Hemocytes of Bulinus truncatus rohlfsi (Mollusca: Gastropoda)

Two basic cell types occur in the hemolymph of Bulinus truncatus rohlfsi: granulocytes and hyalinocytes. Granulocytes are divided into three subtypes: (1) Granulocytes I, which account for 19% of the hemocytes, are small, young amoebocytes with 1–20 filopodia and small numbers of cytoplasmic granules, including some lysosomes; (2) granulocytes II, which account for 78% of the cells, are large, fully developed amoebocytes that possess 1–20 filopodia and many granules, both acidophilic and basophilic, including numerous lysosomes, phagosomes, and mitochondria; and (3) spent granulocytes, which are rare, have few filopodia, large accumulations of glycogen granules and prominent vacuoles in addition to lysosomes in the cytoplasm. These three subtypes of granulocytes probably represent ontogenetic stages within a single cell line. In addition, granulocytes with 40 or more filopodia and little ectoplasm, found in only 1 of 45 snails examined, probably reflect a pathologic condition. Hyalinocytes, which account for 3% of all hemocytes, are similar in size to mature granulocytes, but have few or no cytoplasmic granules and lack filopodia and glycogen granules. Total hemocyte concentration in hemolymph is 328,000 ± 188,000 cells/ml.     

Recognition of foreignness by blood cells of the freshwater snail Lymnaea stagnalis, with special reference to the role and structure of the cell coat

The structure and function of the cell coat of the blood cells (amoebocytes) of the freshwater snail Lymnaea stagnalis were studied with ultrahistochemical tests, including concanavalin A (Con A) labeling, and with in vitro phagocytosis experiments. The cell coat is intensely stained by ruthenium red and tannic acid. The cells possess binding sites for Con A. Proteolytic enzymes destroy the receptors for Con A and totally inhibit the phagocytic activity of amoebocytes. Incubation experiments with proteases, carbohydrases, and inhibition sugars revealed that (1) the Con A binding sites are anchored in the plasma membrane by proteins, and (2) glucose, fructose, mannose, and to a lesser extent N-acetylglucosamine and N-acetylgalactosamine, inhibit the binding of Con A to amoebocytes, suggesting that these carbohydrates might form part of these binding sites.     

Separation of oyster hemocytes by density gradient centrifugation and identification of their surface receptors

Glutaraldehyde-fixed hemocytes of Crassostrea virginica were subjected to differential centrifugation on a 5, 10, 15, and 25% discontinous sucrose gradient. Five subpopulations of cells were separated by this technique. Subpopulation 1 coincides with the small granulocytes, subpopulation 2 is comprised of hyalinocytes, subpopulation 3 of medium-sized granulocytes, subpopulation 4 of large granulocytes, and subpopulation 5 of a mixture of very large granulocytes and aggregates of small cells. By using several plant and animal lectins, it was ascertained that cells of subpopulations 1, 3, and 4 were agglutinated with Con A and extracts of the albumin glands of Helix pomatia and Cepaea nemoralis while those of subpopulation 2 were agglutinated by the same three lectins as well as wheat germ agglutinin. By applying the Con A-peroxidase cytochemical technique, it was determined that approximately 20% of the granulocytes of subpopulations 1 and 3 do not possess Con A-binding sites and only 18% of the large cells comprising subpopulation 5 possess such sites. These results suggest that the subpopulations of C. virginica granulocytes distinguishable by their dimensions and densities may be further subdivided by differences in specific surface binding sites.     

Experimentally induced elevations in acid phosphatase activity in hemolymph of Biomphalaria glabrata (Mollusca).

The levels of acid phosphatase activity in the hemocytes and serum of the following four groups of the pulmonate gastropod Biomphalaria glabrata were ascertained: (1) those injected with heat-killed Bacillus megaterium, (2) those challenged with sterile distilled water, (3) those shaminjected, and (4) those left untampered. It was determined that challenge with heat-killed bacteria sesulted in significant elevations in acid phosphatase activity in both cells and serum at 1, 2, and 4 hr postinjection, with the level being highest at 2 hr. Injection with water resulted in a significant elevation in cellular enzyme level at 1 hr postinjection but not at 2 and 4 hr; however, there were significant elevations at 2 and 4 hr in serum. This is interpreted to indicate that the elevated intracellular enzyme was subsequently released into serum. Sham injection resulted in significant elevations in acid phosphatase levels in hemocytes at 2 and 4 hr postinjection but no increase of enzyme activity in serum during the course of the experiment. This is interpreted to mean that this hydrolase was not released as a result of sham injection. The source of acid phosphatase was apparently the cytoplasmic granules of granulocytes, which are true lysosomes.     

Concanavalin A-induced receptor redistribution on Biomphalaria glabrata hemocytes: Characterization of capping and patching responses

Exposure of rounded, glass-adherent hemocytes from a Schistosoma mansoni-susceptible (PR albino) and S. mansoni-refractory (10-R2) stock of snails, Biomphalaria glabrata, to fluoresceinlabeled concanavalin A induces a redistribution of surface membrane Con A receptors. Receptor redistribution (patching and capping) on hemocytes from both snail stocks can be characterized as (1) rapid, with maximum cap formation occurring within 15 min of lectin treatment at 22°C, (2) sodium azide sensitive, but only at relatively high inhibitor concentrations (100–200 mm^?N₃ for capping and 200 mm^?N₃ for patching inhibition), (3) pronase sensitive (partial), but trypsin resistant, and (4) generally unaffected by exposure of snails to S. mansoni miracidia 60 or 180 min prior to extraction of hemolymph (hemocyte) samples for Con A testing. Although differences in the time course of receptor redistribution are exhibited between PR albino and 10-R2 snail hemocytes, the results of experiments involving sodium azide, proteolytic enzymes, and schistosome exposure strongly suggest that Con A-binding determinants and their associated membrane components on rounded hemocytes are very similar in both susceptible and refractory Biomphalaria stocks. It is concluded that if schistosome recognition in refractory 10-R2 snails is mediated through specific hemocyte membrane components, those components associated with Con A reactivity probably are not directly involved in the recognition process.     

Hemolymph functions in Mytilus californianus: The cytochemistry of hemocytes and their responses to foreign implants and hemolymph factors in phagocytosis

The hemocytes of Mytilus californianus are of three types: small and large basophils and large granular acidophils. The basophils contain lysosomal enzymes and phagocytose colloidal carbon. Agglutinins for yeast and human A Rh+ve erythrocytes are present in plasma, but are not needed for effective phagocytosis; in vitro both acidophilic and basophilic hemocytes rapidly phagocytose these particles. Plasma proteins, analyzed electrophoretically, are under strong homeostatic control. When Mya arenaria mantle is placed orthotopically on M. californianus mantle, the implant is invaded by host hemocytes in a manner consistent with that described in other published reports on molluscan graft rejection. Steady state is achieved by 26 days postimplant. Second- and third-set implants are rejected more rapidly than are first-set implants, but this is not a specific response. Third-set implants elicit a host cellular response that is more localized than the response to first-set implants. These data do not permit conclusions with respect to memory in these molluscan immune responses, but do imply a qualitative “improvement” in this quasi-immune response of M. californianus.     

Chemotactic attraction between hemocytes of the oyster,Crassostrea virginica,and bacteria

Experiments were conducted to ascertain whether there is chemotactic attraction by Bacillus megaterium and Micrococcus varians, both Gram-positive species, and Escherichia coli and Vibrio parahaemolyticus, both Gram-negative species, for hemocytes of the American oyster, Crassostrea virginica. It was ascertained quantitatively that oyster hemocytes are attracted to live E. coli, B. megaterium, and M. varians but not to heat-killed bacteria. Furthermore, oyster cells are not attracted to either live or heat-killed V. parahaemolyticus. It is concluded that the chemoattractant is some molecule emitted by living vegetative cells of certain Gram-positive as well as Gramnegative bacteria.     

Concanavalin A increases the strength of baby hamster kidney cell attachment to substratum

The strength of attachment of normal and transformed baby hamster kidney cells was markedly increased when attached cells were treated with concanavalin A (Con A). The cells became less sensitive to detachment by physical shear or by treatment with trypsin or EDTA; however, their morphology, as observed by phase contrast microscopy, did not change. The effects of Con A were prevented by the simultaneous addition of either D-glucose or α-methyl-D-glucoside with the Con A. Also addition of these reagents to the attached cells after Con A treatment partially reversed the effects caused by Con A. Pre-treatment of the culture flasks with Con A before cell attachment resulted in an increase in the strength of cell attachment to the culture flasks as compared to untreated controls.     

Attachment of the Yeast Rhodosporidium toruloides Is Mediated by Adhesives Localized at Sites of Bud Cell Development

The basidiomycetous yeast Rhodosporidium toruloides (anamorph, Rhodotorula glutinis) is a common phylloplane epiphyte with biocontrol potential. To understand how R. toruloides adheres to plant surfaces, we obtained nonadherent fungal mutants after chemical mutagenesis with methane-sulfonic acid ethyl ester. Sixteen attachment-minus (Att⁻) mutants were identified by three methods: (i) screening capsule-minus colonies for loss of adhesive ability; (ii) enrichment for mutants unable to attach to polystyrene; and (iii) selection for reduced fluorescence of fluorescein isothiocyanate-concanavalin A (Con A)-stained cells by fluorescence-activated cell sorting. None of the 16 mutants attached to polystyrene or barley leaves. The lectin Con A eliminated adhesion in all of the wild-type isolates tested. Hapten competition assays indicated that Con A bound to mannose residues on the cell surface. Adhesion of wild-type R. toruloides was transient; nonadhesive cells subsequently became adhesive, with bud development. All Att⁻ mutants and nonattaching wild-type cells lacked polar regions that stained intensely with fluorescein isothiocyanate-Con A and India ink. Lectin, enzyme, and chemical treatments showed that the polar regions consisted of alkali-soluble materials, including mannose residues. Tunicamycin treatment reduced wild-type adhesion, indicating that the mannose residues could be associated with glycoproteins. We concluded that compounds, including mannose residues, that are localized at sites of bud development mediate adhesion of R. toruloides to both polystyrene and barley leaf surfaces.     

Toxoplasma gondii: lymphocyte function during acute infection in mice

T-cell function during acute Toxoplasma gondii infection was evaluated in murine models. Blastogenic response to the T-cell mitogen concanavalin A (Con A) was not depressed during infection with either the C37 or the C56 strain of T. gondii in either $\text{BALB}\text{c}$ or $\text{C}\text{57}\text{BL}\text{6}\text{J}$ mice that were inoculated either intravenously or intraperitoneally with varying doses of tachyzoites 7, 14, or 30 days earlier. In evaluation of lymphocytes from individual mice, utilization of a range of concentrations of Con A was found to be important for correct interpretation of results. There was variability in the magnitude of response of individual mice and in the concentration of mitogen that produced an optimal response among the inbred mice. The T-cell-dependent, primary antibody response to sheep red blood cells (SRBC) was not depressed in $\text{BALB}\text{c}$ mice infected with the C37 strain of Toxoplasma 1 and 8 days prior to inoculation with SRBC. A lower blastogenic reponse to Con A of lymphocytes from $\text{C}\text{57}\text{BL}\text{6}\text{J}$ mice compared with that of $\text{BALB}\text{c}$ mice appeared to correlate with increased susceptibility of $\text{C}\text{57}\text{BL}\text{6}\text{J}$ mice to low-challenge inocula of T. gondii.     

Divergent actions of monomeric, dimeric, and tetrameric concanavalin A on lymphocyte traffic

The autoradiographic analysis of the localization of [³H]adenosine-labeled cells exposed to concanavalin A (Con A) in vitro has confirmed that the altered migration of Con A-treated lymphocytes is a consequence of their slower rate of migration and delay in normal areas of traffic (5, 6). The mechanisms through which Con A alters cell migration were further investigated by studying the effects of several derivatives of Con A on the distribution of ⁵¹Cr-labeled lymph node cells. The results obtained show that the monomeric and dimeric forms of Con A were unable to modify cell traffic, a condition that was partially reversed when succinyl Con A-treated cells were exposed to divalent antibodies to Con A. This suggests that Con A may alter lymphocyte recirculation by actively modifying the membrane fluidity or the surface lateral transport of the lymphocyte. Whatever the exact mechanisms responsible for the altered migration are, they probably involve complex active processes that can be related to the heterogeneity of Con A receptors, the existence of subsets of cells with different reactivities to the lectin, or simply the result of a passive phenomenon dependent on the presence of Con A on the cell surface.     

Pathology and host responses induced by Schistosomatium douthitti in the freshwater snail Lymnaea catascopium.

Most Schitosomatium douthitti miracidia penetrated the esophageal wall of Lymnaea catascopium without provoking amoebocyte encapsulation responses or extensive pathological changes. Amoebocytes frequently attached to developing mother and daughter sporocysts, but did not encapsulate or destroy them. Pressure resulting from extensive growth of mother sporocysts ruptured the transverse membrane of some snails. After releasing daughter sporocysts, mother sporocysts in some snails were destroyed by amoebocytes. Many migrating cercariae became trapped in the tissues of L. catascopium, particularly in the posterior portion of the foot, and were encapsulated and destroyed. Large increases in numbers of amoebocytes in the anterior portion of the lung roof of infected snails were noted, even before cercarial production had been initiated. Atrophy of the digestive gland occurred in infected snails.     

Haemocyte morphology and function in the Akoya Pearl Oyster, Pinctada imbricata

The morphology and cytochemistry of Pinctada imbricata haemocytes were studied in vitro. Three distinct blood cell types were identified; hyalinocytes, granulocytes, and serous cells. Haemocytes were classified based on the presence/absence of granules, and nucleus to cytoplasm ratio. Granulocytes were the most common cell type (62 ± 2.81%), followed by hyalinocytes (36 ± 2.35%), and serous cells (2 ± 0.90%). Granulocytes, and hyalinocytes were found to be immunologically active, with the ability to phagocytose Congo red stained yeast. Of the cells involved in phagocytosis, granulocytes were the most active with 88.8 ± 3.9% of these haemocytes engulfing yeast. Cytochemical stains (phenoloxidase, peroxidase, superoxide, melanin, neutral red) showed that enzymes associated with phagocytic activity were localised in granules within granulocytes. Based on their affinities for Giemsa/May-Grünwald stain, haemocytes were also defined as either acidic, basic or neutral. Hyalinocytes and serous cells were found to be eosinophilic, whilst granulocytes were either basophilic (large granulocytes), eosinophilic (small granulocytes) or a combination of the two (combination granulocytes). Light, differential interference contrast and epi-fluorescence microscopy identified three sub-populations of granulocytes based on size and granularity; small (4.00-5.00 μm in diameter, with small granules (0.05-0.5 μm in diameter), large (5.00-9.00 μm in diameter, with large granules (0.50-2.50 μm in diameter) and combination (5.00-9.00 μm in diameter, with both large and small granules). These observations demonstrate that P. imbricata have a variety of morphologically and functionally specialized haemocytes, many of which maybe associated with immunological functions.     

Study of real-time lectin-carbohydrate interactions on the surface of a quartz crystal microbalance

A quartz crystal microbalance (QCM) biosensor system for lectin-carbohydrate interactions has been developed. Yeast mannan was immobilised on polystyrene-coated quartz crystals, and interactions tested with the lectin concanavalin A (Con A). The biosensor could be easily operated, where mannan immobilisation and all binding analyses were performed in real-time using a flow-through system. The apparent binding constant for yeast mannan to Con A was estimated to be 0.4 microM, well in accordance to reported literature values. In addition, the effective concentration values (EC50-values) for a series of mannose/mannoside ligands, acting as competitors to the mannan/Con A interaction, were determined to range from 0.18 to 5.3 mM, in good correlation with a related enzyme-labelled lectin assay (ELLA) protocol.     

The attachment to human buccal epithelial cells by Candida albicans: An in vitro kinetic study using concanavalin A

The early in vitro kinetics of Candida albicans attachment to human buccal epithelial cells was studied with the aid of an adhesion assay and solutions of concanavalin A (Con A), a lectin which is capable of inhibiting yeast adhesion. Various saccharides and putative receptor analogues were also tested. Solutions of each single reagent were added to tubes containing aliquots of mucosal cells and germinated yeasts at the beginning of a 1-hour incubation period (time O) or at 10 minute intervals during the assay. The number of yeasts attached to 200 mucosal cells was subsequently determined microscopically. Yeast adhesion remained constant following addition of phosphate-buffered saline (PBS) at time 0 or at any time thereafter. However, addition of Con A at 0, 10 or 20 minutes of incubation decreased adhesion significantly to 38%, 45% and 63% of control values. This inhibitory effect dwindled as time of incubation prior to lectin addition increased and Con A could not inhibit adhesion significantly after twenty minutes. Results obtained with Con A using live germinated yeasts were similar to those obtained with formalin-killed C. albicans. The other reagents tested failed to decrease adhesion significantly. These included the putative receptor analogues fibronectin, N-acetyl-d-glucosamine and d-galactose, and several non-specific saccharides such as -d-methylglucopyranoside, d-ribose and d-xylose. It is suggested that in vitro attachment to human mucosal cells by C. albicans is inhibitable up to a defined point in time by a lectin with affinity for mannosecontaining surface moieties, but becomes non-reversible thereafter. This experimentally-observed irreversibility is independent of yeast cell viability.     

Adoptive transfer of experimental allergic encephalomyelitis with activated spleen cells: Comparison of in vitro activation by concanavalin A and myelin basic protein

Adoptive transfer of experimental allergic encephalomyelitis (EAE) was carried out in Lewis rats using splenic lymphocytes incubated in vitro with either concanavalin A (Con A) or myelin basic protein (MBP). Requirements were established for sensitization of donors, culture conditions, numbers of transferred cells, and incubation period of EAE in recipients. These were strikingly similar whether Con A or MBP was used. In addition, cellular proliferation in vitro was not required in either system, but proliferation after transfer to the recipient was essential for the development of clinical signs and histological lesions. These methods have potential value for analyzing mechanisms of immune induction in this classic model of autoimmune disease.     

Separation of mitogen-induced suppressor cells of human antibody-producing cells

Human antibody-forming cells were demonstrated by a plaque in agar technique following in vitro stimulation with either pokeweed mitogen or Cowan I strain of protein A-positive Staphylococcus aureus bacteria. We evaluated the effects on this antibody formation caused by the addition of cells which had been stimulated with PH A or Con A. Both Con A and PHA cells harvested after 3 days showed strong inhibition of pokeweed-induced plaque formation. The majority of the suppression could be accounted for by a blast fraction separated on 1g sedimentation gradients from the Con A or PHA cultures. Small cells from such cultures showed inhibition of PFC when added at high ratios (1:2), but this suppressive activity diluted out much more rapidly than that of the blast cells. No helper activity was noted with either small cells or blasts. Our studies indicate a T-cell blast as the suppressive fraction in Con A- or PHA-stimulated human lymphoid cells. While this T-cell suppression applies to T-dependent responses such as antibody stimulation with pokeweed mitogen, it does not have a substantial effect on Cowan I-induced plaque-forming responses. The finding that Cowan I-induced plaques could not be inhibited by Con A or PHA blasts indicates the T independence of this response.     

Cellular immune responses of spruce budworm larvae to Entomophthora egressa protoplasts and other test particles

The protoplast stage of two isolates of Entomophthora egressa developed normally and eventually produced conidiophores when injected into larvae of the spruce budworm, Choristoneura fumiferana. The spruce budworm hemocytes never made long-term contact with the protoplasts either in vivo or in vitro. The protoplasts made active, short-term contact with spruce budworm granulocytes both in vivo and in vitro. Total larval hemocyte counts (THC) initially declined when larvae were injected with protoplasts, growth medium (MGM), or Escherichia coli. The recovery rate to THC control levels was similar for MGM and protoplasts and supports the concept of nonrecognition of protoplasts by the hemocytes. The granulocytes were important in both nodulation and phagocytosis of E. coli and Bacillus cereus, whereas the plasmatocytes were important in phagocytosis. In in vitro studies, spruce budworm granulocytes did not adhere to rod-shaped hyphal bodies, spherical hyphal bodies, or germinating spherical hyphal bodies of E. egressa, whereas the granulocytes readily encapsulated the hyphae. There was no evidence for the production by the protoplasts of metabolites which might interfere with hemocyte adhesion. When protoplasts contacted Tenebrio molitor granulocytes, the protoplasts reacted by increasing the number of protoplasmic extensions and by granule discharge. The process of granule discharge may be an active protoplast defense mechanism. The sporangiospores of Absidia repens and Rhizopus nigricans adhered to spruce budworm granulocytes; however, the number of A. repens spores per granulocyte and the level of granulocytes with spores decreased in the presence of phenylthiourea. The adhesion of A. repens spores to granulocytes was enhanced by N-acetylglucosamine, whereas glucosamine, sucrose, fucose, fructose, arabinose, and galactose either had no effect on or reduced spore adhesion. Thus, the chitin (or its subunits) in the hyphal wall may initiate the granulocyte response.     

Control of Root-knot Nematodes on Tomato by Lectins

Significant control of tomato root knot was achieved by applications of the lectins Concanavalin A (Con A) and Limax flavus agglutinin in greenhouse, growth chamber, and microplot trials. Four consecutive weekly applications at lower concentrations of Con A yielded better control than single applications at a higher total concentration. The present state of knowledge on binding of Con A to soil nematodes and the in vitro effect of this lectin in chemotactic behavior are discussed. The mode of action of Con A on root-knot control is unknown.     

Mitogen Synergism in low-responding CBA/CaJ mice.

Although neither phytohemagglutinin (PHA) nor concanavalin A (Con A) stimulated blood cultures in vitro from low-responding CBA/CaJ mice effectively, a mixture of PHA and Con A over a range of concentrations stimulated a response from CBA/CaJ mouse blood that was greater than the sum of the responses produced by using PHA or Con A individually. This synergistic effect was expressed as the percentage by which the responses to the PHA and Con A mixture exceeded the sum of the responses to PHA alone and Con A alone. When the mitogen concentrations that gave maximum responses individually were used, the synergistic effect averaged 319% in cultures of blood from low-responding CBA/CaJ mice. Apparently simultaneous exposure to PHA and Con A stimulates DNA synthesis in white blood cells of CBA/CaJ mice that fail to respond to either mitogen alone.