Abstracts: Poster Session,Group A – Adhesion Angiogenesis and Motility

Past studies have shown that carbohydrate residues reactive with the Griffonia simplicifolia isolectin B₄ (GS I-B4) are present on the surface of highly-malignant murine sarcoma cells but are lacking or expressed in much lower amounts on the surface of low-malignant cells isolated from the same parent tumors (Am J Pathol 111: 27; J Nat Cancer Inst 71: 1281). In the present study it is shown that an antibody which recognizes the trisaccharide Galα1-3Galβ1-4GlcNAc- is reactive with the highly-malignant cells but is non-reactive with the low-malignant cells. Further studies show that the high-malignant cells not only bind GS I-B₄ but also bind Evonymus europaea lectin (which like GS I-B₄ recognizes terminal galactose in α1-3 linkage) and Erythina crystagalli lectin (which recognizes sub-terminal galactose in the β1-4 linkage – e.g., Galβ1-4GlcNAc). In contrast, the low malignant cells bind Erythina crystagalli lectin as efficiently as the high malignant cells but do not bind (or bind much smaller amounts of) either GS I-B₄ or Evonymus europaea lectin. The present studies also show that there is no significant difference between high- and low-malignant cells in expression of α-galactosidase activity. In contrast, the high-malignant cells express high levels of α-galactosyl transferase activity while this enzyme is virtually undetectable in low-malignant cells. Taken together, these studies indicate that differential expression of a single monosaccharide residue distinguishes high- and low-malignant murine sarcoma cells. These studies also identify a mechanism to account for surface carbohydrate differences between the high- and low-malignant cells.     

Species-restricted effects of human and mouse lymphokines on macrophages

Lymphokines produced by antigenic or mitogenic stimulation of human, guinea pig and mouse lymphocytes were tested for their effects on monocytes or macrophages of the same and heterologous species, to determine whether there is any species restriction in their reactivity. Supernatants from lymphocyte cultures were tested for migration inhibitory activity in an indirect agarose microdroplet assay and for their ability to augument cytolytic activity of macrophages or monocytes in a [3H]thymidine-release assay. Supernatants of human peripheral blood mononuclear cells, stimulated with phytohemagglutinin or purified protein derivative of tuberculin (PPD) were able to strongly inhibit the migration of human monocytes and guinea pig peritoneal exudate cells (PEC), but had no detectable effect on mouse PEC. The human supernatants could also significantly augment the cytolytic activity of human monocytes, but had no effect on cytotoxicity by mouse peritoneal macrophages. Conversely, supernatants of PPD-stimulated spleen cells from mice immune to bacillus Calmette-Guérin (BCG) strongly inhibited the migration of, and significantly augmented cytolysis by, mouse PEC, but had no detectable effects on human monocytes. Moreover, supernatants of concanavalin A-stimulated lymph node cells from guinea pigs inhibited the migration of guinea pig PEC and human monocytes, but had no effects on mouse PEC. The migration inhibitory effects of the human and mouse supernatants did not appear to be mediated by interferon (IF), since partially purified type-1 IF had no detectable effect. In addition, supernatants of human lymphocytes stimulated by Corynebacterium parvum strain 5888, that induced little or no IF, were able to inhibit the migration of, and augmented cytolysis by, human monocytes. These C.parvum supernatants also showed migration inhibitory activity on mouse PEC but did not induce cytolytic activity in these cells.     

Lymphokine-mediated fusion and migration inhibition of alveolar macrophages

Guinea pigs were shown to produce a lymphokine termed macrophage fusion factor (MFF) which mediated the fusion of 70–80% of guinea pig or rabbit alveolar macrophages, but not guinea pig peritoneal macrophages. In the conventional migration inhibitory factor (MIF) assay, guinea pig aveolar macrophages were inhibited in their migration and large numbers of giant cells were present. There appeared to be a correlation between the titer of MFF and migration inhibition of alveolar macrophages but not with MIF titer as expressed on the peritoneal macrophage. Guinea pig MFF production was erratic and its absence from lymphokine supernatant fluids correlated with an absence of migration inhibitory activity for the alveolar macrophage. Guinea pig MIF production was more constant and high titers were invariably present. Rabbit crude lymphokine supernatant fluids containing MFF also inhibited the migration of their alveolar macrophages when measured at 24 and 48 hr during the MIF assay. Extensive numbers of giant cells were observed in the cell fan whenever migration inhibition was present. α-l-Fucose, which is known to block the receptor sites of MIF, failed to block giant cell formation in either the MFF or the MIF assay and also failed to block migration inhibition of the alveolar macrophages. The results suggest that lymphokines other than MIF can inhibit the migration of alveolar macrophages in the standard MIF assay and that the lymphokine responsible for migration inhibition and fusion of alveolar macrophages is the same lymphokine, MFF.     

Cytophilic antibody: correlation of its distribution with activation of basophils and macrophages

Immunoglobulin determinants on the surface of guinea pig basopils were detected using mono- and divalent antiglobulin reagents conjugated with fluorscein isothiocyanate. The fluorescent pattern obtained with monovalent antiglobulin was always that of uniformly stained rings. Divalent antiglobulin agglutinated immunoglobulin determinates to form small fluorescent sports subsequently collected at one end of the cell (“cap” formation). The formation of caps is temperature-dependent and is inhibited by sodiu azide and cytochalasin B. When basophils were incubated at 37 °C with divalent antiglobulin, morphological changes occurred, indicating that they had been activated. The same effect could be obtained with monovalent Fab?, but at least one hundred times more of it was required than the corresponding F(ab?)₂. Cytophilic antiboidies on the surface of guinea pig peritoneal macrophages were also detected by immunofluorescence. In this case again, divalent antiglobulin reagents agglutinated the determinants to form small fluorescent sports. Cap formation was not observed and pinocytosis started as the first aggregates were formed. It was also found that cytophilic antibodies were not stable on the macrophages' surface and were easily eluted at 37 °C. A reaction with cross-linking agents (divalent antiglobulin or antigen) stabilized the cytophilic antibody and pinocytosis was initiated.     

Cell-Mediated Immunity in Guinea Pig and Man to a Salmonella typhi Glycoprotein Complex Assessed by Migration Inhibition Methods

A Salmonella typhi glycoprotein preparation in concentrations of 5 to 10 _μg/ml strongly inhibited the migration of peritoneal exudate cells (PEC), obtained from guinea pigs immunized with S. typhi vaccine in Freund's complete adjuvant (FCA) PEC from animals injected with saline in FCA were also inhibited but to a lesser extent, whereas cells from guinea pigs that had received only S. typhi vaccine or normal guinea pigs showed variable and weaker inhibition. PEC from all guinea pigs receiving FCA were strongly inhibited by 5 to 15 _μg PPD/ml. These PPD concentrations had no inhibitory effect on exudate cells from guinea pigs receiving S. typhi vaccine only. The S.typhi glycoprotein concentration (5 and 10 _μg/ml) primarily used in the migration inhibition studies had negligible inhibitory effect on the phytohemagglutinin (PHA)-induced DNA, RNA, and protein syntheses in guinea pig lymph node cells. In the human system only blood leukocytes of donors who had received their third or fourth booster of heat-killed S. typhi vaccine within the latest 2 years were inhibited in their in vitro migration by the S. typhi glycoprotein complex.     

Expression and distribution of sialic acid influenza virus receptors in wild birds

Avian influenza (AI) viruses have been detected in more than 105 wild bird species from 12 different orders but species-related differences in susceptibility to AI viruses exist. Expression of α2,3-linked (avian-type) and α2,6-linked (human-type) sialic acid (SA) influenza virus receptors in tissues is considered one of the determinants of the host range and tissue tropism of influenza viruses. We investigated the expression of these SA receptors in 37 wild bird species from 11 different orders by lectin histochemistry. Two isoforms of Maackia amurensis (MAA) lectin, MAA1 and MAA2, were used to detect α2,3-linked SA, and Sambucus nigra lectin was used to detect α2,6-linked SA. All species evaluated expressed α2,3-linked and α2,6-linked SA receptors in endothelial cells and renal tubular epithelial cells. Both α2,3-linked and α-2,6-linked SA receptors were expressed in respiratory and intestinal tract tissues of aquatic and terrestrial wild bird species from different taxa, but differences in SA expression and in the predominant isoform of MAA lectin bound were observed. With a few possible exceptions, these observed differences were not generally predictive of reported species susceptibility to AI viruses based on published experimental and field data.     

Further screening of Aspergillus species for occurrence of lectins and their partial characterization

Fifteen species of Aspergillus were screened for occurrence of lectins. Nine of them (A. sydowii, A. candidus, A. allahabadi, A. terricola, A. ficuum, A. sparsus, A. carneus, A. pulvinus and A. aculeatus) were found to possess lectin activity. None of the species elaborated lectin in culture supernatant. All the lectins agglutinated rat, pig and rabbit erythrocytes. A. sydowii, A. candidus, A. allahabadi, A. terricola, A. ficuum, A. sparsus, A. carneus and A. aculeatus lectins agglutinated all human type erythrocytes equally, while A. pulvinus lectin specifically agglutinated human type A and O erythrocytes. Neuraminidase and protease treatment to erythrocytes substantially augmented lectin titres manyfold. Lectins showed specificity to mucin and asialofetuin and all of them were specific to L‐arabinose except that of A. carneus. Lectins from A. sydowii, A. ficuum, A. sparsus and A. carneus displayed remarkable specificities to D‐xylose. Maximum lectin activity was expressed by 11 day old cultures of A. sydowii (titre 32), A. ficuum (titre 64) and A. sparsus (titre 1024). Lectins from A. aculeatus, A. candidus and A. terricola were expressed by 7–10 days, 6–9 days and 5–11 days old cultures, respectively. A. allahabadi cultures exhibited maximum lectin activity (titre 32) after 8–10 days of cultivation. A. carneus and A. pulvinus expressed optimal titres of 32 and 8, respectively on the 9^th day. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Sequential appearance of γ/δ- and α/β-bearing T cells in the peritoneal cavity during an i.p. infection with Listeria monocytogenes

To search for a potential role of T cell antigen receptor (TcR) γ/β-bearing cells in host-defense against Listeria monocytogenes, we analyzed the sequential appearance of γ/δ and α/β T cell in the peritoneal exudate cells (PEC) during an i.p. infection with sublethal dose (2 × 10³) of viable Listeria organisms in mice. The PEC on day 1 after the infection consisted of 48% macrophages and 50% lymphocytes, most of which were surface IgM⁺ (B) cells. The number of PEC increased to the maximal level by day 3. The PEC at this stage contained an appreciable number of CD3⁺ T cells in addition to a large number of macrophages. Of the CD3⁺ cells, the proportion of CD4^?CD8^? cells, most of which expressed no TcR α/β, increased to the maximal level on day 3 after the infection. In correlation with an increased number of CD3⁺CD4^?CD8^?TcR α/β^? cells, high level of TcR γ/δ chain gene messages was detected in the nonadherent population of the PEC on this stage. On the other hand, the PEC on day 8 contained an increased number of CD4⁺CD8^? and CD4^?CD8⁺ cells which expressed TcR α/β chain on their surface. These results suggest that the γ/δ T cells precede the α/β T cells in appearance during listerial infection. The γ/β T cells may be involved at the first line of the host-defense against Listeria.     

Lectin-induced agglutination of trophozoites of different species and strains ofEntamoeba

In vitro agglutinability of trophozoites of threeEntamoeba histolytica strains, cultivated under axenic conditions in the presence of concanavalin A (Con A), was shown to be related to the degree of their pathogenicity for experimental animals and of the concentration of Con A. Seven strains ofE. invadens tested also agglutinated in the presence of Con A, and the degree of agglutination was proportional to the concentration of the lectin. Three strains ofE. histolytica-like Laredo-type amebae, a strain ofE. terrapinae, and a strain ofE. moshkovskii agglutinated very slightly, only in the presence of the highest concentration of Con A tested.     

Bovine adenovirus serotype 3 utilizes sialic acid as a cellular receptor for virus entry

Bovine adenovirus serotype 3 (BAd3) and porcine adenovirus serotype 3 (PAd3) entry into the host cells is independent of Coxsackievirus adenovirus receptor and integrins. The role of sialic acid in BAd3 and PAd3 entry was investigated. Removal of sialic acid by neuraminidase, or blocking sialic acid by wheat germ agglutinin lectin significantly inhibited BAd3, but not PAd3, transduction of Madin–Darby bovine kidney cells. Maackia amurensis agglutinin or Sambucus nigra (elder) agglutinin treatment efficiently blocked BAd3 transduction suggesting that BAd3 utilized α(2,3)-linked and α(2,6)-linked sialic acid as a cell receptor. BAd3 transduction of MDBK cells was sensitive to sodium periodate, bromelain, or trypsin treatment indicating that the receptor sialoconjugate was a glycoprotein rather than a ganglioside. To determine sialic acid-containing cell membrane proteins that bind to BAd3, virus overlay protein binding assay (VOPBA) was performed and showed that sialylated cell membrane proteins in size of approximately 97 and 34 kDa bind to BAd3. The results suggest that sialic acid serves as a primary receptor for BAd3.     

THE INTERACTION OF SELECTIVE PLANT LECTINS WITH NEURAMINIDASE-TREATED AND UNTREATED IgA1 FROM THE SERA OF IgA NEPHROPATHY PATIENTS

A study on the binding interaction of lectins from Artocarpus heterophyllus (jacalin), Glycine max and Sambucus nigra with standardised quantity of IgA from the IgA nephropathy patients and normal controls was performed. The Glycine max lectin demonstrated higher affinity towards the serum IgA of IgAN patients as compared to normal controls. However, the affinity binding was lower in cases of jacalin and the Sambucus nigra lectin. When serum samples were treated with neuraminidase, the differential jacalin affinity binding between IgA1 of patients and normal controls was abrogated. Our data are in support of the view that the O-linked oligosaccharide moieties of the patients IgA1 were generally lacking in galactose and sialic acid residues.     

Guinea pig S19 ribosomal protein as precursor of C5a receptor-directed monocyte-selective leukocyte chemotactic factor

Objective: To reveal the C5a receptor-mediated monocyte-selective chemoattraction of the homo-dimer of guinea pig S19 ribosomal protein (RP S19), and to study the topological relationship between the RP S19 and C5a receptor genes.Methods: cDNA cloning and nucleotide sequencing, leukocyte chemotaxis measurement, and fluorescent in situ hybridization (FISH) were performed in the guinea pig.Results: The amino acid sequence of the guinea pig RP S19 deduced from the cDNA nucleotide sequence was identical to the human protein. The dimer of a recombinant RP S19 attracted guinea pig monocytes but suppressed neutrophil chemotactic movement. Both effects were C5a receptor-mediated. In the FISH analysis, the signals denoting the guinea pig RP S19 gene and C5a receptor gene completely overlapped each other.Conclusions: The guinea pig RP S19 dimer possessed a dual ligand effect, agonistic to the monocyte C5a receptor and antagonistic to the neutrophil receptor. The RP S19 and C5a receptor genes co-localized on the same chromosome.Received 24 April 2004; returned for revision 14 June 2004; accepted by M. Katori 21 June 2004     

Preparative Fractionation of T and B Lymphocytes of the Syrian Golden Hamster with Soybean Agglutinin

T and B lymphocytes of the Syrian golden hamster were separated from spleen cell preparations on the basis of their differential agglutinability with the lectin soybean agglutinin. Only B lymphocytes were agglutinated by this lectin, and they could be separated from the unagglutinated T lymphocytes by sedimentation through 50% heat inactivated calf serum at unit gravity. The B lymphocyte aggregates could be dissociated into single cells that were viable and functional after treatment with 0.5 M galactose. The isolated cell fractions were characterized by their blastogenic response to various T cell and B cell specific mitogens and by the presence or absence of cell surface IgG.     

Human peritoneal macrophages in culture: a model for studying inflammatory disorders in vitro

In this study we characterized a model of human peritoneal macrophages maintained in culture for up to 48 h that can be used to study different functions of this cell population in vitro. The cells remained viable and functionally active over time, with well-preserved phagocytic properties. They expressed a macrophage marker, CD14. Once in culture, human peritoneal macrophages secreted C1q and nitric oxide in a pattern described in murine, guinea pig, and rat peritoneal macrophages . The described model can be used to study physiology and pathophysiology of peritoneal macrophages in vitro, offering all the advantages of the use of a human cell population. Received: 8 November 2002 / Accepted: 11 February 2003 Correspondence to D. Faust     

Lectin-binding properties of different Leishmania species

Carbohydrate cell-surface residues on stationary promastigotes of 19 isolates of Leishmania were studied with a panel of 27 highly purified lectins, which were specific for N-acetyl-D-glucosamine, D-mannose, L-fucose, D-galactose, N-acetyl-D-galactosamine, and sialic acid. The specificity of the cell-surface carbohydrates was analyzed by agglutination and radioiodinated lectin-binding assays. L. (L.) amazonensis and L. (L.) donovani were agglutinated by 12 and 10 of the 27 lectins used, respectively. Artocarpus integrifolia lectin (Jacalin) was incapable of agglutinating the tested species of the donovani complex, and this result was confirmed by radioiodinated Jacalin-binding assays. Jacalin had an average of 3.8 × 10⁶ receptors/L. (L) amazonensis promastigote and bound with an association constant of 5 × 10⁶ M ⁻¹. Received: 14 September 1998 / Accepted: 27 January 1999     

Determination of attachment sites for N-linked carbohydrate groups of class II histocompatibility α-chain and analysis of possible O -linked glycosylation of α- and γ -chains

Two glycopeptide fractions were isolated from a tryptic digest of the human class II antigen α-subunit, by chromatography on Lens culinaris and Ricinus communis lectin columns, respectively. Partial NH₂-terminal sequence analysis of radiochemically labelled glycopeptide fractions allowed alignment with two stretches of the deduced DRα sequence, each encompassing a signal for N-linked glycosylation, i.e. Asn-X-Thr(Ser). The fraction displaying affinity for L. culinaris lectin was susceptible to the action of endo-β-N-acetylglucosaminidase H whereas the fraction adsorbed to R. communis was not. Since DRα is known to carry only one N-linked carbohydrate chain in the high-mannose form, this glycan could thereby be mapped to Asn 78. Accordingly, the complex N-linked carbohydrate is attached to Asn 118. Moreover, analysis of material released from class II antigens and γ-chains upon mild alkaline hydrolysis, indicates the presence of O-linked sugars on the α- and γ- but not the β-subunits.     

Sialidase-enhanced lectin-like mechanism for Actinomyces viscosus and Actinomyces naeslundii hemagglutination

Laboratory strains representing six numerical taxonomy clusters and fresh isolates of human Actinomyces viscosus and Actinomyces naeslundii were studied by standard flocculation slide tests for the ability to hemagglutinate erythrocytes (RBC) from various animal species. Human AB and horse RBC were agglutinated more frequently and rapidly than others; guinea pig RBC were agglutinated by only a few strains. Human AB RBC were selected for studies of hemagglutination mechanisms. Treatment of RBC with clostridial neuraminidase (NTRBC) greatly enhanced hemagglutination for almost all strains. In hapten inhibition experiments in which various concentrations of sugars were used, beta-galactosides were the most effective inhibitors of hemagglutination for both RBC and NTRBC; inhibition of NTRBC agglutination required higher concentrations. Soybean lectin agglutinated both RBC and NTRBC but not Actinomyces cells. NTRBC agglutinated at a 125-fold-lower concentration. Hemagglutination was sensitive to ethylenediaminetetraacetate for one strain tested. Hemagglutination reactions were reversible by addition of beta-galactosides. The ability of Actinomyces strains to "prime" RBC for hemagglutination by removing sialic acid to expose more penultimate beta-galactoside sites was studied by recycling Actinomyces-agglutinated RBC which were dispersed with a lactose solution and washed free of bacteria (primed RBC). Priming in this manner augmented subsequent hemagglutination by indicator Actinomyces strains and made the RBC more sensitive to agglutination by soybean lectin. The priming ability of Actinomyces strains generally correlated with the amount of sialic acid removed from primed RBC. Strains representing the numerical taxonomy clusters differed in both their hemagglutinating and priming activities. Cluster 5 strains (typical A. naeslundii) were good agglutinators of RBC, NTRBC, and primed RBC but were poor primers. Cluster 3 strains (atypical A. naeslundii) were the weakest hemagglutinators but could prime RBC adequately for subsequent agglutination by other strains. Together, these data indicate that Actinomyces hemagglutination proceeds via a two-step mechanism: (i) neuraminidase removal of terminal sialic acid and (ii) lectin-like binding to exposed beta-galactoside-associated sites on the RBC. Strains differ in the extent to which they can perform the two functions, and this specificity may relate to their taxonomic classification.     

Comparative studies ofTrypanosoma cruzi andT. cruzi-like stocks from different South American countries using lectins

The agglutination behaviour of four-day-old epimastigote culture forms of 34Trypanosoma cruzi, andT. cruzi-like stocks from Brazil, Chile, Colombia, Ecuador and Peru were tested with 15 carbohydratespecific lectins. We distinguished intraspecifically two groups of agglutination reactions: Group 1 includes stocks which react withTriticum vulgaris andAaptos papillata II (wheat germ agglutinin: WGA-type). Group 2 includes stocks agglutinated byArachis hypogaea andAaptos papillata II (peanut lectin: PNA-type). The agglutination reactions with lectins fromTriticum vulgaris andAaptos papillata II correlate with the presence of N-acetylneuraminic acid on the cell surface. After treatment with neuraminidase, the WGA-type is agglutinated by PNA but not by lectins fromTriticum vulgaris andAaptos papillata II. Further results demonstrate that a certain zymodeme pattern can be correlated with carbohydrate determinants.     

Partial separation and functional characterization of guinea pig basophil-stimulating factor

We have previously described T lymphocyte dependent guinea pig basophil growth from bone marrow precursors in vitro. In the current studies, basophil-stimulating factor (BSF) present in mitogen-stimulated splenic conditioned medium (CM) has been functionally characterized, utilizing an assay for BSF on nonadherent bone marrow target cells. BSF was found to be heat stable, nondialyzable, and inactivated by proteases. Monosaccharides known to inhibit guinea pig lymphokines yielded a unique profile of inhibition of BSF activity and nonidentity of BSF with guinea pig migration inhibition factor, interleukins 1 or 2, and granulocyte-macrophage colony-stimulating activity, from which BSF could be separated after gel filtration. BSF-containing CM also had no detectable interleukin-3 activity as measured in a murine assay. An inverse relation was found between interleukin 2 and BSF production by peritoneal exudate T cells (PEL) stimulated with antigen. Fractionation of serum-containing and serum-free CM demonstrated a molecular size for BSF of 50,000-65,000 daltons. Guinea pig BSF is a distinct T cell dependent lymphokine with an active protein moiety which may interact with target bone marrow cells through a cell surface carbohydrate receptor.     

Migration inhibition experiments with mixtures of human peripheral blood lymphocytes and guinea pig peritoneal exudate cells

Migration inhibition using mixtures of human peripheral blood lymphocytes and non-sensitized guinea pig peritoneal exudate cells was demonstrable with purified protein derivative of tuberculin (PPD) and Candida albicans extract (CAE) when lymphocytes were obtained from individuals with a delayed type skin reaction to these antigens. No migration inhibition occurred when lymphocytes were used from skin test negative individuals. Furthermore it was found that lymphocytes from some skin test negative persons, which did not induce migration inhibition, showed, however, an increased DNA synthesis when cultured in vitro with PPD. This indicates that a dissociation between the results of migration inhibition, skin test and lymphocyte transformation can occur not only in patients with immune disorders, as has been described by other investigators, but also in healthy individuals.