasmic tyrosine kinase Lyn and Syk. Addition of CD60 b and c mAb together with anti-IgM/IL-4 to in vitro cultivated tonsillar B cells resulted in a costimulatory effect. During spontaneous and staurosporine-induced apoptosis a distinct population of activated annexin V+/CD60 b+/CD60 c- B cells was observed. CD60 b and c are also found on cells of the extrafollicular T cell area. On tonsillar T cells, CD60 b mAb had a costimulatory effect together with PHA while CD60 c mAb alone was sufficient to induce proliferation. In further contrast to B cells, during apoptosis a distinct CD60 b+ T cell subpopulation was not observed. Together, surface-expressed CD60 b and c are differently expressed on tonsillar B and T cells and may be involved in the regulation of activation and apoptosis of lymphocytes in secondary lymphatic tissue.

A panel of sulfated oligosaccharides was tested for antimalarial activity and inhibition of adhesion to the placental malaria receptor chondroitin-4-sulfate (CSA). The heparan sulfate mimetic PI-88, currently undergoing phase II anticancer trials, displayed the greatest in vitro antimalarial activity against Plasmodium falciparum (50% inhibitory concentration of 7.4 microM) and demonstrated modest adhesion inhibition to cell surface CSA.

The immune system works through leukocytes interacting with each other, with other cells, with tissue matrices, with infectious agents, and with other antigens. These interactions are mediated by cell-surface glycoproteins and glycolipids. Antibodies against these leukocyte molecules have provided powerful tools for analysis of their structure, function, and distribution. Antibodies have been used widely in hematology, immunology, and pathology, and in research, diagnosis, and therapy. The associated CD nomenclature is commonly used when referring to leukocyte surface molecules and antibodies against them. It provides an essential classification for diagnostic and therapeutic purposes. The most recent (8th) Workshop and Conference on Human Leukocyte Differentiation Antigens (HLDA), held in Adelaide, Australia, in December 2004, allocated 95 new CD designations and made radical changes to its aims and future operational strategy in order to maintain its relevance to modern human biology and clinical practice.

Carbohydrates are implicated in many of the invasive and adhesive interactions that occur between Plasmodium falciparum malaria parasites and human host cells, including invasion of sporozoites into hepatocytes, entry of merozoites into new host erythrocytes during asexual blood-stage replication, adhesion of infected erythrocytes to uninfected erythrocytes (rosetting) and to a number of host endothelial receptors including ICAM-1, CD36 and chondroitin-4-sulphate. In addition to increasing our understanding of host-parasite interactions, the investigation of carbohydrates with differing levels and patterns of sulphation as inhibitors may contribute to the development of novel therapeutics targeting malaria. Here we show that three polysaccharides derived from seaweed (carrageenans) with differing sulphation levels and patterns can inhibit the in vitro erythrocytic invasion and growth of both drug sensitive and drug resistant P. falciparum lines and the adhesion of parasitized erythrocytes to the human glycoprotein CD36.

Adhesion of erythrocytes infected with the malaria parasite Plasmodium falciparum to human host receptors is a process associated with severe malarial pathology. A number of in vitro cell lines are available as models for these adhesive processes, including Chinese hamster ovary (CHO) cells which express the placental adhesion receptor chondroitin-4-sulphate (CSA) on their surface. CHO-745 cells, a glycosaminoglycan-negative mutant CHO cell line lacking CSA and other reported P. falciparum adhesion receptors, are often used for recombinant expression of host receptors and for receptor binding studies. In this study we show that P. falciparum-infected erythrocytes can be easily selected for adhesion to an endogenous receptor on the surface of CHO-745 cells, bringing into question the validity of using these cells as a tool for P. falciparum adhesin expression studies. The adhesive interaction between CHO-745 cells and parasitized erythrocytes described here is not mediated by the known P. falciparum adhesion receptors CSA, CD36, or ICAM-1. However, we found that CHO-745-selected parasitized erythrocytes bind normal human IgM and that adhesion to CHO-745 cells is inhibited by protein A in the presence of serum, but not in its absence, indicating a non-specific inhibitory effect. Thus, protein A, which has been used as an inhibitor for a recently described interaction between infected erythrocytes and the placenta, may not be an appropriate in vitro inhibitor for understanding in vivo adhesive interactions.

Adhesion of Plasmodium falciparum-infected erythrocytes to placental chondroitin 4-sulfate (CSA) has been linked to the severe disease outcome of pregnancy-associated malaria. Soluble polysaccharides that release mature-stage parasitized erythrocytes into the peripheral circulation may help elucidate these interactions and have the potential to aid in developing therapeutic strategies. We have screened a panel of 11 sulfated polysaccharides for their capacities to inhibit adhesion of infected erythrocytes to CSA expressed on CHO-K1 cells and ex vivo human placental tissue. Two carrageenans and a cellulose sulfate (CS10) were able to inhibit adhesion to CSA and to cause already bound infected erythrocytes to de-adhere in a dose-dependent manner. CS10, like CSA and in contrast to all other compounds tested, remained bound to infected erythrocytes after washing and continued to inhibit binding. Both carrageenans and CS10 inhibited adhesion to placental tissue. Although highly sulfated dextran sulfate can inhibit CSA-mediated adhesion to CHO cells, this polysaccharide amplified adhesion to placental tissue severalfold, demonstrating the importance of evaluating inhibitory compounds in systems as close to in vivo as possible. Interestingly, and in contrast to all other compounds tested, which had a random distribution of sulfate groups, CS10 exhibited a clustered sulfate pattern along the polymer chain, similar to that of the undersulfated placental CSA preferred by placental-tissue-binding infected erythrocytes. Therefore, the specific anti-adhesive capacity observed here seems to depend not only on the degree of charge and sulfation but also on a particular pattern of sulfation.

Complex glycosylated glycoproteins, glycolipids and proteoglycans are expressed on the cell surface and are also found as constituents of the extracellular matrix (ECM). Interactions of the carbohydrate moiety of these macromolecules with specific receptors (lectins) are involved in many functions of immune cells such as cell-cell or cell-ECM adhesion, recognition, and neutralization of pathogens and regulation of apoptosis. For studies on live cells mAbs recognizing distinct oligosaccharide structures are useful tools because in contrast to other analytical methods of carbohydrate biochemistry they are able to react with glycans in the complex sterical context of the cell surface. In general expression patterns of carbohydrate mAbs depend on (i) the number and type of carriers to which the glycans are linked (glycoproteins, glycolipids), (ii) the steric situation on the cell surface, and (iii) modifications of the basic glycotope (different branching, chain length, masking by sialylation, sulphation or fucosylation).

Sialic acids as terminal residues of oligosaccharide chains play crucial roles in several cellular recognition events. Exploiting the selective affinity of Achatinin-H toward N-acetyl-9-O-acetylneuraminic acid-alpha2-6-GalNAc, we have demonstrated the presence of 9-O-acetylated sialoglycoproteins (Neu5,9Ac(2)-GPs) on lymphoblasts of 70 children with acute lymphoblastic leukemia (ALL) and on leukemic cell lines by fluorimetric HPLC and flow cytometric analysis. This study aims to assess the structural aspect of the glycotope of Neu5,9Ac(2)-GPs(ALL) and to evaluate whether these disease-specific molecules can be used to monitor the clinical outcome of ALL. The Neu5,9Ac(2)-GPs(ALL) were affinity-purified, and three distinct leukemia-specific molecular determinants (135, 120, and 90 kDa) were demonstrated by SDS-PAGE, western blotting, and isoelectric focusing. The carbohydrate epitope of Neu5,9Ac(2)-GPs(ALL) was confirmed by using synthetic sialic acid analogs. The enhanced presence of anti-Neu5,9Ac(2)-GP(ALL) antibody in ALL patients prompted us to develop an antigen-ELISA using purified Neu5,9Ac(2)-GPs(ALL) as coating antigens. Purified antigen was able to detect leukemia-specific antibodies at presentation of disease, which gradually decreased with treatment. Longitudinal monitoring of 18 patients revealed that in the early phase of the treatment patients with lower anti-Neu5,9Ac(2)-GPs showed a better prognosis. Minimal cross-reactivity was observed in other hematological disorders (n = 50) like chronic myeloid leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia, and non-Hodgkin's lymphoma as well as normal healthy individuals (n = 21). This study demonstrated the potential of purified Neu5,9Ac(2)-GPs(ALL) as an alternate tool for detection of anti-Neu5,9Ac(2)-GP antibodies to be helpful for diagnosis and monitoring of childhood ALL patients.

Surface expressed negatively charged sialoglycans contribute to the regulation of adhesive cellular interactions and are thus involved in the growth and differentiation of hematopoietic progenitor cells. In particular, the cell surface sialylation state may govern the liberation of CD34+ hematopoietic precursors from bone marrow stroma cells and extracellular matrix. In order to assess the overall surface sialylation of live human CD34+ hematopoietic precursor cells, we applied a previously described flow cytometric enzyme assay. Cells with and without sialidase pretreatment were incubated in the presence of fluorescent CMP-sialic acid and exogenous ST6GalI. Thus sialylation of surface-expressed lactosamine residues was analysed. We demonstrated that surface lactosamines of CD34+ precursors derived from bone marrow and peripheral blood are over 95% sialylated, predominantly in alpha2-6 linkage. These results are in accordance with flow cytometric analysis of surface lectin staining. Sialic acid specific lectins MAA and SNA were strongly bound whereas SBA, VVA, and PNA became reactive only after sialidase pretreatment. CD34+ leukemia cell lines TF1 and KG1a also showed a high degree of surface sialylation, whereas cell line KG1 expressed to the largest extent free lactosamines. In these cell lines, alpha2-6 and alpha2-3 sialylated residues were present in equal amounts. In a variation of the flow cytometric enzyme assay, live cells were incubated without exogenous STGal I to measure the activity of endogenous ecto-sialyltransferase. Ecto sialyltransferase activity was observed in all CD34+ cells which was able to resialylate major surface glycoproteins such as HLA Class I, CD45, CD43, and CD34. The ecto-sialyltransferase may serve to maintain or increase surface sialylation rapidly without de novo synthesis.

Sialic acids as terminal residues of oligosaccharide chains play a crucial role in several cellular recognition events. The presence of sialic acid on promastigotes of Leishmania donovani, the causative organism of Indian visceral leishmaniasis, was demonstrated by fluorimetric high-performance liquid chromatography showing Neu5Ac and, to a minor extent, Neu5,9Ac2. The presence of Neu5Ac was confirmed by GC/MS analysis. Furthermore, binding with sialic acid-binding lectins Sambucus nigra agglutinin (SNA), Maackia amurensis agglutinin (MAA), and Siglecs showed the presence of both alpha2,3- and alpha2,6-linked sialic acids. No endogenous biosynthetic machinery for Neu5Ac could be demonstrated in the parasite. Concomitant western blotting of parasite membranes and culture medium with SNA demonstrated the presence of common sialoglyconjugates (123, 90, and 70 kDa). Similarly, binding of MAA with parasite membrane and culture medium showed three analogous sialoglycans corresponding to 130, 117, and 70 kDa, indicating that alpha2,3- and alpha2,6-linked sialoglycans are adsorbed from the fetal calf serum present in the culture medium. L. donovani promastigotes also reacted with Achatinin-H, a lectin that preferentially identifies 9-O-acetylated sialic acid in alpha2-->6 GalNAc linkage. This determinant was evidenced on parasite cell surfaces by cell agglutination, ELISA, and flow cytometry, where its binding was abolished by pretreatment of cells with a recombinant 9-O-acetylesterase derived from the HE1 region of the influenza C esterase gene. Additionally, binding of CD60b, a 9-O-acetyl GD3-specific monoclonal antibody, corroborated the presence of terminal 9-O-acetylated disialoglycans. Our results indicate that sialic acids (alpha2-->6 and alpha2-->3 linked) and 9-O-acetyl derivatives constitute components of the parasite cell surface.

Summary: Sialyltransferases sialylate plasma glycoproteins in hepatocytes and may (as hepatic key enzymes) constitute markers for liver diseases. We examined expression of the prevalent alpha2,6 sialyltransferase (ST6Gal I) and sialoglycans in normal liver, cirrhotic liver, and hepatocellular carcinoma (HCC) using a new ST6Gal I-specific mAb and recombinant fusion proteins of CD22 and sialoadhesin recognizing alpha2,6- or alpha2,3-sialylated glycans in immunohistology and flow cytometry. In normal and cirrhotic liver, ST6Gal I and sialoglycans were localized in the Golgi region of hepatocytes surrounding the bile canaliculi and along the bile canaliculi, respectively. Sialoglycans were additionally recognized in Kupffer cells, bile ducts, endothelial cells, and oval cells. Well-differentiated and moderately differentiated HCC showed Golgi and diffuse cytoplasmic staining of ST6Gal I and sialoglycans, whereas the cytoplasmic staining for ST6Gal I and sialoglycans was decreased or even absent in poorly differentiated HCC. Detection of sialoglycans by the recombinant fusion proteins in Western blots of cell lysates derived from cell lines revealed two major double bands of sialoglycoproteins at 65 and 120 kDa for hepatocytes, three major bands at 54, 49, and 44 kDa for colonic epithelial cells, and one band at 60 kDa for endothelial cells. Our results describe the expression patterns of ST6Gal I and sialoglycans in various liver tissues and demonstrate an altered expression of these structures between benign and malignant hepatocellular lesions.

Intercellular adhesion molecule 3 (ICAM-3) is a leukocyte-specific receptor involved in primary immune responses. We have investigated the interaction between ICAM-3 and ezrin/radixin/moesin (ERM) proteins and its role in LFA-1-induced cell-cell interactions and membrane positioning of ICAM-3 in polarized migrating lymphocytes. Protein-protein binding assays demonstrated a phosphatidylinositol 4,5-bisphosphate-induced association between ICAM-3 and the amino-terminal domain of ERM proteins. This interaction was not essential for the binding of ICAM-3 to LFA-1. Dynamic fluorescence videomicroscopy studies of cells demonstrated that moesin and ICAM-3 coordinately redistribute on the plasma membrane during lymphocyte migration. Furthermore, overexpression of the amino-terminal domain of moesin, which lacks the consensus moesin actin-binding site, caused the subcellular mislocalization of ICAM-3. A CD4 chimerical protein containing the cytoplasmic tail of ICAM-3 was targeted to the trailing edge. Point mutation of Ser(487), Ser(489), and Ser(496) to alanine in the juxtamembrane region of ICAM-3 significantly impaired both ERM binding and polarization of ICAM-3. ERM-directed polarization of ICAM-3 was also impaired by phosphorylation-like mutation of Ser(487) and Ser(489), but not of Ser(496). Our results underscore the key role of specific serine residues within the cytoplasmic region of ICAM-3 for its ERM-directed positioning at the trailing edge of motile lymphocytes.

The most recent Human Leucocyte Differentiation Antigen Workshop ("HLDA7") took place in 2000 in Harrogate, UK and the proceedings are about to be published (Leucocyte Typing VII). New Sections were introduced in this Workship (Dendritic cells, Stem/progenitor cells, Erythroid cells and Carbohydrate Structures) and monoclonal antibodies were selected for which at least some molecular data were already available (to avoid "blind" screening of reagents against known specificities). A total of more than 80 new CD specificities were established (previously the average was less than 30 new CD specificities per Workshop) and these are listed in this article. There is already evidence for the existence of many new leucocyte surface molecules for study at the next HLDA Workshop (in Adelaide in 2004), and we have listed in this article a number of such potential CD candidates (identified following the production of monoclonal antibodies or via gene cloning). There are also today an increasing number of lineage- and/or stage-restricted leucocyte-associated molecules localised within the cell cytoplasm (or nucleus): they will certainly prove of intense in the future for many laboratories studying human haematopoietic cells (regardless of whether a new "intracellular CD" categorisation scheme is devised for such molecules).