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PubMed1984

Protochordate immunity--II. Diverse hemolymph lectins in the solitary tunicate Styela clava.

Hemolymph lectins (agglutinins) of the tunicate Styela clava were analyzed by agglutination, cross-absorption and carbohydrate-hemagglutination inhibition using several vertebrate erythrocytes. Lectin activity was heat labile, dependent on divalent cations and refractory to neuraminidase-treated erythrocytes. Four lectins with different carbohydrate specificities were found. Carbohydrate specificities included L-rhamnose, D-glucuronolactone, maltose, D-galactosamine, D-mannosamine, D-galactose, hyaluronic acid and bacterial lipopolysaccharide. Since S. clava lectins can be inhibited by carbohydrates found in the extracellular capsule or cell wall of most bacteria, we propose that the lectins may be part of the tunicate immuno-defense system.

Animals↗

PubMed2003

Changes in N-glycosylation of human stromal cells by telomerase expression.

It was established that remarkable changes in the N-glycosylation are induced in immortalized cancer cells. Whether changes were induced in human stromal cells immortalized by transfection with the human telomerase catalytic subunit (hTert) cDNA was examined by lectin blot analysis. Morphological appearance and growth rate of the gene-transfected stromal cells were not changed significantly. However, lectin blot analysis of membrane glycoprotein samples showed that bindings of Ricinus communis agglutinin-I (RCA-I) and of leuko-agglutinating phytohemagglutinin to glycoprotein bands increase significantly in the gene-transfected cells. No lectin binding was observed when blotted filters were treated with diplococcal beta-1,4-galactosidase or N-glycanase prior to incubation with RCA-I. In contrast, no changes in Coomassie brilliant blue-staining and in binding of concanavalin A were obtained between the primary and gene-transfected stromal cells. These results indicate that the highly branched N-glycosylation with augmented galactosylation is induced in human stromal cells immortalized by the telomerase expression.

Bone Marrow Cells↗

PubMed1996

Identification of CD8 as a peanut agglutinin (PNA) receptor molecule on immature thymocytes.

Differentiation of most T lymphocytes occurs within the thymus and is characterized by variable expression of CD4/CD8 coreceptor molecules, increased surface density of T cell antigen receptor (TCR) alpha beta proteins, and decreased expression of glycan chains recognized by the galactose-specific lectin peanut agglutinin (PNA). Although appreciated for several decades that PNA agglutination is useful for the physical separation of immature and mature thymocyte sub-populations, the identity of specific PNA-binding glycoproteins expressed on immature thymocytes remains to be determined. In the current report, we studied the expression of PNA-specific glycans on immature and mature T cells and used lectin affinity chromatography and immunoprecipitation techniques to characterize PNA-binding glycoproteins on thymocytes. Our data demonstrate that PNA-specific glycans are localized on a relatively small subset of thymocyte surface proteins, several of which were specifically identified, including CD43, CD45, and suprisingly, CD8 molecules. CD8 alpha and CD8 alpha' proteins bound to PNA in the absence of CD8 beta expression showing that O-glycans on CD8 beta glycoproteins are not necessary for PNA binding and that glycosylation of CD8 alpha and CD8 alpha' proteins proceeds effectively in the absence of CD8 beta. Finally, we demonstrate that PNA binding of CD8 is developmentally regulated by sialic acid addition as CD8 proteins from mature T cells bound to PNA only after sialidase treatment. These studies identify CD8 as a PNA receptor molecule on immature thymocytes and show that PNA binding of CD8 on immature and mature T cells is developmentally regulated by sialic acid modification.

Animals↗

PubMed1997

Fractionation of feline bone marrow with the soybean agglutinin lectin yields populations enriched for erythroid and myeloid elements: transplantation of soybean agglutinin-negative cells into lethally irradiated recipients.

Feline bone marrow cells treated with the soybean agglutinin (SBA) lectin are separated into two populations, the agglutinated SBA(+) fraction containing predominantly cells of myeloid origin and the nonagglutinated SBA(-) fraction consisting of cells primarily of the erythroid lineage. FACScan analyses revealed a clear distinction of the cells based on their light scattering properties, i.e., large cells and cells with high granularity were found in the SBA(+) fraction, whereas cells having a low forward light scatter and side light scatter were found in the SBA(-) fraction. Colony-forming assays showed colony-forming unit-granulocyte/monocyte (CFU-GM) cells to have a strong affinity for SBA because these were found almost entirely in the SBA(+) fraction; in contrast, burst-forming unit-erythroid (BFU-E)-forming cells were concentrated in the SBA(-) fraction. When the marrow was fractionated by counterflow centrifugal elutriation (CCE), a differential binding to SBA among the CFU-GM forming cells was found. The SBA(-) fractions of cells collected at 21 and 25 ml/min contained primarily BFU-E forming cells, similar to that observed with whole marrow; the later CCE fractions, those collected at 32 ml/min and the rotor off fraction, when treated with SBA showed a small but significant number of CFU-GM cells in the SBA(-) fraction. T lymphocytes were found predominantly in the SBA(+) fractions of whole bone marrow and the CCE fractions. Successful autologous marrow transplants were performed with the early CCE SBA(-) fractions. The latter cells were used for our initial transplant attempts because ongoing studies in our laboratory had shown these cells to be free of any viral-containing cells when the marrow had been obtained from animals infected with the feline immunodeficiency virus. In summary, although SBA treatment of feline marrow yields a marked separation of CFU-GM and BFU-E progenitors, select CCE SBA(-) fractions contain stem cells capable of providing hematopoietic reconstitution of lethally irradiated animals.

Animals↗

PubMed1976

Serological relationships among some Pichia species.

Antigenic analyses of five species of the genus Pichia were carried out for taxonomic study by the slide agglutination method using monospecific and absorbed antisera and the agglutinin absorption technique. Comparative studies were also performed with a few strains of each of the same species and their classifications are discussed with respect to the antigenic structures and the patterns of proton magnetic resonance (PMR) spectra of their cell wall polysaccharides. ichia delftensis and Pichia zaruensis possessed thermostable antigens 1,2,5 and 11, and the former had also thermoabile antigen m. Both species were closely related to Candida krusei. Pichia toletana possessed thermostable antigens 1,2,5,11,17 and 49. Pichia bovis contained thermostable antigens 1,2,14,15,16,20 and 21, and it was related to most species of the genus Hansenula, although assimilation of potassium nitrate was negative. Finally, Pichia etchellsii possessed thermostable antigens 1,2,3,4,9 and 14, and was closely related to Pichia vini. Patterns of PMR spectra of mannans of these species also supported their serological relationships. Therfore, P. delftensis, P. zaruensis and P. etchellsii are considered to be the synonyms of Pichia fluxuum, Pichia dispora and P. vini respectively, although P. toletanan and P. bovis are independent species.

Antigens, Fungal↗

PubMed1987

Monoclonal antibodies against murine neonatal and pregnancy-associated natural suppressor cells induce resorption of the fetus.

Pregnant and neonatal/fetal mice have been shown to harbour naturally occurring inhibitory cells of both T and non-T type. Non-T suppressor cells present in the spleen of primiparous pregnant and newborn animals inhibit proliferative responses in autologous and allogeneic mixed lymphocyte reactions. Such cells can be positively selected for by agglutination with the B cell-specific lectin soybean agglutinin (SBA). We generated rat IgG monoclonal antibodies against unique cell surface structures on the non-T inhibitory cells, and cytotoxic pretreatment of spleen cells from pregnant or neonatal/fetal mice largely abrogates their suppressive activity on proliferative responses. Furthermore, in vivo administration of such antibodies to pregnant inbred and outbred mice results in termination of the pregnancy or decreased litter size. Since injection of anti-T cell IgG monoclonal antibodies does not interfere with the delivery of normal sized litters it is evident from these studies that the non-T immunoregulatory cells, in contrast to T-inhibitory cells, are of great importance in ensuring immunological homeostasis in the fetal-placental environment during pregnancy in mice.

Animals↗

PubMed1983

Naturally occurring non-T suppressor cells in pregnant and neonatal mice: some functional and phenotypic characteristics.

The mammalian fetus thrives in the presence of a maternal immune system which is considered capable of initiating an aggressive reaction against alloantigens expressed on fetal cells. The control of harmful immunological reactions during pregnancy may be due in part to the combined action of soluble immunoregulatory factors and suppressor cells in the maternal/fetal environments. In the present investigation, a comparison has been made between naturally occurring splenic suppressor cells isolated from neonatal and pregnant adult CBA/J mice. Functional analysis of inhibitory activity was carried out using conventional one-way allogeneic mixed lymphocyte reactions (MLR) consisting of adult CBA/J spleen cells responding against mitomycin C-inactivated BALB/cJ spleen cells. Suppressor cells in the spleen of pregnant animals as well as a population of inhibitory cells in newborn spleen could be shown to be highly resistant to cytotoxic pretreatment with anti-T cell serum plus complement. Both newborn and pregnant non-T suppressor cells were shown to be agglutinated by the B cell-specific lectin soybean agglutinin. The densities of these two populations of non-T inhibitory cells, as determined by Percoll gradient centrifugation, were demonstrated to be very similar (ie, within the range of 1.067 to 1.043 g/ml). The striking parallels in the functional and phenotypic characteristics of the non-T suppressor lymphocytes found in the spleen of pregnant and neonatal animals suggest a common mechanism for their induction.

Animals↗

PubMed1975

[Study of Salmonella cultures. IV. Immunogenicity of Salmonella resistant to antibiotics].

Three avirulent and 5 slightly virulent for birds mutants of Salmonella enteritidis and Salmonella braenderup, resistant to streptomycin (S), chlorteracyclin (CT), and chloramphenicol (Ch) were used in the production of live vaccines. The latter were tested for immunogenicity on albino mice and birds. On the eighth and 15th day following vaccination the protein fractions in the blood serum of the birds were followed up through electrophoresis on agar gel, while the agglutinin titer was determined by means of test tube agglutination. The experimental results were mathematically processed with the use of statistical methods. All slightly virulent and resistant to antibiotics mutants showed strong immunogenic properties and gave protection (preventing death) to 76.2-81.82 per cent of the mice. Of the avirulent mutants one showed moderate virulence giving protection to 71.43 per cent of the animals. Tested on 110-day-old chickens the avirulent mutants gave protection to 80 per cent of them. The average titers of the blood serum antibodies of birds vaccinated with immunogenic mutants, and the average percent values of the gamma-globulin fractions were higher on the eighth than on the 15th day. The protein fractions showed changes in the albumin and the gamma-globulin. The rate of correlation between these indices, as shown by the coefficient of correlation, is high (rxy = - 0.843). In this case the negative value of rxy in the equation points to the negative link between the characters.

Animals↗

PubMedDate not supplied

Regulation of lymphocyte responses in vitro. VII. - "Cell contact inhibition", a possible regulatory mechanism for lymphocyte activation.

Potentiation by cytochalasin B (CB) of (3H)thymidine incorporation in lymphoid cell suspensions stimulated by phytohemagglutinin (PHA) is counteracted by adherent cells. The drug has no effect on the production of the macrophage product lymphocyte activating factor (LAF) and does not interfere with its potentiating activity. CB appears to inhibit agglutination of lymphocytes. Other factors (absence of serum, macrophages, mechanical disturbance of the cultures) diminishing the size of aggregates increase the response of lymphocytes. Responses to less agglutinating mitogens are less or not enhanced by CB. Factors increasing agglutination or/and cell contacts (high cell density, wheat germ agglutinin) diminish lymphocyte activation and make it susceptible to CB potentiating effect. The results suggest that topoinhibition (cell contact inhibition) is an important mechanism regulating the lymphocyte response to mitogens in culture. Its effect could be modulated by macrophages and certain serum factors, and CB would potentiate these responses by reducing contact inhibition.

Animals↗

PubMed2002

Lectins as markers for blood grouping.

Lectins are unique proteins of varying biological importance. They are characterized by specific binding to carbohydrate residues, whether monosaccharides, disaccharides or polysaccharides. The sugar heads on the surface of the erythrocyte specify the different blood groups. Lectins, as an antigenic determinant of blood group, have come to be an important tool in the identification of different blood groups. A handful of lectins may be considered excellent reagents for anti-A, anti-B, anti-N etc, but the anti-A and anti-M are not yet regarded as commercially suitable antisera. Lectin from Vicia cracca has been proved to be a good anti-A, lectin from Dolichus biflorus can be used as anti-A1, and lectin from Griffonia simplicifolia as anti-B. Lectin from Vicia graminea is said to be a good typing reagent as Anti-N. On the other hand, the lectins involved in polyagglutination are absolutely essential as the reagent of choice and these cannot as yet be replaced by antibodies of any kind. Erythrocytes with exposed cryptantigens are significantly more sensitive to agglutination by certain lectins than by polyclonal antibodies. Peanut agglutinin (PNA), Polybrene, and Glycine max lectins are frequently used for the identification of different cryptantigens. The application of lectins as an anti-B reagent has proven to be as useful as human polyclonal or mouse monoclonal antibodies. Besides their specificity, lectins are excellent reagents because of their lower cost and indigenous production. The importance of various lectins used as markers for blood grouping is discussed.

ABO Blood-Group System↗

PubMed1987

A spleen-derived maturational factor allows immature thymocytes, prepared as cells bearing low amounts of surface sialic acid, to become cytotoxic T cells.

A population of immature mouse thymocytes bears low levels of surface sialic acid and can be separated from the more mature high sialic acid-bearing thymocytes by selective agglutination with the sialic acid-specific lectin, lobster agglutinin 1. These immature thymocytes do not proliferate in response to concanavalin A (Con A). They do not produce interleukin 2 (IL-2), do not provide T cell help to B cells for an in vitro antibody response, and as shown here, do not become cytotoxic T lymphocytes when polyclonally stimulated with Con A + IL-2. We describe here a spleen-derived maturational factor which stimulates these immature thymocytes, in the presence of Con A and IL-2, to become cytotoxic T lymphocytes. The maturational factor is a protein secreted by Con A-stimulated mouse or rat spleen cells; it is apparently neither interleukin 1, IL-2, interleukin 3, gamma-interferon, nor combinations of these cytokines, because these materials do not replace the maturational factor. The active material in Con A-stimulated mouse spleen cell supernatant was recovered from a G-75 column in the 33,000-48,000 m.w. range. These experiments suggest that within the lobster agglutinin 1-negative thymocyte population there are cells which can mature under the influence of a spleen-derived factor. It is possible that these cells represent the small subpopulation of immature cells destined to become immunocompetent peripheral T cells. On the other hand, the factor may be rescuing cells destined to die in the thymus.

Animals↗

PubMed1984

[Antibody titers in the serum of patients suspected of typhoid using the classical Widal and microagglutination tests].

Blood serum samples taken from typhoid fever suspected patients during the last typhoid fever epidemic in Ankara in 1981 were tested comparatively with classical Gruber-Widal test and microagglutination technique using safranin-stained Salmonella typhi O and H antigens. Results showed that microagglutination technique was more reliable, less time and antigen consuming and permitted a more objective evaluation of agglutinin titers.

Agglutination Tests↗

PubMed1976

Structure and toxicity of pure ricinus agglutinin.

Highly purified ricinus agglutinin was found to inhibit protein synthesis in HeLa cells. This effect could be prevented by the addition of the specific antiricinus agglutinin serum, whereas specific anti-ricin serum did not protect the cells, demonstrating that the toxic effect of ricinus agglutinin is not due to contamination with ricin. After reduction of ricinus agglutinin with 2-mercaptoethanol in the presence of 0.5 M galactose the constituent peptide chains were separated by chromatography on a DE-52 column. The B'-chain passed through the column, whereas the A'-chain bound and was eluted with a salt gradient. The B'-chain was further purified by chromatography on a CM-52 column. The shortest chain, the A'-chain, was found to inhibit cell-free protein synthesis whereas the B'-chain did not have this ability. On the other hand, the B'-chain was able to induce agglutination of erythrocytes when tested together with anti-ricinus agglutinin serum indicating that the "b'-chains bind to the cells. Ouchterlony immunodiffusion tests with crude anti-ricin and anti-ricinus agglutinin sera revealed that the two constituent chains of ricinus agglutinin are immunologically partial identical and that they also show reaction or partial identity with both chains of the toxic lectin ricin. The data indicate that a similar structure-function relationship exists in ricinus agglutinin as in ricin. The reason for the much lower toxicity of ricinus agglutinin than of ricin in living animals is discussed.

HeLa Cells↗

PubMed1975

Lectin agglutinability of non-neoplastic and neoplastic human lymphoid cells in vitro.

Agglutination by two lectins, Concanavalin A (Con A) and Ricinus communis agglutinin (RCA), has been investigated in a human lymphoid cell system. The main conclusions of this study are: (1) no systematic correlation exists between the neoplastic state and sensitivity to Con A or RCA; (2) cells of neoplastic lines vary unsystematically in their surface properties as evaluated by Con A agglutination, with the possible exception that presence of Epstein-Barr virus (EBV) is associated with a high degree of agglutination and (3) cells of diploid lymphoblastoid lines and phytohemagglutinin (PHA)-stimulated lymphoctes agglutinate similarly and significantly better than unstimulated T- or B-lymphocytes. The relatively simple Con A agglutination assay can be used as an adjunct in classification of human lymphoid cell lines.

Agglutination↗

PubMed1987

Further characterization of the cold agglutinin from the snail Achatina fulica.

The cold agglutinin from the albumin gland of the snail Achatina fulica was purified to homogeneity by using sheep gastric mucin-Sepharose 4B as affinity column followed by gel filtration on Bio-Gel P-300. The homogeneity was checked by alkaline gel electrophoresis, immunodiffusion and immunoelectrophoresis. The purified cold agglutinin is a glycoprotein of native M2 220,000 consisting of three non-covalently bound subunits of Mr 84,000, 74,000 and 62,000 and having a pI value of 4.5. The predominant amino acids are aspartic acid and glutamic acid (or amides) and serine, which account for 39% of the residues. About 3% of the residues are half-cystine. The lectin is a glycoprotein with about 30.7% carbohydrate, the most abundant sugars being galactose, N-acetylgalactosamine and N-acetylglucosamine. Mannose, xylose and fucose are also present. The inhibition of agglutination of human umbilical-cord erythrocytes by the cold agglutinin is specific for methyl beta-D-galactoside and also for glycolipids present on cord erythrocytes. The c.d. data show only negative ellipticity values in the far-u.v. region for the protein at various concentrations and temperatures and also in the presence of the hapten lactose (at different concentrations), indicating the presence of a random-coil conformation in the agglutinin that varies according to temperature.

Agglutinins↗

PubMed1985

Natural killer (NK) cell activating factor released from murine thymocytes stimulated with an anti-tumor streptococcal preparation, OK-432.

Natural killer (NK) activity of mouse splenocytes was significantly augmented when the splenocytes were incubated for 3 to 4 hr with culture supernatants of mouse thymocytes stimulated by OK-432, an antitumor preparation from the Streptococcus pyogenes SU-strain. Antiviral activity was also detected in the culture supernatants, but IL 2 activity was not. When the culture supernatants of thymocytes stimulated by OK-432 were fractionated on a column of Blue Sepharose CL-6B, NK enhancing activity and antiviral activity were observed in partly overlapping fractions that bound to the column. However, the antiviral activity in the Blue Sepharose-bound fraction was neutralized completely by treatment with anti-IFN (alpha, beta) antiserum, whereas significant NK cell enhancing activity was still observed after treatment with anti-IFN (alpha, beta) antiserum. When the Blue Sepharose-bound fraction was subjected to gel filtration, the NK cell enhancing activity was detected in the 25,000 to 35,000 and 40,000 to 67,000 m.w. regions, but antiviral activity was observed in the over 67,000 m.w. region. These results indicate that a new kind of lymphokine, called natural killer cell activating factor (NKAF), distinct from IFN and IL 2, was found. The NKAF was found to have the following properties: its pI value is between pH 5.5 and 6.5, it binds to concanavalin A- and lentil agglutinin-Sepharose, and it is stable with pH 2-24 hr treatment. In addition, NKAF-producing cells were peanut agglutinin (PNA)-thymocytes when thymocytes were fractionated by the agglutination-sedimentation method with the use of PNA.

Animals↗

PubMed1992

Sexual agglutination in budding yeasts: structure, function, and regulation of adhesion glycoproteins.

The sexual agglutinins of the budding yeasts are cell adhesion proteins that promote aggregation of cells during mating. In each yeast species, complementary agglutinins are expressed by cells of opposite mating type that interact to mediate aggregation. Saccharomyces cerevisiae alpha-agglutinin and its analogs from other yeasts are single-subunit glycoproteins that contain N-linked and O-linked oligosaccharides. The N-glycosidase-sensitive carbohydrate is not necessary for activity. The proposed binding domain of alpha-agglutinin has features characteristic of the immunoglobulin fold structures of cell adhesion proteins of higher eukaryotes. The C-terminal region of alpha-agglutinin plays a role in anchoring the glycoprotein to the cell surface. The S. cerevisiae alpha-agglutinin and its analogs from other species contain multiple subunits; one or more binding subunits, which interact with the opposite agglutinin, are disulfide bonded to a core subunit, which mediates cell wall anchorage. The core subunits are composed of 80 to 95% O-linked carbohydrate. The binding subunits have less carbohydrate, and both carbohydrate and peptide play roles in binding. The alpha-agglutinin and alpha-agglutinin genes from S. cerevisiae have been cloned and shown to be regulated by the mating-type locus, MAT, and by pheromone induction. The agglutinins are necessary for mating under conditions that do not promote cell-cell contact. The role of the agglutinins therefore is to promote close interactions between cells of opposite mating type and possibly to facilitate the response to phermone, thus increasing the efficiency of mating. We speculate that they mediate enhanced response to sex pheromones by providing a synapse at the point of cell-cell contact, at which both pheromone secretion and cell fusion occur.

Agglutinins↗

PubMedDate not supplied

Serologic diagnosis of human brucellosis: analysis of 214 cases by agglutination tests and review of the literature.

The serum agglutination test (SAT) and 2-mercaptoethanol (2ME) agglutination were used in studies of the sera of 214 patients in whom brucellosis was suspected. On the basis of historical, epidemiologic, clinical, and serologic data, four groups were identified: group I (108 cases) had negative agglutination reactions, and brucellosis was considered unlikely; group II (57 cases) had positive agglutination reactions, and active brucellosis was diagnosed; group III (37 cases) had positive agglutination reactions, but other factors--notably, a history of prior infection--made inactive brucellosis likely; and group IV (12 cases) had positive agglutination reactions, but insufficient data were available for further classification. Most patients with active brucellosis had agglutinin titers of greater than or equal to 160; however, no single titer was always diagnostic. Although more sensitive tests are available, agglutination reactions provide data sufficient to differentiate active from inactive disease when other factors are considered and follow-up sera are tested. This article discusses individual cases and reviews the literature on the diagnosis of brucellosis.

Adolescent↗