The effect of schistosome excretory-secretory products on Biomphalaria glabrata hemocyte motility

Excretory-secretory (E-S) products contained in supernatants from in vitro cultured Schistosoma mansoni primary sporocysts were assayed for their effects on the in vitro motility of Biomphalaria glabrata hemocytes. Both whole (unfractionated) and fractionated E-S products were tested in modified Boyden chemotaxis chambers. E-S product fractionation was accomplished using both membrane ultrafiltration (MF) and high-pressure liquid chromatography (HPLC). Transformation (Tr) products, but not those products released by 8-day sporocysts, significantly inhibited the random motility of hemocytes from an S. mansoni susceptible strain (M-Line) of B. glabrata. This activity was found in both high and low MF fractions of Tr but not in an intermediate MF fraction. In an effort to isolate the active component(s) of the high MF fraction, HPLC was used to separate components based on size exclusion. Although each of four HPLC fractions displayed some inhibitory activity, the greatest consistent activity was found in fraction 3, which was composed, predominantly, of a 108-kDa protein. In contrast to the response of M-Line cells to Tr E-S products, the motility of hemocytes from an S. mansoni-resistant strain (10-R2-OK) of B. glabrata was not significantly reduced from controls. The high MF fraction, however, elicited a slight positive chemokinetic response, while the low MF fraction reduced 10-R2-OK hemocyte motility slightly but not significantly. While three HPLC fractions significantly reduced 10-R2-OK hemocyte motility, this effect was significantly less than that produced by the same HPLC fractions on M-Line hemocyte motility. These data suggest that S. mansoni sporocyst Tr E-S products differentially affect the random motility of M-Line and 10-R2-OK snail hemocytes. Although the significance of this differential effect on the in vivo defenses of B. glabrata is not known, it could be important in the host-parasite interaction which leads to either resistance or susceptibility.     

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.     

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.     

In vitro effect of larval Schistosoma mansoni excretory-secretory products on phagocytosis-stimulated superoxide production in hemocytes from Biomphalaria glabrata

The in vitro production of the reactive oxygen metabolite superoxide (O2-) was confirmed in hemocytes from the schistosome intermediate host Biomphalaria glabrata. Active forms of the enzyme superoxide dismutase (SOD) inhibited reduction of nitroblue tetrazolium (NBT) to formazan in cells that had phagocytozed zymosan particles, whereas an inactivated form of SOD did not. Moreover, based on the prevalence of O2(-)-positive hemocytes and the relative intensity of NBT staining reactions, hemocytes from the Schistosoma mansoni-resistant 10-R2 strain of B. glabrata possessed an overall greater capacity for generating superoxide than did those from S. mansoni-susceptible M-line snails. Schistosoma mansoni excretory-secretory (E-S) products, released during in vitro transformation of miracidia to sporocysts, inhibited phagocytosis of zymosan particles and superoxide activity in hemocytes from both snail strains, but 10-R2 hemocytes maintained higher levels of phagocytosis and superoxide production than did M-line hemocytes. The dose-dependent decreases in phagocytosis observed in both snail strains in the presence of E-S products could not account fully for the concomitant decrease in superoxide levels detected, indicating that either a single E-S factor differentially affects phagocytosis and superoxide production, or that different E-S factors are involved in the specific interference of each of these hemocyte functions.     

Schistosoma mansoni modulation of phagocytosis in Biomphalaria glabrata

Both short-term (3 hr) exposure of Biomphalaria glabrata snails (M-line and 13-16-R1) to Schistosoma mansoni (PR1) miracidia and in vitro incubation of parasite sporocysts with host hemolymph components altered host phagocytic ability. Hemocytes obtained from susceptible (M-line) snails that had been exposed to parasite miracidia for 3 hr showed reduced levels of phagocytosis of yeast cells in vitro compared to hemocytes from unexposed individuals. Incubation of whole hemolymph with sporocysts in vitro also reduced yeast phagocytosis in this susceptible strain. In contrast, resistant (13-16-R1) hemocytes showed increased levels of yeast phagocytosis after in vitro incubation with the parasite, and the opsonic properties of 13-16-R1 plasma were greater after exposure of snails to miracidia. These strain-specific effects of S. mansoni on host hemocyte phagocytosis and plasma opsonization were seen only when both plasma and hemocytes were present at the time of exposure to the parasite.     

Effects of exogenous interleukin-1β on primary sporocysts of Schistosoma mansoni (Trematoda) incubated with plasma and hemocytes from schistosome-susceptible and resistant Biomphalaria glabrata (Gastropoda)

Abstract. The cytokine interleukin-1β (IL-1β) mediates interactions of immune and inflammatory cells in mammals. Previous reports also have linked plasma (cell-free hemolymph) levels of IL-1β in the snail Biomphalaria glabrata to resistance against Schistosoma mansoni . In the present study, fluorescent probes were used to study larval schistosome and snail hemocyte viability during in vitro encounters. Hemolymph (plasma and hemocytes) from schistosome-susceptible (M-line) and resistant (13–16-R1) B. glabrata was added to sporocysts of S. mansoni and the viability of hemocytes and parasites was assessed. Next, IL-1β was added to sporocyst-hemolymph samples, the viability of sporocysts and hemocytes determined and then compared to control assays. The number of live sporocysts present after incubation for 1 h with hemolymph from M-line snails was significantly greater than the number seen when hemolymph from 13–16-R1 snails was tested. Nearly all sporocysts survived the 1 h incubation with M-line hemolymph, and most of the hemocytes attached to sporocysts were dead. In contrast, nearly all sporocysts were dead when hemolymph from 13–16-R1 snails was tested, and most attached hemocytes were alive. Addition of IL-1β to M-line hemolymph resulted in a dramatic increase in sporocyst death. Addition of IL-1β to 13–16-R1 hemolymph produced a small but significant increase in the rate of sporocyst death. These results indicate that the concentration of IL-1β present in hemolymph from B. glabrata is directly related to the ability of this snail to kill S. mansoni sporocysts in vitro.     

A comparative study of mechanisms underlying digenean-snail specificity: in vitro interactions between hemocytes and digenean larvae

A panel of 4 digenetic trematode species (Echinostoma paraensei, E. trivolvis, Schistosoma mansoni, and Schistosomatium douthitti) and 5 snail species (Biomphalaria glabrata, Helisoma trivolvis, Lymnaea stagnalis, Stagnicola elodes, and Helix aspersa) was examined to determine if known patterns of host specificity could be explained by the tendency of digenean larvae to be bound by snail hemocytes, or by the ability of larvae to influence the spreading behavior of hemocytes. In short-term (1 hr) in vitro adherence assays, there was no overall pattern to suggest that sporocysts were more likely to be bound by hemocytes from incompatible than compatible snails. Compared with the other parasites, sporocysts of E. paraensei were less likely to be bound by hemocytes from any of the snail species tested. All rediae examined, including those of another species Echinoparyphium sp., were also remarkably refractory to binding by hemocytes from any of the snails. Of all the larvae examined, only sporocysts and young daughter rediae of E. paraensei caused hemocytes to round up in their presence. This was true for hemocytes from the compatible species B. glabrata and the incompatible lymnaeid species S. elodes and L. stagnalis. The patterns of host specificity shown by this particular panel of parasites and snails were not predicted by either the extent of hemocyte adherence to digenean larvae or by the ability of larvae to affect hemocyte spreading behavior. The results of this study suggest that a role for hemocytes, although likely, may require different assays, possibly of a more prolonged nature, for its detection. Also, different parasite species (notably E. paraensei) and intramolluscan stages have distinctive interactions with host hemocytes, suggesting that the determinants of specificity vary with the host-parasite combination, and with the parasite life cycle stage.     

Killing of Schistosoma mansoni sporocysts by hemocytes from resistant Biomphalaria glabrata: role of reactive oxygen species

The fate of Schistosoma mansoni (Trematoda) sporocysts in its molluscan host Biomphalaria glabrata (Gastropoda) is determined by circulating phagocytes (hemocytes). When the parasite invades a resistant snail, it is attacked and destroyed by hemocytes, whereas in a susceptible host it remains unaffected. We used 3 inbred strains of B. glabrata: 13-16-R1 and 10-R2, which are resistant to the PR-1 strain of S. mansoni, and M-line Oregon (MO), which is susceptible to PR-1. In an in vitro killing assay using plasma-free hemocytes from these strains, the rate of parasite killing corresponded closely to the rate by which S. mansoni sporocysts are killed in vivo. Hemocytes from resistant snails killed more than 80% of S. mansoni sporocysts within 48 hr, whereas sporocyst mortality in the presence of hemocytes from susceptible snails was <10%. Using this in vitro assay, we assessed the involvement of reactive oxygen species (ROS) produced by resistant hemocytes, during killing of S. mansoni sporocysts. Inhibition of NADPH oxidase significantly reduced sporocyst killing by 13-16-R1 hemocytes, indicating that ROS play an important role in normal killing. Reduction of hydrogen peroxide (H2O2) by including catalase in the killing assay increased parasite viability. Reduction of superoxide (O2-), however, by addition of superoxide dismutase or scavenging of hydroxyl radicals (*OH) and hypochlorous acid (HOCl) by addition of hypotaurine did not alter the rate of sporocyst killing by resistant hemocytes. We conclude that H2O2 is the ROS mainly responsible for killing.     

Schistosoma mansoni: lectin-dependent cytotoxicity of hemocytes from susceptible host snails, Biomphalaria glabrata

Normally benign hemocytes from a strain (M-line) of the snail, Biomphalaria glabrata, susceptible to Schistosoma mansoni, became cytotoxic toward the sporocyst stage if the parasite was first treated with the lectin, concanavalin A. Concanavalin A binding was inhibitable with alpha-methyl mannoside and killing was dose-dependent. Maximal levels of concanavalin A-induced cytotoxicity were comparable with levels observed when hemocytes from a resistant snail strain (13-16-R1) encountered untreated sporocysts. Induction of the cytotoxic response did not occur if hemocytes alone were pretreated with the lectin. A unique method incorporating ultraviolet microscopy and the vital fluorescent dye, eosin Y, was used for discriminating between live and dead sporocysts. This model may prove useful in understanding mechanisms used by invertebrate effector cells in recognition and killing of invading organisms.     

Involvement of nitric oxide in killing of Schistosoma mansoni sporocysts by hemocytes from resistant Biomphalaria glabrata

In strains of the snail Biomphalaria glabrata (Gastropoda) that are resistant to the parasite Schistosoma mansoni (Trematoda), hemocytes in the hemolymph are responsible for elimination of S. mansoni sporocysts. The defensive role of reactive nitrogen species was investigated in in vitro interactions between hemocytes derived from the resistant 13-16-R1 strain of B. glabrata and the parasite. The nitric oxide synthase (NOS) inhibitor N(omega)-nitro-L-arginine methylester (L-NAME) and the nitric oxide (NO) scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide reduced cell-mediated killing of S. mansoni sporocysts. To determine if peroxynitrite (ONOO-) is involved in killing, assays were run in the presence of the ONOO- scavengers uric acid and deferoxamine. These did not influence the rate of parasite killing, indicating that NO is directly responsible for mediating cytotoxicity, but ONOO- is not. The combination of the NOS inhibitor L-NAME and catalase, an enzyme that detoxifies hydrogen peroxide (H2O2), reduced average sporocyst mortality to a greater extent than L-NAME alone. Killing of the sporocysts was, however, not totally inhibited. It is suggested that NO and H2O2 are both involved in hemocyte-mediated toxicity of 13-16-R1 B. glabrata against S. mansoni sporocysts.     

Characterization of excretory-secretory proteins synthesized in vitro by Schistosoma mansoni primary sporocysts

Excretory-secretory (E-S) products released by larval schistosomes have been implicated in the interference of host snail defense systems. Because of the potentially important role that E-S products play in the parasite-host relationship, total and newly synthesized E-S proteins from in vitro-cultured Schistosoma mansoni primary sporocysts were characterized using incorporation of [35S]methionine followed by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography. Total E-S protein decreased more than 5-fold from day 1 to day 3 of culture and remained constant until day 8 when protein concentrations began to increase. Release of newly synthesized protein, however, increased from day 1 through day 8. Both silver staining and fluorography of SDS-PAGE-separated E-S products revealed a wide variety of polypeptides ranging in Mr from 13 to greater than 200 kDa. The dynamics of the release of individual polypeptides, both total and newly synthesized, varied over time. Although certain polypeptides decreased in concentration, others remained constant or increased with time in culture. Culture conditions were found to be important for sporocyst viability and growth, and for release of newly synthesized proteins. Sporocysts cultured in medium containing fetal bovine serum (complete) grew significantly larger and had a significantly greater viability than did sporocysts cultured in medium lacking serum (incomplete). Also, sporocysts cultured in complete medium synthesized and released significantly more protein than did sporocysts cultured in incomplete medium. These sporocysts continued to produce a 54-kDa polypeptide, whereas sporocysts in incomplete medium stopped producing this protein by day 3 of culture. The present study has shown that S. mansoni primary sporocysts, cultured in vitro, synthesize and secrete a wide variety of glycoproteins and that the type and quantity of glycoproteins released are dependent on culture conditions.     

Echinostoma paraensei and Schistosoma mansoni: adherence of unaltered or modified latex beads to hemocytes of the host snail Biomphalaria glabrata.

Hemocytes derived from a strain (13-16-R1) of Biomphalaria glabrata resistant to Schistosoma mansoni were significantly more likely to bind untreated latex beads than hemocytes from the schistosome-susceptible M line strain. Beads preincubated in 13-16-R1 plasma were more readily bound by both 13-16-R1 and M line hemocytes than beads preincubated in M line plasma. Beads preincubated in plasma derived from snails of either strain infected with the trematode Echinostoma paraensei were more readily bound by hemocytes than beads preincubated in plasma from control snails of the corresponding strain. Plasma from snails exposed to S. mansoni did not have a similar effect. Throughout these experiments, beads receiving a particular treatment were consistently bound at higher rates by 13-16-R1 than M line hemocytes. SDS-PAGE of plasma components eluted from beads revealed differences between treatments, particularly in diffuse bands falling into two groups, of 75-130 and 150-220 kDa. The results indicate that both hemocytes and plasma components from the two host strains differ and identify plasma molecules deserving of additional study as possible modulators of hemocyte effector functions. Also, S. mansoni and E. paraensei provoked different responses in the same host snail.     

Differential binding of hemolymph proteins from schistosome-resistant and -susceptible Biomphalaria glabrata to Schistosoma mansoni sporocysts

Humoral factors have been associated with resistance of Biomphalaria glabrata to infection by Schistosoma mansoni. The goal of this study was to determine which serum (cell-free hemolymph) proteins bind to the surface of S. mansoni sporocysts. For this, 125I-labeled serum from schistosome-resistant (10-R2) and -susceptible (M-line) B. glabrata was incubated with sporocysts, washed, and then subjected to SDS-PAGE and autoradiography. Other samples examined included radiolabeled 10-R2 and M-line serum, sporocysts incubated with unlabeled serum followed by incubation with radiolabeled serum, and radiolabeled sporocysts. Results indicated that many polypeptides in the serum from both strains of B. glabrata were radiolabeled. Dominating both profiles were bands in the 90-210-kDa range. However, some differences between the serum of the 2 snail strains were observed with M-line serum having several radiolabeled polypeptides in the 31-40- and 66-85-kDa range that were absent in serum from 10-R2 B. glabrata. When sporocysts were incubated with radiolabeled serum, 3 polypeptides (116, 180, 210 kDa) from both snail strains bound to the surface of the parasite. Further, a 55-kDa polypeptide bound to sporocysts incubated with 10-R2 serum but did not bind to those parasites incubated with M-line serum. Preincubation of sporocysts with unlabeled serum prior to incubation with radiolabeled serum significantly inhibited the uptake of radiolabeled proteins. This differential binding of serum polypeptides from different strains of B. glabrata may be important in determining resistance or susceptibility of the snail to larval schistosome infection.     

Echinostoma paraensei: hemocytes of Biomphalaria glabrata as targets of echinostome mediated interference with host snail resistance to Schistosoma mansoni

Earlier in vivo work by Lie et al. (1977) indicated that the innate resistance of the 10R2 strain of Biomphalaria glabrata to PR1 Schistosoma mansoni could be interfered with if the snails were infected previously with another trematode, Echinostoma paraensei. We have studied this interference phenomenon using in vitro methods in an attempt to understand its mechanistic basis. Hemolymph, derived from 10R2 snails infected with E. paraensei for 14-28 days, killed 25% of S. mansoni sporocysts in vitro, significantly less (P less than 0.001) than the 90% killing rate observed with hemolymph from uninfected, control 10R2 snails. Hemolymph from the infected 10R2 snails and from schistosome susceptible M line snails did not differ significantly (P greater than 0.1) in their relative inability to kill S. mansoni sporocysts in vitro. The defect in sporocyst killing exhibited by echinostome infected 10R2 snails was traced to the cellular, rather than the humoral, component of the hemolymph. Preparations containing uninfected 10R2 snail hemolymph and echinostome daughter rediae exhibited significantly less (P less than 0.001) killing of S. mansoni sporocysts than did controls containing only 10R2 hemolymph and S. mansoni sporocysts. Our results suggest that echinostome larvae release factors that interfere with the ability of B. glabrata hemocytes to kill S. mansoni sporocysts.     

On the origin of the Biomphalaria glabrata hemocytes

A histologic, morphometric and ultrastructural study performed on Biomphalaria glabrata submitted to infection with Schistosoma mansoni miracidia failed to provide significant evidences that the so-called amebocyte-producing organ (APO) is really the central organ for hemocyte production. In infected snails no general reactive changes appeared in the APO, the mitoses were seen only occasionally, and the possibility of cellular hyperplasia was ruled out by morphometric measurements. Under the electron microscope the APO cells presented an essentially epithelial structure, without features indicative of transition toward hemocytes. On the other hand, the present findings pointed to a multicentric origin for the mollusc hemocytes, as earlier studies had indicated. Dense foci of hemocyte collections appeared sometimes around disintegrating sporocysts and cercariae in several organs and tissues of the infected snails, including a curious accumulation of such cells inside the ventricular cavity of the heart. In the heart and other sites, features suggestive of transformation of vascular space endothelial lining cells into hemocytes were apparent. To some extent, the postulated multicentric origin for B. glabrata hemocytes recapitulates earlier embryologic findings in vertebrates, when mesenchymal vascular spaces generate the circulating and phagocytic blood cells.     

Schistosoma mansoni: passive transfer of resistance by serum in the vector snail, Biomphalaria glabrata

Passive transfer of natural resistance to Schistosoma mansoni (PR-1 strain) has been successfully accomplished in the snail intermediate host, Biomphalaria glabrata (PR albino, M-line strain). Injection of serum (cell-free hemolymph) from a naturally schistosome-resistant strain of B. glabrata (10-R2) into PR albino snails induced a complete protection from a primary infection with the parasite in 29 of 48 snails (60.4%). In comparison, inoculation of homologous PR albino serum or heterologous proteins (fetal calf serum) had no effect. Moreover, this protection could be induced 24 hr prior to, or 24 hr after, exposure to the parasite, although heating of 10-R2 serum to 70 C for 30 min destroyed its protective ability. When in vitro transformed sporocysts were preincubated in 10-R2 or PR albino serum and then were injected into susceptible snails, a high level of infection (88.5 and 83.3%, respectively) was produced in both groups. Thus, the 10-R2 serum factor does not appear to be mediating specific parasite recognition by host hemocytes. Alternatively, our results suggest that 10-R2 serum possesses a heat-labile factor which specifically activate B. glabrata hemocytes to encapsulate and destroy sporocysts whereas PR albino serum lacks this factor.     

Lysosomal enzyme activities in susceptible and refractory strains of Biomphalaria glabrata during the course of infection with Schistosoma mansoni

The distribution and abundance of the lysosomal enzyme markers, acid phosphatase (AP), peroxidase (PO), and nonspecific esterase (NE), within circulating blood cells (hemocytes) were examined in a schistosome-susceptible (PR albino M-line) and a resistant (10-R2) strain of Biomphalaria glabrata during the course of infection with Schistosoma mansoni. The dynamics of serum (cell-free hemolymph) AP activities and total hemocyte numbers in infected snails also were investigated. Hemocyte subpopulations, as determined by these enzyme markers, responded differently to parasite infection between snail strains. Generally, the hemocyte subpopulations within PR albino snails remained largely unchanged, whereas the same subpopulations in 10-R2 snails fluctuated considerably. The distribution of AP in the hemocytes of 10-R2 snails decreased by 1 hr postexposure (PE) to the parasite and remained low through 12 hr before increasing to control values at 24 hr and 2 wk PE. In comparison, PO activity increased by 1 hr PE and peaked at 12 hr before dropping to 0 hr values by 2 wk PE. The NE activity exhibited still another pattern with the percentage of NE-positive cells decreasing from 0 to 12 hr PE followed by a recovery to 0-hr values by 24 hr. The abundance of these hemocyte enzymes followed a similar pattern to that of their distribution, although some differences were observed. Serum AP values varied little in PR albino snails except for a significant increase at 2 wk PE, indicating a possible response to tissue damage resulting from migrating daughter sporocysts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dynamics of the leukocytic response of Biomphalaria glabrata during the larval development of Schistosoma mansoni and Echinostoma liei

The daily evolution of the number of hemocytes in Biomphalaria glabrata was ascertained under three conditions: uninfected snails, snails infected with Schistosoma mansoni, and snails infected with Echinostoma liei. The Results show differences between the three experiments as well as in the average hemocyte density over the whole experimental period, as in the temporal dynamics of circulating hemocyte number. Specifically, it appears that the development of E. liei in B. glabrata induces a density of circulating hemocytes greater than that in uninfected B. glabrata or in snails infected with S. mansoni. The hemocyte dynamics observed in both experimental groups might best be interpreted by taking into account differences in the immunogenic stimulating capacity of the two trematodes and different physiological functions of the hemocytes brought into play during the infection: wound repair, nutrient digestion and transport, and excretion.     

Respiratory burst of Biomphalaria glabrata hemocytes: Schistosoma mansoni-resistant snails produce more extracellular H2O2 than susceptible snails

The production of reactive oxygen species by hemocytes from the gastropod Biomphalaria glabrata has been linked to their ability to kill the trematode parasite Schistosoma mansoni. For 2 laboratory strains of the snail, 1 resistant (13-16-R1) and 1 susceptible (MO) to the PR1 strain of S. mansoni, we compared hemocyte production of extracellular hydrogen peroxide when stimulated with the protein kinase C agonist phorbol myristate acetate (PMA). The time course of the PMA-induced response is similar in both strains with respect to onset, peak production, and termination of the respiratory burst. However, the magnitude of the response differs between strains, in that hemocytes from resistant snails generate significantly more hydrogen peroxide. These findings suggest that the capacity to produce hydrogen peroxide could be critical in determining susceptibility or resistance to S. mansoni.     

Larval Schistosoma mansoni excretory-secretory glycoproteins (ESPs) bind to hemocytes of Biomphalaria glabrata (Gastropoda) via surface carbohydrate binding receptors

Flow cytometric analysis of circulating blood cells (hemocytes) of Biomphalaria glabrata, molluscan intermediate host of Schistosoma mansoni, revealed the presence of 2 overlapping hemocyte subpopulations, designated R1 and R2. R1 hemocytes are characterized by their smaller size, reduced granularity, and the presence of the BGH1 surface epitope, whereas R2 cells are larger, more granulated, and generally lack the BGH1 cell marker. Both hemocyte subpopulations bound fluorescent dye (Oregon Green)-conjugated excretory-secretory glycoproteins (fESPs), although the specific fESP binding signal (geometric mean value), after correction for cellular autofluorescence, was greater in the R1 hemocyte subpopulation compared to that of the R2 subset. Partial inhibition of fESP binding to hemocytes consistently was achieved using various glycoconjugates (mucin, asialo-mucin, asialo-fetuin, heparin) and polysaccharides (fucoidan, dextran sulfate 8000), suggesting the involvement of hemocyte carbohydrate-binding receptors (CBRs) in reactions with ESP-associated glycans. Although sulfation of carbohydrate ligands contributed significantly to ESP blocking activity of some inhibitory polysaccharides and heparin, other sulfated proteoglycans (chondroitins A and B, heparan sulfate) were noninhibitory, indicating that charge alone was not solely responsible for the observed inhibition of hemocyte binding by fESPs. A similar blocking effect by desialylated glycoproteins (asialo-mucin, asialo-fetuin) further supports the contention that ESP-hemocyte interactions are mediated primarily through CBRs. The glycoconjugate inhibitors of ESP binding were only partially effective over a range of concentrations and their glycan moieties (oligosaccharides or long-chain polymers) comprised a diversity of major sugar groups, suggesting that hemocyte CBRs and S. mansoni larval ESPs likely represent a multiple receptor-ligand system. Previously reported findings of differential effects of ESPs on a variety of in vitro hemocyte functions are consistent with such a hypothesis.