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At least 19 recordsLinked to original sources
PubMedDate not supplied

Monoclonal antibodies against Manduca sexta hemocytes bind Aedes aegypti hemocytes: characterization of six monoclonal antibodies that bind hemocytes from both species.

The mechanisms by which hemocytes mediate a mosquito's defense response to parasites or pathogens are not well understood. This is due in part to difficulty in collecting intact mosquito hemocytes for experiments and to a lack of reagents, such as antibodies. Our objectives were to collect adult Aedes aegypti hemocytes under conditions suitable for immunofluorescence microscopy, and to test whether monoclonal antibodies, generated against larval Manduca sexta hemocytes, bind adult Ae. aegypti hemocytes. We present immunofluorescence micrographs of M. sexta and Ae. aegypti hemocytes stained by six monoclonal antibodies. Two antibodies, MS11 and MS32, immunolocalized to hemocyte nuclei in both species. On Western blots, these antibodies generate one signal at approximately 40 kDa and four others between 10 and 25 kDa. Immunofluorescence staining patterns of the other four antibodies were more complex. That these antibodies bind hemocytes from both species suggests significant molecular similarities exist between hemocytes from evolutionarily divergent species.

Aedes↗
PubMed1989

Differential adherence of M line Biomphalaria glabrata hemocytes to Schistosoma mansoni and Echinostoma paraensei larvae, and experimental manipulation of hemocyte binding.

The ability of M line strain Biomphalaria glabrata hemocytes to adhere to mother sporocysts (MS) of PR1 Schistosoma mansoni or to MS or daughter rediae (DR) of Echinostoma paraensei was studied using an in vitro hemocyte adherence assay. Hemocytes were significantly more likely to bind to S. mansoni MS than to E. paraensei MS or DR. Hemocyte adherence to E. paraensei MS or DR was significantly increased if glutaraldehyde-fixed larvae were used as targets. Also, E. paraensei MS pretreated with the lectin concanavalin A (Con A) were more likely to be bound by hemocytes than MS pretreated with Con A in the presence of the competing sugar, alpha-methyl mannoside. Pretreatment of hemocytes with Con A increased their ability to bind E. paraensei sporocysts, but the effect was small compared to that achieved by pretreatment of MS with Con A. The lectin probably did not function as a bridging molecule between hemocytes and MS but, rather, altered the MS surface in a way that facilitated adherence. Similarly, adherence to E. paraensei MS was significantly increased if the MS were pretreated with cell-free M line plasma prior to use in adherence assays. Our results indicate that the two parasites provoke fundamentally different responses from M line hemocytes in vitro and that the living tegument can be modified by host humoral factors and by lectins such that hemocyte binding is significantly increased.

Animals↗
PubMed1994

Immunochemical identification of insect hemocyte populations: monoclonal antibodies distinguish four major hemocyte types in manduca sexta.

We have made 140 monoclonal antibodies to hemocytes (insect blood cells) from Manduca sexta. Four of these antibodies, when used in immunofluorescent microscopy of fixed hemocytes, distinguish the four main morphologically distinct hemocyte types. Plasmatocytes, granular cells, and oenocytoids are each recognized by a unique antibody specific to that type; spherulocytes are recognized by an antibody that also binds to plasmatocytes. When used in flow cytometry with nonfixed hemocytes, three of the four antibodies bind their respective cells; the oenocytoid marker failed to bind to any hemocytes. This set of four monoclonal antibodies may be useful for labeling individual cell types and for separating the different hemocyte types for further study of hemocyte functions.

Animals↗
PubMed1984

Hemocyte lysate enhancement of fungal spore encapsulation by crayfish hemocytes.

Crayfish hemocytes exhibited a stronger encapsulation reaction to fungal blastospores of Beauveria bassiana coated with hemocyte lysate, than to blastospores treated with plasma or buffer, indicating an opsonic function of hemocyte lysate proteins. Five proteins of the prophenoloxidase activating system in the hemocytes were attached to foreign surfaces (including the blastospores) after activation and it is suggested that these attaching proteins (one being phenoloxidase) are responsible for the opsonic function of the hemocyte lysate on crayfish blood cells.

Animals↗
PubMed1988

Monoclonal antibody recognized hemocyte subpopulations in juvenile and adult Lymnaea stagnalis: functional characteristics and lectin binding.

The mouse monoclonal antibody LS1 recognizes a membrane epitope present on circulating hemocytes of the gastropod mollusc Lymnaea stagnalis. In both juvenile and adult pond snails, LS1+ (LS1 positive) hemocytes have the morphology of immature cells. The percentage of LS1+ hemocytes is higher in juveniles (ca. 39%) than it is in adults (ca. 14%). Functional characteristics of LS1+ hemocytes and lectin binding to these cells were studied. In both age groups, the proliferative activity, as measured by the incorporation of deoxybromouridine, is much higher for LS1+ hemocytes than it is for LS1- (LS1 negative) cells. LS1+ hemocytes are phagocytically less active and have a lower lysosomal enzyme (peroxidase) content as compared to hemocytes that lack the epitope. Histochemical staining of the total population of circulating hemocytes shows that the lectins DBA, BS-l-A4 and BS-l-B4, PNA, SBA and ECA do not react with the hemocytes. LTA, APA, WGA, Con A and LCA bind to all hemocytes. RCA and STA recognize surface carbohydrate moieties present on subpopulations of hemocytes only. The LS1+ hemocyte population virtually lacks the carbohydrate residues recognized by STA, whereas the LS1- population never shows binding of RCA. Our results support the findings that the LS1 epitope is a membrane marker of less differentiated hemocytes in both juvenile and adult L. stagnalis. Furthermore, they suggest a correlation between the presence of the LS1 epitope and the absence of STA binding, whereas absence of the LS1 marker may correlate with the presence of a sugar recognized by RCA.

Animals↗
PubMed1989

Phagocytic activity of hemocytes of M-line Biomphalaria glabrata snails: effect of exposure to the trematode Echinostoma paraensei.

The phagocytic activity of hemocytes from 6-8-mm M-line Biomphalaria glabrata snails was studied in an in vitro assay using glutaraldehyde-fixed sheep erythrocytes (SRBC) as target cells. For individual snails, the percentage of hemocytes ingesting SRBC during a 1-hr interval, termed the phagocytic activity index (PAI), was determined. Hemocytes from snails infected for 1 day with Echinostoma paraensei had a slightly elevated PAI, but at both 8 and 30 days postexposure (DPE), hemocytes from infected snails had a significantly lower PAI than controls. Hemocytes taken from snails at 8 DPE also had a low PAI using rabbit erythrocytes and yeast as target cells. The low PAI at 8 DPE is attributed to the presence of large numbers of poorly spreading hemocytes with low phagocytic activity. Hemocytes from snails with 30-day infections were well spread but nonetheless had a low PAI. The presence of plasma from 8-day infected snails did not alter the PAI of hemocytes from control snails, nor was the PAI of hemocytes from infected snails changed by plasma from control snails. SRBC preincubated for 60 min in plasma from various groups of M-line snails did not elicit an increase in PAI when presented to hemocytes from control snails; in some cases, as with plasma from 6-8-mm control snails, such preincubation significantly reduced the PAI below levels obtained using SRBC preincubated in culture medium. As compared to hemocytes from snails with normally developing, 8-day-old intraventricular sporocysts (IS), hemocytes from snails exposed to infection but subsequently lacking IS had a significantly higher PAI.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Secretory protein biosynthesis in snail hemocytes: in vitro modulation by larval schistosome excretory-secretory products.

Circulating hemocytes of the snail, Biomphalaria glabrata, synthesize and secrete a variety of polypeptides when maintained in vitro in serum-free medium containing [35S] methionine. SDS-PAGE/fluorographic analysis of supernatants from resistant snail (10-R2-OK strain) hemocyte cultures revealed the presence of numerous labeled polypeptides ranging in Mr from 220 to 14 kDa. Most of these same proteins were also produced by hemocytes of a susceptible B. glabrata strain (M-line), but the overall rate of secretory protein synthesis was reduced from that of resistant snail cells. In addition, excretory-secretory (ES) products contained in supernatants from Schistosoma mansoni miracidial transformation and 1-day primary sporocyst cultures stimulated increases in the synthesis of various polypeptides. Particularly striking was a 3-fold increase in the synthesis of a 66-kDa secretory polypeptide by hemocytes of both snail strains, and a concomitant increase in M-line hemocytes and decrease in 10-R2-OK cells of a 63-kDa polypeptide. Overall, however, the level of ES product-induced secretory protein synthesis was greater in 10-R2-OK snail hemocytes than in those of the M-line strain. Exposure of a nonhemocytic B. glabrata cell line to parasite culture supernatants had no stimulatory/inhibitory effect on labeled protein ouput, suggesting that the observed hemocyte response may be snail cell-type specific. Finally, the larval ES components responsible for modulating hemocyte protein metabolism are mainly concentrated in a heat-stable fraction composed of molecules of greater than 30 kDa. However, the loss of the ability of heated parasite products to stimulate synthesis of certain hemocyte proteins and the presence of minor stimulating activity in a low molecular weight fraction (less than 10 kDa) implies the possible existence of multiple larval components affecting formation of specific hemocyte secretory polypeptides. It is concluded that snail hemocytes are capable of in vitro synthesis and secretion of a variety of methionine-containing polypeptides, and that ES products of early larval schistosomes can modulate (i.e., stimulate or inhibit) this metabolic process. A differential response of susceptible vs. resistant hemocytes to larval products suggests that the degree to which these cells can be metabolically activated may determine their cytotoxic effectiveness.

Animals↗
PubMed1994

A time-lapse study of interactions between Echinostoma paraensei intramolluscan larval stages and adherent hemocytes from Biomphalaria glabrata and Helix aspersa.

In vitro interactions between intramolluscan stages (sporocyst, daughter rediae, and metacercariae) of the trematode parasite Echinostoma paraensei and adherent hemocytes from the gastropods Biomphalaria glabrata (intermediate host) and Helix aspersa (non-host) were visualized by time-lapse videomicroscopy. Hemocytes of either species not exposed to E. paraensei displayed extensive mobility and activity of cellular extensions. Image analysis disclosed no significant change in the total surface area occupied by hemocytes in a selected field of view over 2 hr. Echinostoma paraensei exerted life stage-specific effects on the behavior of B. glabrata hemocytes; the cells moved away from sporocysts and daughter rediae but not encysted metacercariae. In the presence of sporocysts, hemocytes rounded up, whereas hemocytes adjacent to rediae assumed a stringy, beady appearance. Hemocytes close to the parasite were affected more rapidly than more distant cells. In 2 hr, a hemocyte-free "halo" formed around the parasite larvae, significantly reducing the hemocyte-occupied surface area (to 43% by sporocysts and to 70% by rediae). The changes induced by sporocysts and rediae are similar to those noted in both in vivo and in vitro studies of the B. glabrata-E. paraensei model system and are interpreted as manifestations of parasite-mediated interference with host hemocyte function. Helix aspersa (non-host) hemocytes were not affected, suggesting that E. paraensei-mediated effects on hemocytes exhibit a degree of specificity.

Analysis of Variance↗
PubMed1986

Lectin-dependent recognition of foreign cells by hemocytes of the mussel, Mytilus edulis.

Phagocytosis of human erythrocytes (rbc) by hemocytes of the mussel Mytilus edulis was found to be influenced by four heterologous lectins. The effects were examined in the absence of Ca++ ions under three experimental conditions: when the lectins were bound to 1) both hemocytes and rbc, 2) only hemocytes, but not to rbc, and 3) only rbc, but not to hemocytes. The lectins used included: albumen gland agglutinin from Helix pomatia (HPA), wheat germ agglutinin (WGA), Ricinus-120 (Ric-120) and Concanavalin-A (Con A). HPA, WGA and Ric-120, for which both hemocytes and A-rbc possess receptors, strongly enhanced uptake of A-rbc. This lectin-mediated phagocytosis was abolished by addition of specific sugars either to lectin-pretreated rbc (HPA, WGA) or to a pretreated hemocyte monolayer (Ric-120); this indicated the stimulation of phagocytosis by the binding of lectin to carbohydrate determinants at the surface of hemocytes and target cells. On the other hand, HPA which binds to hemocytes, but not to O-rbc, did not influence phagocytosis of these rbc; and Con A which binds to A-rbc, but not to hemocytes, also failed to stimulate phagocytosis. These findings reveal the importance of carbohydrate determinants on the surface of hemocytes as well as on target cells in recognition and in lectin-mediated phagocytosis of foreign cells by Mytilus hemocytes.

Bivalvia↗
PubMed1995

Hemocytes of schistosome-resistant and -susceptible Biomphalaria glabrata recognize different antigens on the surface of Schistosoma mansoni sporocysts.

A cytoadherence assay was used to determine whether antibodies to plasma and hemocyte components of Schistosoma mansoni-susceptible (M-line) and -resistant (10-R2, 13-16-R1) strains of Biomphalaria glabrata affected attachment of hemocytes to chemically fixed schistosome sporocysts differentially. Experiments used purified, intact IgG and purified Fab fragments of each antibody. Indirect fluorescent antibody tests confirmed that the intact purified IgG to plasma and hemocytes from the 3 strains of snails bound to sporocysts. There was no qualitative difference in fluorescence among any of the antibodies to snail components, although the various antibodies affected adherence of hemocytes to the parasite differentially. Adherence assays revealed that antibodies to plasma components inhibited binding of hemocytes from the snail strain to which the antibody was generated. Additionally, antibody to plasma from resistant strains of B. glabrata inhibited binding of hemocytes from the homologous and heterologous resistant strains but not hemocytes from susceptible snails. Antibody to hemocytes from M-line snails did not inhibit binding of hemocytes from any of the snail strains. Since results of assays using intact IgG correlated well with assays using their Fab fragment counterparts, it was concluded that hemocytes from the 3 snail strains utilize specific antigen-binding sites on sporocysts. Results of these assays also indicate that, with regard to the mechanism of hemocyte binding to sporocysts, M-line and 13-16-R1 snails are more dissimilar than M-line and 10-R2 or 10-R2 and 13-16-R1 B. glabrata.

Animals↗
PubMed1994

Surface membrane polypeptides associated with hemocytes from Schistosoma mansoni-susceptible and -resistant strains of Biomphalaria glabrata (Gastropoda).

Previous studies have shown that hemocytes from inbred susceptible and resistant strains of the snail, Biomphalaria glabrata, differentially react to primary sporocysts of the blood fluke, Schistosoma mansoni. It has been hypothesized that this differential reactivity may be due to differences in the expression of hemocyte surface determinants serving as parasite recognition and/or hemocyte activating factors. In order to begin addressing this hypothesis the hemodynamics and surface polypeptides of circulating hemocytes from susceptible and resistant B. glabrata strains were compared in two morphologically distinct cellular subpopulations that differed in their substrate adherence properties. When compared to a S. mansoni-resistant snail strain, two susceptible strains exhibited overall lower total circulating cell numbers and a consistently lower proportion of nonadherent hemocytes. Exposure of snails to S. mansoni miracidia induced significant increases in total circulating cell number in all strains by 2 days postexposure (PE). However, the proportion of adherent and nonadherent hemocytes remained constant in each snail strain at 2 and 4 days PE. A sensitive technique involving biotin labeling of polypeptides at the surface of living hemocytes, followed by nonreducing SDS-PAGE separation and electrotransfer to nitrocellulose, revealed a similar pattern of hemocyte surface polypeptides ranging in apparent molecular weights from 210 to 30 kDa in the three B. glabrata strains. One exception was the presence of a 66-kDa polypeptide expressed at the surface of both adherent and nonadherent hemocytes in the two susceptible snail strains, which was only weakly expressed or absent from hemocytes of the resistant 13-16-R1 B. glabrata strain.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1983

Identification of antigenically distinct hemocyte subpopulations in Biomphalaria glabrata (Gastropoda) using monoclonal antibodies to surface membrane markers.

Five monoclonal antibodies (mABs) against surface antigens on circulating, glass-adherent hemocytes of the snail, Biomphalaria glabrata, were produced by somatic cell hybridization methods. Two mABs (IID2.6-Bg and IID4.8-Bg) are pan-hemocytic, reacting uniformly with epitopes shared by all adherent hemocytes. Determinants recognized by these mABs also are present in soluble form and appear to be associated with a hemoglobin-depleted ultracentrifuged fraction of snail hemolymph. Hybridoma-derived mABs IIC6.8-Bg and VB10.3-Bg recognize hemocyte surface epitopes expressed by only 50-60% of the adherent cell population. These mABs also are reactive with soluble hemolymph antigens but apparently recognize determinants which are different from the IID2.6-Bg and IID4.8-Bg reactive sites. Another antigenically distinct hemocyte subpopulation is recognized by mAB IID7.1-Bg. Epitopes that are reactive with this mAB differ from the previously described determinants by their asymmetrical distribution on the surface of positive cells and the absence of soluble antigenic components in hemolymph. Furthermore, unlike the other mABs, the prevalence of hemocytes staining with IID7.1-Bg antibodies differed between two strains of B. glabrata. Results of this study clearly demonstrate that circulating B. glabrata hemocytes, consisting of a single, predominant population of adherent cells, is composed of several distinct antigenic subpopulations based on the specific binding of anti-hemocyte mAB probes. Our successful application of hybridoma techniques to the study of molluscan hemocyte surface antigens underscores further the great potential usefulness of this method in analysing the molecular basis of hemocyte reactivity.

Animals↗
PubMed1991

Aedes aegypti: characterization of hemocyte polypeptide synthesis during wound healing and immune response to inoculated microfilariae.

Hemocytes from adult, female Aedes aegypti, intrathoracically inoculated with microfilariae (mf) of the nematode Dirofilaria immitis, were compared to saline-inoculated and uninoculated controls using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), 125I-labeling, and wheat germ agglutinin (WGA) binding techniques. Activation of wound healing and/or melanotic encapsulation responses by the inoculation of saline or mf into the host hemocoel induced alterations in the hemocyte activity of these mosquitoes. Protein assays of whole hemocyte lysates revealed that hemocytes from saline- and mf-inoculated mosquitoes had higher protein concentrations than uninoculated controls. Many polypeptides were seen within all three hemocytes preparations when stained with silver nitrate, but there was an overall increase in protein synthesis in hemocytes from inoculated mosquitoes. In addition, a 200-kDa polypeptide was uniquely expressed in hemocytes from inoculated mosquitoes. There were several prominent surface proteins labeled with 125I, and several of these increased dramatically in intensity during wound healing and/or a melanotic encapsulation response. Similar results were seen in two-dimensional separations. A set of basic polypeptides comigrated with an acidic polypeptide resulting in a surface protein of approximately 80-90 kDa that increased in inoculated mosquitoes. Hemocytes from inoculated mosquitoes exhibited a group of three acidic polypeptides, whereas hemocytes from uninoculated mosquitoes exhibited only one of these protein fragments. Three surface polypeptides bound 125I-labeled WGA, and binding of WGA to hemocyte surface polypeptides was successfully inhibited by the incubation of cells with the lectin and its competing sugar.

Acetylglucosamine↗
PubMedDate not supplied

Suppression of chemiluminescence of eastern oyster (Crassostrea virginica) hemocytes by the protozoan parasite Perkinsus marinus.

Experiments were conducted to determine the ability of the protistan parasite, Perkinsus marinus, to inhibit chemiluminescence of hemocytes from the eastern oyster, Crassostrea virginica. Luminol-enhanced chemiluminescence (CL) was used to measure the production of reactive oxygen intermediates (ROI) generated by oyster hemocytes using zymosan as a stimulant. To determine whether P. marinus suppresses ROI evoked from zymosan-stimulated hemocytes, live or heat killed P. marinus in filtered estuarine water (YRW) (salinity = 20 ppt) were added to (1) zymosan-stimulated hemocytes after CL reached its peak, or (2) hemocytes at the same time as zymosan, and reduction of CL responses were recorded. In both tests, controls received only estuarine water. Live P. marinus meronts significantly suppressed ROI production by zymosan-stimulated hemocytes. The suppression of ROI production was dose dependent. Suppression of ROI production from zymosan-stimulated hemocytes by heat killed P. marinus was significantly less than by live P. marinus. Similarly, CL of hemocytes was reduced, though not significantly when hemocytes were exposed to YRW preincubated with P. marinus. When P. marinus meronts were used as a stimulant, no CL response was elicited. Results of this study suggest that P. marinus cells are able to suppress ROI release from oyster hemocytes, thus evading this component of the host's defense.

Animals↗