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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 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.

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.

Short Title Polyelectrolyte nanoparticles and vascular endothelial cells. Abstract 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.

Endoplasmic reticulum (ER) stress results from protein unfolding/misfolding during cellular maturation, which requires a coordinated action of several chaperones and enzymes and Ca2+ signalling. ER-stress possibly has a positive effect on survival of pancreatic cancer cell. Therefore, detailed insights into this complex signaling network are urgently needed. Here, we systematically analyzed the impact of ER stress-mediated unfolded protein response (UPR) and Ca2+-signaling cross-talk for the survival of pancreatic adenocarcinoma (PDAC) cells. We observed enhanced ER activity and initiation of UPR signaling induced by a carbazole alkaloid (mahanine). This event triggers a time-dependent increase of intracellular Ca2+ leakage from ER and subsequently Ca2+ signaling induced by enhanced reactive oxygen species (ROS) produced by this pro-oxidant agent. In addition, we observed an altered glycosylation, in particular with regard to reduced linkage-specific sialic acids possibly due to decreased sialyltransferase activity. Changes in sialylation entailed enhanced expression of the ganglioside GD3 in the treated cells. GD3, an inducer of apoptosis, inhibited pancreatic xenograft tumor. Taken together, our study describes a molecular scenario how PDAC cells are driven into apoptosis by mahanine by UPR-driven ER stress-associated and ROS-mediated calcium signaling and possibly defective sialylation.

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.

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.

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 αvβ3 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.

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.

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.

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.

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.

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.

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.

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.

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

aThe Nuffield Department of Clinical Laboratory Sciences, University of Oxford, Oxford, United Kingdom; bCentre d'Immunologie, Parc Scientifique de Luminy, Cedex, France; cFaculte de Medicine de Creteil, Cedex, France; dDivision of Immunity and Infection, MRC Center for Immune Regulation, Department of Rheumatology, University of Birmingham, Edgbaston, Birmingham, United Kingdom; eJohns Hopkins Comprehensive Cancer Center, Baltimore, Maryland, USA; fDepartment of Microbiology, University of Washington, Seattle, Washington, USA; gCLB, Department of Experimental Immunohematology, Amsterdam, The Netherlands; hLaboratory of Molecular Hematology/Division of Molecular Medicine, North Shore University Hospital, Manhasset, New York, USA; iKinderklinik, Medizinische Hochschule Hannover, Hannover, Germany; jMater Medical Research Institute, Aubigny Place, Mater Hospital, Queensland, Australia; kInstitute of Molecular Genetics AS CR, Prague, Czechoslovakia; lInstitut fur Immunologie der Univ. Heidelberg, Heidelberg, Germany, mProfessor of Biochemistry, University of Illinois College of Medicine, Urbana, Illinois, USA; nGerman Cancer Research Center, Heidelberg, Germany; oNational Institute of Health, Bethesda, Maryland, USA; pDirector of Biology Celltech RD qInstitute for Research in Biomedicine, Bellinzona, Switzerland; rUniversity of Adelaide, Department of Pediatrics, Women's and Children's Hospital, South Australia, Australia

This paper reviews the Seventh Human Leucocyte Differentiation Antigen (HLDA7) workshop. Due to the limitations of “blind” antibody screening, which had been evident at the previous meeting in 1996, participants at HLDA7 adopted a more selective approach to the choice of antibodies by identifying new CD specificities. This resulted in the addition of more than 80 new CD specificities. Plans for the eighth and subsequent workshops are also previewed.

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.

Sialic acid, as a terminal saccharide residue on cell surface glycoconjugates, plays an important role in a variety of biological processes. In this study, we investigated subclones of the human B lymphoma cell line BJA-B for differences in the glycosylation of cell surface glycoconjugates, and studied the functional implications of such differences. With respect to the expression level of most of the tested B cell-associated antigens, as well as the presence of penultimate saccharide moieties on oligosaccharide chains, subclones were phenotypically indistinguishable. Marked differences among subclones, however, were found in the overall level of glycoconjugate sialylation, involving both alpha-2,6 and alpha-2,3-linked sialic acid residues. Accordingly, subclones were classified as highly- (group I) or hyposialylated (group II). The function of two sialic acid-dependent receptor-mediated processes is correlated with the sialylation status of BJA-B subclones. Susceptibility to and binding of the B lymphotropic papovavirus (LPV) was dependent on a high sialylation status of host cells, suggesting that differential sialylation in BJA-B cells can modulate LPV infection via its alpha-2,6-sialylated glycoprotein receptor. CD95-mediated apoptosis, induced by either the human CD95 ligand or a cytotoxic anti-CD95 monoclonal antibody, was drastically enhanced in hyposialylated group II cells. An increase in endogenous sialylation may be one antiapoptotic mechanism that converts tumor cells to a more malignant phenotype. To our knowledge, this is the first report demonstrating that differential sialylation in a clonal cell line may regulate the function of virus and signal-transducing receptors.

We found that human peripheral B and T cells differed in the surface expression of alpha2-6 sialylated type 2 chain glycans. In contrast to B cells, T cells expressed only sialoglycans with repeated N-acetyllactosamine (Galss1-4GlcNAc) disaccharides. This finding was based on the specificity of the monoclonal antibodies HB6, HB9 (CD24), HD66 (CDw76), FB21, and CRIS4 (CDw76) with the alpha2-6 sialylated model gangliosides IV6NeuAcnLc4Cer (2-6 SPG), VI6NeuAcnLc6Cer (2-6 SnHC), VIII6NeuAcnLc8Cer (2-6 SnOC), and X6NeuAcnLc10Cer (2-6 SnDC). We found that, in addition to their common requirement of an alpha2-6 bound terminal sialic acid for binding, the antibodies displayed preferences for the length of the carbohydrate backbones. Some of them bound mainly to 2-6 SPG with one N-acetyllactosamine (LacNAc) unit (HB9, HD66); others preferentially to 2-6 SnHC and 2-6 SnOC, with two and three LacNAc units, respectively (HB6 and FB21); and one of them exclusively to very polar alpha2-6 sialylated type 2 chain antigens (CRIS4) such as to 2-6 SnOC and even more polar gangliosides with three and more LacNAc units. These specificities could be correlated with the cellular binding of the antibodies as follows: whereas all antibodies bound to human CD 19 positive peripheral B cells, their reactivity with CD3 positive T cells was either nearly lacking (HD66, HB9), intermediate (about 65%: HB6, FB21) or strongly positive (CRIS4, 95%). Thus, the binding of the antibodies to 2-6 sialylated glycans with multiple lactosamine units appeared to determine their binding to T-cells.

SUMMARYAge-related changes in functional subsets of lymphocytes may influence the potential to build up immune responses. In particular, the capacity of tonsillar lymphocytes to counter infections may be altered during ageing. In order to address this question we investigated the proportional distribution of several subsets of tonsillar T and B cells with regard to ageing. Tonsils were derived from 119 patients between 2 and 65 years of age. Lymphocyte subsets were monitored by three-colour fluorescence of relevant CD markers in flow cytometry. As a general tendency the percentage of CD3+ T cells steadily increased whereas that of CD19+ B cells decreased at the same time. No significant differences were observed between lymphocytes of patients with and without inflammatory history of the tonsils. The percentage of CD8+ T cells declined whereas that of CD4+ T cells increased during the same time span. CD45RA+ T cells increased during the first two decades of life and gradually decreased thereafter. In contrast, CD45RO+ T cells showed an opposite trend. No differences were seen in the population of CD3−/CD56+ natural killer (NK) cells. The mature B cell marker CD40 showed no significant changes during ageing. However, CD38+ B cells, representing B cells of late maturation stages, dramatically declined up to the age of 65. In a similar manner the CD5+ subpopulation of B cells decreased during ageing. Substantial changes in major tonsillar T and B cell populations as shown in this study may have an impact on the ageing process of the immune system.

Chemokines as well as the signaling through the adhesion molecules intercellular adhesion molecule (ICAM)-3 and CD43 are able to induce in T lymphocytes their switching from a spherical to a polarized motile morphology, with the formation of a uropod at the rear of the cell. We investigated here the role of CD43 in the regulation of T-cell polarity, CD43-cytoskeletal interactions, and lymphocyte aggregation. Pro-activatory anti-CD43 monoclonal antibody (MoAb) induced polarization of T lymphocytes with redistribution of CD43 to the uropod and the CCR2 chemokine receptor to the leading edge of the cell. Immunofluorescence analysis showed that all three ezrin-radixin-moesin (ERM) actin-binding proteins localized in the uropod of both human T lymphoblasts stimulated with anti-CD43 MoAb and tumor-infiltrating T lymphocytes. Radixin localized at the uropod neck, whereas ezrin and moesin colocalized with CD43 in the uropod. Biochemical analyses showed that ezrin and moesin coimmunoprecipitated with CD43 in T lymphoblasts. Furthermore, in these cells, the CD43-associated moesin increased after stimulation through CD43. The interaction of moesin and ezrin with CD43 was specifically mediated by the cytoplasmic domain of CD43, as shown by precipitation of both ERM proteins with a GST-fusion protein containing the CD43 cytoplasmic tail. Videomicroscopy analysis of homotypic cell aggregation induced through CD43 showed that cellular uropods mediate cell-cell contacts and lymphocyte recruitment. Immunofluorescence microscopy performed in parallel showed that uropods enriched in CD43 and moesin localized at the cell-cell contact areas of cell aggregates. The polarization and homotypic cell aggregation induced through CD43 was prevented by butanedione monoxime, indicating the involvement of myosin cytoskeleton in these phenomena. Altogether, these data indicate that CD43 plays an important regulatory role in remodeling T-cell morphology, likely through its interaction with actin-binding proteins ezrin and moesin. In addition, the redistribution of CD43 to the uropod region of migrating lymphocytes and during the formation of cell aggregates together with the enhancing effect of anti-CD43 antibodies on lymphocyte cell recruitment suggest that CD43 plays a key role in the regulation of cell-cell interactions during lymphocyte traffic.

Summary Carcinoma of the breast is the second most frequent tumour in African females. Breast carcinomas in African females appear about a decade earlier and follow a more aggressive clinical course than those in developed countries. To elucidate this difference we investigated 63 biopsied benign lesions of the female breast for their potential to malignant progression. We also performed histologic typing and grading of 184 female breast carcinomas received at the Muhimbili University Hospital in Dar es Salaam, Tanzania. Fibrocystic disease and fibroadenomas were the most frequent lesions. The majority of patients with fibrocystic disease had no proliferative lesion and thus were not at a significantly increased risk of developing breast carcinomas. For fibroadenomas, no indication for precancerous lesions was found. The vast majority of breast carcinomas investigated were invasive. As a striking feature, the majority of those studied (66%) were of the non-special type (NST), displaying a more aggressive behaviour than the remaining tumours of the special type (ST). In the group of ST tumours, cribriform types constituted 41 % of the cases which may be a special feature of the carcinomas in African females. Among the NST, the tumours were either of grade II or grade III, whereas in ST, 25% of the cases were of grade I. Since histology observed in this study is comparable to that seen in patients from the Western society, late hospital presentation with advanced tumour stages may be a major reason for differences in clinical behaviour between African and Western females. A genetic factor, however, may be an important contributing factor.

Gangliosides with a terminal 9-O-acetylated disialosyl group (CDw60 structures) show a restricted surface expression on human leukocytes. Hithereto, they have only been detected on subpopulations of human T lymphocytes. Using the defined CDw60 antibody UM4D4 and two new antibodies with preferential CDw60 activities, F6 and Z17, we demonstrate for the first time that CDw60 is an activation marker on human B lymphocytes. In vitro phorbol ester-stimulated human peripheral blood B lymphocytes as well as in vivo activated tonsillar B lymphocytes became CDw60 positive. CDw60 expression of these cells exceeds that of resting and activated T-lymphocytes.

Proteoglycans interact with soluble proteins such as growth factors and thereby regulate extracellular signals. During B lymphocyte maturation, secretion of proteoglycans may be functionally related to the different requirements of the respective maturation stage. In order to address this question we compared structures of proteoglycans released by three B lymphocyte lines which correspond to different maturation stages. Plasma-cell type U266 cells secreted the largest proteoglycans (150 kDa), followed by mature B cells JOK-1 (130 kDa) and pre-B cells Nalm 6 (90 kDa). On average, secreted proteoglycans carried four glycosaminoglycan chains with molecular masses ranging each from 32 kDa (U266) to 23 kDa (Nalm 6). All three cell lines secreted more than 90% of their proteoglycans possessing chondroitin sulfate chains having chondroitin-4-sulfate (delta Di-4S) as the prevalent disaccharide unit. In these proteochondroitin sulfates, unsulfated chondroitin (delta Di-0S) was present in smaller quantities and chondroitin-6-sulfate (delta Di-6S)-containing proteoglycan was released only by Nalm 6 and U266 cells. Cell line Nalm 6 exclusively produced proteochondroitin sulfate, whereas in culture medium of JOK-1 and U266 a small amount of proteoheparan sulfate was found also. In all three cell lines, treatment with chondroitinase ABC released a protein of 30 kDa and chemical deglycosylation resulted in a core protein of 21 kDa. In addition to pure proteochondroitin sulfate, a small portion of proteoheparan sulfate with a protein moiety of 30 kDa was detected after heparitinase treatment in supernatants of JOK-1 and U266. Thus, our results indicate that released proteoglycans may undergo modulations in their glycosaminoglycan moieties during B-cell differentiation. This may have functional consequences at the level of growth factor regulation.

The L1 adhesion molecule is a member of the immunoglobulin superfamily which is expressed by neural and hematopoietic cells. L1 is primarily a cell surface molecule but in its released form it becomes embedded in the extracellular matrix. In addition to the established L1-L1 homotypic interaction, L1 can bind to alpha v beta 3 in the human. The 6th Ig-like domain is critical for this function. We now demonstrate that a fusion protein containing the 6th Ig-like domain of L1 (6.L1-Fc) can support the migration of human MED-B1 (alpha v beta 3+) but not of Nalm-6 cells (alpha 5 beta 1+). The migration was blocked in the presence of a mab to alpha v beta 3 and was not seen on a 6.L1-Fc in which the RGD site was mutated. Activation of human T lymphocytes in the presence of PHA and PMA led to the induction of alpha v beta 3 and alpha v beta 5 expression and concomitantly induced migration of the cells on 6.L1-Fc. The migration was blocked by mabs to alpha v beta 3 but not to alpha v beta 5. Our results suggest that L1 exposed at the cell surface or as a matrix constituent can serve as a potent substrate for alpha v beta 3 mediated cell migration.

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.

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.

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. P20652 (VLID)1679798

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.

The alpha 5 beta 1 integrin (CD49e/CD29), a heterodimeric membrane protein, is the "classical" fibronectin receptor on many cell types. During B-cell ontogeny, expression of the alpha 5-subunit is developmentally regulated. The alpha 5 beta 1 is decisive for migration on fibronectin substrate and very likely cooperates with other adhesion molecules in transvascular trafficking. To test whether alpha 5 beta 1 influences local growth vs. disseminative spread of neoplastic B-cells in vivo, human B-cell lines mimicking different maturational stages were transferred s.c. into severe combined immunodeficiency (SCID) mice and examined for alpha 5 beta 1 expression and for adherence on fibronectin substrate in vitro and ex vivo. All cell lines were locally tumorigenic. Dissemination was observed in all animals carrying Nalm-6 tumors, in one animal with a BL 60 and in 2 mice carrying a Raji tumor. By contrast, Daudi, BJAB and U266 tumors did not disseminate. As evidenced by immunohistochemistry and flow cytometry, all lines and their tumors were to various extents beta 1-positive but showed considerable differences in alpha 5 expression. The functional surface expression of alpha 5 beta 1 correlated with fibronectin adherence of the lines. Daudi expressed alpha 5 beta 1 in a non-functional configuration which was rendered functional only upon applying high concentrations of Mg++ and Mn++. B-cell lines functionally expressing alpha 5 beta 1 at high or moderate levels disseminated in SCID mice while alpha 5-negative lines and Daudi did not. These results support the conclusion drawn from an earlier in situ analysis of human B-cell lymphomas/leukemias that the alpha 5 beta 1 integrin contributes to the disseminative phenotype of malignant B cells.

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.

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.

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.

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.

The expression of sialic acids (Sia) is highly conserved in deuterostomes, i.e., from echinoderms to humans. They constitute components of cell surface glycoproteins and gangliosides, where they occupy mainly the terminal position as individual monosaccharides and, more rarely, as oligo- or polymers. They are frequently found in secreted glycoconjugates and in oligosaccharides, mainly of blood serum, milk, and mucus secretions [1, 2].

The expression of sialic acids (Sia) is highly conserved in deuterostomes, i.e., from echinoderms to humans. They constitute components of cell surface glycoproteins and gangliosides, where they occupy mainly the terminal position as individual monosaccharides and, more rarely, as oligo- or polymers. They are frequently found in secreted glycoconjugates and in oligosaccharides, mainly of blood serum, milk, and mucus secretions [1, 2].