Polysialic acid as an antigen for monoclonal antibody HIgM12 to treat multiple sclerosis and other neurodegenerative disorders.

CNS regeneration is a desirable goal for diseases of brain and spinal cord. Current therapeutic strategies for the treatment of multiple sclerosis (MS) aim to eliminate detrimental effects of the immune system, so far without reversing disability or affecting long-term prognosis in patients. Approachable molecular targets that stimulate CNS repair are not part of the clinical praxis or have not been identified yet. The purpose of this study was to identify the molecular target of the human monoclonal antibody HIgM12. HIgM12 reverses motor deficits in chronically demyelinated mice, a model of MS. Here, we identified polysialic acid (PSA) attached to the neural cell adhesion molecule (NCAM) as the antigen for HIgM12 by using different NCAM knockout strains and through PSA removal from the NCAM protein core. Antibody binding to CNS tissue and primary cells, antibody-mediated cell adhesion, and neurite outgrowth on HIgM12-coated nitrocellulose was detected only in the presence of PSA as assessed by western blotting, immunoprecipitation, immunocytochemistry, and histochemistry. We conclude that HIgM12 mediates its in vivo and in vitro effects through binding to PSA and has the potential to be an effective therapy for MS and neurodegenerative diseases. The human antibody HIgM12 stimulates neurite outgrowth in vitro and promotes function in chronically demyelinated mice, a model of multiple sclerosis. The cellular antigen for HIgM12 was undetermined. Here, we identified polysialic acid attached to NCAM (neural cell adhesion molecule) as the cellular target for HIgM12. This includes glial fibrillary acidic protein (GFAP)-positive mouse astrocytes (GFAP, red; HIgM12, green; DAPI, blue) among other cell types of the central nervous system. These findings indicate a new strategy for the treatment of neuro-motor disorders including multiple sclerosis.

Comparative study of hemagglutination and lectin activity in Australian medicinal mushrooms (higher Basidiomycetes).

Fifteen Australian mushroom species (higher Basidiomycetes) were assessed for hemagglutination and lectin activity. Hemagglutination activity was evaluated using both neuraminidase treated and untreated rabbit and human A, B, and O erythrocytes. Lectin activity was determined by the ability of various mono- and oligosaccharides to inhibit hemagglutination activity. Of the mushrooms evaluated, seven contained lectin activity. However, five (Agaricus bitorquis, Chlorophyllum brunneum, Coprinus comatus, Cortinarius sp. TWM 1710, and Omphalotus nidiformis) expressed lectin activity in only one of two collections tested. The two remaining lectin active mushroom species (Phlebopus marginatus and Psathyrella asperospora) possessed lectin activity with the same sugar specificity in both collections. Although lectins were identified with diverse specificity, lactose-specific lectin activity was most frequently identified, being present in Agaricus bitorquis, Copronus comatus, Omphalotus nidiformis, and Phlebopus marginatus. In contrast, Psathyrella asperospora, Cortinarius sp. TWM 1710, and Chlorophyllum brunneum were found to possess lectin activity specific for N-acetyl-D-glucosamine, galactose, and N-acetyl-neurammic acid, respectively. Significantly, the galactose-specific lectin activity identified in Cortinarius sp. TWM 1710 and the lactose-specific lectin activity in Phlebopus marginatus have not been previously reported.

A safety evaluation of DAS181, a sialidase fusion protein, in rodents.

DAS181 is a novel inhaled drug candidate blocking influenza virus (IFV) and parainfluenza virus (PIV) infections through removal of sialic acid receptors from epithelial surface of the respiratory tract. To support clinical development, a 28-day Good Laboratory Practices inhalation toxicology study was conducted in Sprague-Dawley rats. In this study, achieved average daily doses based on exposure concentrations were 0.47, 0.90, 1.55, and 3.00 mg/kg/day of DAS181 in a dry powder formulation. DAS181 was well tolerated at all dose levels, and there were no significant toxicological findings. DAS181 administration did not affect animal body weight, food consumption, clinical signs, ophthalmology, respiratory parameters, or organ weight. Gross pathology evaluations were unremarkable. Histological examination of the lungs was devoid of pulmonary tissue damage, and findings were limited to mild and transient changes indicative of exposure and clearance of a foreign protein. DAS181 did not show any cytotoxic effects on human and animal primary cells, including hepatocytes, skeletal muscle cells, osteoblasts, or respiratory epithelial cells. DAS181 did not cause direct or indirect hemolysis. A laboratory abnormality observed in the 28-day toxicology study was mild and transient anemia in male rats at the 3.00 mg/kg dose, which is an expected outcome of enhanced clearance of desialylated red blood cells resulting from systemic exposure with DAS181. Another laboratory observation was a transient dose-dependent elevation in alkaline phosphatase (ALP), which can be attributed to reduced ALP clearance resulting from increased protein desialylation due to DAS181 systemic exposure. These laboratory parameters returned to normal at the end of the recovery period.

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 alpha(2,3)-linked and alpha(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.

Development of liver regenerative therapy using glycoside-modified bone marrow cells.

Several recent studies have reported that bone marrow cells (BMCs) have the ability to generate functional hepatocytes. However, the efficiency at which BMC transplantation generates functional hepatocytes is rather low. We assumed that if BMCs accumulated directly in liver, the functional BMC-derived hepatocytes should increase efficiently. We tried to increase the accumulation of BMCs directly in liver through the interaction between hepatic asialoglycoprotein receptor and desialylated BMCs. Desialylated BMCs were produced with treatment of neuraminidase. Desialylated BMCs that expressed green fluorescent protein (GFP) were injected into Long Evans Cinnamon (LEC) rats, a human Wilson's disease model, intravenously. At 3 and 5 months after transplantation, GFP-expressing hepatocyte nodules appeared in the liver of these BMC-transplanted LEC rats. These findings suggest that the functional BMC-derived hepatocytes can be generated by the direct accumulation of BMCs and that this strategy is new BMC therapy for liver regeneration.

Monoclonal antibodies against tissue-nonspecific alkaline phosphatase. Report of the ISOBM TD9 workshop.

Nineteen monoclonal antibodies (MAbs) against tissue-nonspecific (liver/bone/kidney) alkaline phosphatase (TNALP) were investigated in the ISOBM TD-9 Workshop. These MAbs were generated with antigens obtained from human bone tissue (n = 9), human osteosarcoma cell lines (SaOS-2 and TPX; n = 7) and human liver tissue (n = 3). The evaluation included the following antigen forms: (a) commercially available preparations of human bone ALP (BALP) and liver ALP (LALP); (b) human BALP isoforms, B/I, B1 and B2; and (c) soluble secreted epitope-tagged recombinant human TNALP (setTNALP) expressed in COS-1, osteosarcoma (SaOS-2) and hepatoma (Huh2) cell lines. In addition, 16 TNALP mutant cDNAs corresponding to a wide spectrum of reported hypophosphatasia mutations were used in an attempt to map specific immunoreactive epitopes on the surface of the TNALP molecule. The TD-9 MAbs were evaluated by immunoradiometric (IRMA) assays, cross-inhibition and different enzyme immunoassay designs. No indications of explicit tissue discriminatory immunoreactivities of the investigated MAbs against TNALP were found. However, certain IRMA combinations of MAbs increased the specificity of BALP measurements. All MAbs bound to the three BALP isoforms B/I, B1 and B2, but none of the investigated MAbs were specific for any of the isoforms. Significant differences were, however, found in immunoreactivity between these isoforms, with cross-reactivities ranging from 21 to 109% between the two major BALP isoforms B1 and B2. Desialylation with neuraminidase significantly increased the MAb affinity for the BALP isoforms B/I, B1 and B2, and also decreased the observed differences in cross-reactivity between these isoforms. We suggest, therefore, that the MAb affinity is dependent on the amount/number of terminal sialic acid residues located at the five putative N-glycosylation sites. Based on the overall results, we present a putative three-dimensional model of the TNALP molecule with positioning of the four major antigenic domains (designated A-D) of the investigated MAbs. The TNALP molecule is depicted as a homodimer, hence most, but not necessarily all, epitopes are displayed twice. The antigenic domains were positioned with the following assumptions: domain A was positioned close to the active site since most of these MAbs interfered with the catalytic activity. Interestingly, both MAbs included in the commercial BALP kits were grouped with domain A. Moreover, 4 of the 5 putative N-glycosylation sites (with terminal sialic acid residues) are located within, or with close proximity to, domain A. Domain B was localized at the top flexible loop (crown domain) of the TNALP molecule. Domain C was clearly defined by the IRMA assay combinations and by site-directed mutants of TNALP to be close to residue E281, which is located near the fourth metal binding site, likely to be occupied by a calcium ion. Domain D was positioned close to residues A115, A162 and E174, but this domain was also close to the GPI anchor site. In conclusion, none of the 19 investigated TD-9 MAbs were entirely specific for BALP or LALP, thus indicating that all MAbs bind mainly to epitopes on the common protein core of BALP and LALP and/or common glycosylated epitopes. However, some MAbs (either single or in combination with other MAbs) work sufficiently well to measure BALP when the assayed samples do not contain elevated levels of LALP.

A hematopoietic cell L-selectin ligand that is distinct from PSGL-1 and displays N-glycan-dependent binding activity.

Human hematopoietic progenitor cells express L-selectin and also express PSGL-1, a ligand for all selectins. Using a shear-based adhesion assay, a hematopoietic cell L-selectin ligand (HCLL) that is expressed on the hematopoietic cell line KG1a and on normal human hematopoietic progenitors was previously identified. To characterize the structural biology of HCLL and to define its relationship to PSGL-1, the effects of chemical and enzymatic treatments on HCLL activity of KG1a cells and membrane preparations were analyzed. Protease digestions and chemical treatments of KG1a cells and membranes indicated that HCLL is an integral membrane glycoprotein. Glycosidase digestions of membrane protein preparations and metabolic treatments of KG1a cells with glycosylation processing modifiers revealed that L-selectin binding determinants on HCLL are sialofucosylated structures presented on complex-type N-glycans. Adhesion assays and biochemical studies showed that this glycoprotein is also expressed on circulating blasts in native acute leukemias. HCLL is distinguishable from PSGL-1: (1) KG1a cells sorted for PSGL-1 expression had equivalent HCLL activity; (2) anti-PSGL-1 blocking antibodies and proteases known to eliminate L-selectin binding to PSGL-1 had no effect on HCLL binding activity of KG1a cells; (3) blasts from native leukemias with low expression of PSGL-1 and CD34 display high HCLL activity; and (4) despite high level expression of PSGL-1, HCLL activity was absent on HL60 cells. These data provide first evidence of a naturally expressed membrane L-selectin ligand expressing binding determinant(s) on an N-linked glycoconjugate. This novel ligand may help mediate L-selectin-dependent cell-cell adhesive interactions within the cytoarchitecture of the bone marrow microenvironment. (Blood. 2000;96:2765-2774)

Characterization of factors regulating lamina-specific growth of thalamocortical axons.

During development, most thalamocortical axons extend through the deep layers to terminate in layer 4 of neocortex. To elucidate the molecular mechanisms that underlie the formation of layer-specific thalamocortical projections, axon outgrowth from embryonic rat thalamus onto postnatal neocortical slices which had been fixed chemically was used as an experimental model system. When the thalamic explant was juxtaposed to the lateral edge of fixed cortical slice, thalamic axons extended farther in the deep layers than the upper layers. Correspondingly, thalamic axons entering from the ventricular side extended farther than those from the pial side. In contrast, axons from cortical explants cultured next to fixed cortical slices tended to grow nearly as well in the upper as in the deep layers. Biochemical aspects of lamina-specific thalamic axon growth were studied by applying several enzymatic treatments to the cortical slices prior to culturing. Phosphatidylinositol phospholipase C treatment increased elongation of thalamic axons in the upper layers without influencing growth in the deep layers. Neither chondroitinase, heparitinase, nor neuraminidase treatment influenced the overall projection pattern, although neuraminidase slightly decreased axonal elongation in the deep layers. These findings suggest that glycosylphosphatidylinositol-linked molecules in the cortex may contribute to the laminar specificity of thalamocortical projections by suppressing thalamic axon growth in the upper cortical layers.

Nonspecific binding of IgE to allergens.

Nonspecific IgE binding to allergens was observed in testing myeloma IgEs, namely, IgE-VL and IgE-PS, chimeric IgE (IgE-JW8), and the recombinant IgE Fc epsilon peptide CH1-CH4, in two different immunoassays. This binding was concentration-dependent but detectable only at higher IgE concentration. In RAST inhibition, IgE-allergen interactions could be reduced by using either matching or nonmatching allergens. In order to test whether the nonspecific binding of IgE to allergens was due to carbohydrate interaction, myeloma IgEs and the chimeric IgE were desialized with neuraminidase. Desialized samples were equally well recognized by xenogenic antibodies as native IgEs, but binding of IgE to Fc epsilon receptors on basophils was affected by the treatment, as shown in the histamine-release assay. Desialization of IgE affected also its binding capacity to allergens in RAST: binding of chimeric IgE was reduced, but nonspecific binding of myeloma IgE-VL was enhanced. Hence, nonspecific allergen-IgE binding may be partly due to a lectin-like interaction, but may depend mostly on the tertiary structure of IgE. Thus, nonspecific IgE-allergen interactions might present a problem 1) at high IgE concentration, and 2) depend on the grade of sialization of IgE, which might affect its conformation. This may explain why patients with elevated total IgE levels often have multiple weak positive RASTs with non-cross-reactive allergens.

Structural definition of a potent macrophage activating factor derived from vitamin D3-binding protein with adjuvant activity for antibody production.

Incubation of human vitamin D3-binding protein (Gc protein), with a mixture of immobilized beta-galactosidase and sialidase, efficiently generated a potent macrophage activating factor, a protein with N-acetylgalactosamine as the remaining sugar. Stepwise incubation of Gc protein with immobilized beta-galactosidase and sialidase, and isolation of the intermediates with immobilized lectins, revealed that either sequence of hydrolysis of Gc glycoprotein by these glycosidases yields the macrophage-activating factor, implying that Gc protein carries a trisaccharide composed of N-acetylgalactosamine and dibranched galactose and sialic acid termini. A 3 hr incubation of mouse peritoneal macrophages with picomolar amounts of the enzymatically generated macrophage-activating factor (GcMAF) resulted in a greatly enhanced phagocytic activity. Administration of a minute amount (10-50 pg/mouse) of GcMAF resulted in a seven- to nine-fold enhanced phagocytic activity of macrophages. Injection of sheep red blood cells (SRBC) along with GcMAF into mice produced a large number of anti-SRBC antibody secreting splenic cells in 2-4 days.

Pig plasma alpha-protease inhibitors PI2, PI3 and PI4 are members of the antichymotrypsin family.

Three related alpha-protease inhibitors, PI2 I, PI3 C and PI4 C2, of blood serum of the pig (Sus scrofa) were isolated. PI2 I inhibited both trypsin and chymotrypsin; PI3 C and PI4 C2 strongly inhibited chymotrypsin, but did not significantly inhibit trypsin. By using SDS-PAGE, the three proteins were found to be composed of single polypeptide chains, and molecular weights were 63,000 for PI2 I, 58,000 for PI3 C and 64,000 for PI4 C2. All three proteins were shown to be glycoproteins. In PI3 C, eight sialic acid residues were found, and in PI4 C2 (similarly as in PI2 F) 10-11 residues were found. Amino acid composition as well as N-terminal sequences of the three proteins were very similar, indicating close homology. Comparison of these partial amino acid sequences with the cDNA-deduced amino acid sequence of pig alpha-antichymotrypsin (AACT; Buchman, 1989, GenBank, Accession No. M29508) revealed great similarities, the sequence of PI2 I being virtually identical with the pig AACT. On the basis of all available results, PI2 is proposed to be pig AACT, an orthologue of human AACT.

Datura stramonium agglutinin released histamine from rat peritoneal mast cells that was inhibited by pertussis toxin, haptenic sugar and N-acetylglucosamine-specific lectins: involvement of glycoproteins with N-acetylglucosamine residues.

The N-acetyl glucosamine (GlcNAc)-specific lectin Datura stramonium agglutinin (DSA) rapidly and sugar-specifically released histamine from rat peritoneal mast cells, and pertussis toxin (IAP) inhibited it, suggesting that DSA activated mast cells via an IAP-sensitive G protein pathway. The additive effects of DSA and basic secretagogues such as compound 48/80 that activate IAP-sensitive G protein directly suggest that they shared the same mechanism of action including involvement of the IAP-sensitive G protein. Using lectin-blotting, blots of the corresponding glycoproteins detected by DSA diminished by haptenic sugar or pretreatment of the cells with N-glycosidase F, suggesting that the binding of DSA was responsible for the mast cell activation. The other GlcNAc-specific lectins such as Phytolacca americana mitogen, Solanum tuberosum agglutinin and wheat germ agglutinin (WGA) inhibited the histamine release induced by DSA, suggesting that these lectins were antagonists, but DSA was an agonist. Sialic acid-specific Macckia amurensis mitogen (MAM) inhibited the histamine release, and neuraminidase-treatment decreased mast cell activation induced by DSA. At least four mast cell glycoproteins that have affinity to DSA, WGA and MAM and are sensitive to neuraminidase-treatment were detected by lectin-blotting. Some of them may be binding sites coupled to histamine release including the IAP-sensitive G protein pathway. DSA is a useful tool for studying signal transduction of mast cells including the involvement of the IAP-sensitive G protein.

Polyacrylamides bearing pendant alpha-sialoside groups strongly inhibit agglutination of erythrocytes by influenza A virus: multivalency and steric stabilization of particulate biological systems.

An alpha-sialoside linked to acrylamide by a short connector (5-acetamido-2-O-(N-acryloyl-8-amino-5-oxaoctyl)-2,6-anhydro-3,5-d ideoxy-D-galacto-alpha-nonulopyranosonoic acid, 1) was prepared. Compound 1 formed high molecular weight copolymers with acrylamide, derivatives of acrylamide, and/or vinylpyrrolidone upon photochemically-initiated free radical polymerization. Those copolymers for which the substituents on the acrylamido nitrogen were small inhibited the agglutination of chicken erythrocytes induced by influenza virus (X-31 (H3N2); a recombinant strain of A/Aichi/2/68 (H3N2) and A/Puerto Rico/8/34 grown in chicken eggs). The inhibitory power of the polymers depended strongly on the conditions of polymerization and the sialic acid content of the polymer. The strongest inhibitors were copolymers (poly(1-co-acrylamide)) formed from mixtures of monomer containing [1]/([1] + [acrylamide]) approximately 0.2-0.7; these copolymers inhibited hemagglutination 10(4)-10(5) times more strongly than did similar concentrations of alpha-methyl sialoside (calculated on the basis of the total concentration of individual sialic acid groups in the solution, whether attached to polymer or present as monomers). Samples polymerized in the presence of low concentrations of cross-linking reagents (bis(acrylamido)methane, BIS, and 2,2'-bis(acrylamido)ethyl disulfide, BAC) also showed increased inhibition (10-10(3)-fold relative to monomers), but their use was limited by their poor solubility. Sterically demanding substituents on any position of the acrylamide component (substituents attached to the vinyl group or N-alkyl groups that are larger than hydroxyethyl) reduced the inhibitory power of the polymer. A 1H NMR assay and a fluorescence depolarization assay showed that poly(1-co-acrylamide) bound to a solubilized trimeric form of the viral receptor for sialic acid (bromelain cleaved hemagglutinin, BHA), less tightly than 1, on a per sialic acid basis. A similar result was also obtained with a model system comprising lactic dehydrogenase (a tetramer) and polymeric derivatives of oxamic acid: that is, poly((28, 29, 30, or 31)-co-acrylamide) had a higher inhibition constant for tetrameric lactic dehydrogenase than did the corresponding monomers (28, 29, 30, or 31) on a per oxamate basis. Poly(1-co-acrylamide) is, in principle, capable of inhibiting the agglutination of erythrocytes by several mechanisms: (1) entropically enhanced binding of the polymer (acting as a polyvalent inhibitor) to the surface of the virus; (2) steric interference of the approach of the virus to the surface of the erythrocyte by a water-swollen layer of the polymer on the surface of the virus; (3) aggregation of the virus induced by the polymer.(ABSTRACT TRUNCATED AT 400 WORDS)

Influenza A virus binding to human neutrophils and cross-linking requirements for activation.

Although neutrophils are not viewed as a principal defense against influenza A virus (IAV) infection, their interactions are both complex and clinically relevant. Activation of the neutrophil is distinctive from that described for chemoattractants. To more fully characterize the pathway by which IAV stimulates the human neutrophil, we have examined its binding characteristics. First, inhibition studies with various sialic acid-containing and sialic-free sugars showed that IAV binds to sialic acid residues and activates receptors distinct from those used by Concanavalin-A (Con-A) and formyl-methionyl-leucyl-phenylalanine (FMLP) and that overlap those bound by wheat germ agglutinin (WGA). That viral hemagglutinin (HA) mediates viral binding and activation was shown by preincubating neutrophils with purified monovalent bromelain-released HA (BHA) and showing that IAV-induced membrane depolarization and hydrogen peroxide (H2O2) production were inhibited approximately 95%. However, binding inhibition required significantly higher concentrations of purified HA, suggesting that binding and cell activation have different interactive requirements. Desialation of the neutrophil surface membrane by neuraminidase treatment resulted in a 90.6% +/- 4.4% and 53.1% +/- 8.7% inhibition of IAV activation of neutrophils and viral binding, respectively. Resialation with ganglioside GT1b totally restored viral binding, but did not reverse the inhibition of activation. Thus, although HA was shown to mediate binding and neutrophil activation, viral binding per se was insufficient to stimulate the cell. Having demonstrated the functional role of HA, we sought to establish the mechanism of stimulation. HA in three different forms (BHA, HA-rosettes, and HA-liposomes) failed to activate the cell, although H2O2 production evoked by IAV stimulation was reduced in competitive inhibition studies with each preparation. Upon cross-linking with a monoclonal antibody to HA, activation comparable to that of intact virus was observed. The requirement for cross-linking of functional receptors, as opposed to activation through the neutrophil Fc receptor, was confirmed in experiments using staphylococcal A protein. These studies have shown the chemical specificity of IAV binding to the human neutrophil, the character of the receptor(s) stimulated to activate the IAV-evoked response, and the activation requirement for cross-linking those receptors responsible for stimulating functional responses.

Inhibition of adhesion of S-fimbriated Escherichia coli to buccal epithelial cells by human milk fat globule membrane components: a novel aspect of the protective function of mucins in the nonimmunoglobulin fraction.

We investigated the presence of factors in human milk that inhibit invasion of pathogenic bacteria. The effect of human milk fat globule membrane (HMFGM) components on adhesion of cloned S-fimbriated Escherichia coli to human buccal epithelial cells was analyzed. S fimbriae are a common feature of E. coli strains causing sepsis and meningitis in newborns and are bound to epithelia via sialyl-(alpha-2-3)galactoside structures. Human milk fat globules (HMFG) could be agglutinated by the above-mentioned bacteria. Agglutination could be inhibited by fetuin, human glycophorin, and alpha 1-acid glycoprotein. In addition, pretreatment of HMFG with Vibrio cholerae neuraminidase markedly reduced bacterium-induced agglutinations, indicating the involvement of neuraminic acid-containing glycoproteins. In contrast, lipid droplets of infant formula or artificial lipid emulsions (Intralipid) could not be agglutinated. HMFG were present in stools of breast-fed neonates as shown by indirect immunofluorescence staining with a monoclonal antibody directed against carbohydrate residues present on HMFGM. These HMFG could be agglutinated by bacteria. HMFG inhibited E. coli adhesion to buccal epithelial cells. To further characterize relevant E. coli binding structures, HMFGM components were separated by gel chromatography. The mucin fraction showed the most pronounced inhibitory effect on adhesion of S-fimbriated E. coli to human buccal epithelial cells. Our data suggest that HMFG inhibit bacterial adhesion in the entire intestine and thereby may provide protection against bacterial infection.

Modulation of murine macrophage responses stimulated with influenza glycoproteins.

Previously it was reported that influenza virus stimulated, nonspecific resistance was largely due to its glycoproteins, hemagglutinin (HA) and neuraminidase (NA). The enhancement of natural killer cell activity was the intrinsic property of NA and HA. In the present study, the stimulatory effect of these glycoproteins on the murine peritoneal macrophages was studied. Electrophoretically purified glycoproteins, NA and HA, of influenza virus A/USSR/90/77 (H1N1) were administered intraperitoneally to C3H/HeN mice, with or without stearyl tyrosine (ST). Macrophages were isolated and were restimulated with phorbol myristate acetate. H2O2 secretion was determined by horseradish peroxidase dependent oxidation of phenol red assay. HA enhanced H2O2 secretion only in the presence of ST (60 nmol.mg-1.h-1), whereas NA alone stimulated H2O2 secretion (83 nmol.mg-1.h-1), by 6-fold over control (13 nmol.mg-1.h-1), and this stimulation was further increased (136 nmol.mg-1.h-1) in the presence of ST. Interleukin 1 (IL-1) activity was determined by using D10.G4.1 cells. There was a little stimulation of IL-1 activity (less than 1 U/mL) of macrophages isolated from HA-primed of HA+ST-primed mice restimulated with HA. On the other hand, IL-1 activity of macrophages isolated from NA-primed mice restimulated with NA significantly increased (102 U/mL) over control (less than 1 U/mL), and an additional 2-fold increase (231 U/mL) resulted when macrophages from NA+ST-primed mice were used. Tumor necrosis factor (TNF) activity was examined by using L929 cells. Negligible TNF activity was observed in macrophages isolated from either HA-primed or HA+ST-primed mice restimulated with HA.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of soluble haemagglutinins on adherence of Helicobacter pylori to HEp-2 cells.

In a study of six laboratory strains of Helicobacter pylori, two different modes of bacterial adherence to HEp-2 cells were found. Electronmicroscopy revealed that strains known to possess soluble haemagglutinin adhered intimately to the cell surfaces, with cupping of the plasma membrane and coalescence of glycocalyces at sites of attachment. Strains of H. pylori without soluble haemagglutinin also attached, but did not induce membrane cupping or show glycocalyx fusion. Light microscopy did not distinguish between these patterns of adherence. Bacterial attachment was unaffected by pre-treatment of HEp-2 cells with neuraminidase. Exposure of the bacteria to trypsin or to colloidal bismuth subcitrate (CBS) before being added to HEp-2 cells markedly impaired bacterial adherence. This effect of CBS may contribute to the known efficacy of bismuth therapy in patients with H. pylori-related gastritis.

Hemopoietic progenitor cell binding to the stromal microenvironment in vitro.

Primitive clonogenic progenitor cells in human bone marrow bind to preformed marrow-derived stromal layers in vitro and generate colonies of blast cells. The binding interaction does not require calcium or magnesium ions and occurs equally well in serum-free and serum-supplemented culture medium. It does not appear to involve known cell adhesion molecules (CAMs) for which monoclonal antibodies are available (integrins, N-CAM, LFA-1, and ICAM-1), and we were unable to demonstrate a role for the progenitor cell antigen CD34 in progenitor cell adhesion to cultured stroma. The CAM expressed by the blast colony-forming cells may exist in transmembrane or phosphatidylinositol (PI)-linked forms because it is only partially degraded by exposure to trypsin or to PI-specific phospholipase C. However, binding of these cells to stroma is not prevented in the presence of monoclonal antibodies reacting with known PI-linked structures (Thy-1, CD14, and CD16). It is either masked by neuraminidase-sensitive residues or is no longer expressed as cells mature, respectively, along the granulocytic or erythroid lineages. The properties of the hemopoietic progenitor CAM are discussed with reference to the properties of other CAMs and of hemopoietic progenitor cell markers.

Sugar competition assays reveal high affinity receptors for Erythrina cristigalli lectin on feline monocytes.

An examination of fluorescein-labelled Erythrina cristigalli lectin (FITC-ECL) staining on feline mononuclear cells (MNC) using fluorescent microscopy and a novel sugar titration competition assay revealed that monocytes (MO) were stained brighter by FITC-ECL than were lymphocytes (LYM). When MNC were stained with FITC-ECL in the presence of 400 mM or greater D-galactose, analysis by flow cytometry revealed continued MO staining while LYM were negative. MO expressed a larger quantity of carbohydrate receptors (CHO-R) for ECL than did LYM. The CHO-R expressed on MO were mostly protease-insensitive and uncapped by sialic acid residues. All of the CHO-R on LYM were protease-sensitive and many were capped by sialic acid residues. A combined labelling of MNC for non-specific esterase staining, latex bead ingestion and FITC-ECL staining in the presence of 400 mM D-galactose confirmed that FITC-ECL specifically stains MO in the presence of high sugar competitor concentrations.

Attachment of influenza C virus to human erythrocytes.

Binding experiments with radioactively labelled influenza C virions were carried out to investigate the interaction of the virus with human erythrocytes. The erythrocytes from any of 35 different individuals were found to contain influenza C virus-binding sites though their number was variable among the individuals and was much less than that on mouse, rat and chicken erythrocytes. Attachment of influenza C virus to human erythrocytes was inhibited completely by prior treatment of the virus with anti-HE monoclonal antibody having a strong haemagglutination inhibition activity. Pretreatment of erythrocytes with neuraminidase or the neuraminate-O-acetylesterase of influenza C virus resulted in a marked reduction in the level of virus binding. Thus it appears that human erythrocytes have a low level of O-acetylated sialic acid-containing glycoconjugates that can interact specifically with the HE glycoprotein of influenza C virus. Proteolytic digestion of erythrocytes with ficin, bromelain or V-8 protease inhibited virus binding almost completely, suggesting that the erythrocyte receptor for influenza C virus is a glycoprotein. In contrast to these enzymes, trypsin treatment of erythrocytes reduced virus binding by only about 50%, and alpha-chymotrypsin treatment did not inhibit at all. It was also found that treatment of erythrocytes with monoclonal antibody to the M or N blood group antigen greatly inhibited virus binding to the cells. These results, taken together, suggest that most influenza C virus receptors on human erythrocytes, if not all, reside on glycophorin A which is known to possess the M or N blood group activity.