Amebocyte-producing organs (APOs) of Biomphalaria glabrata were maintained in nonnutritive saline... more Amebocyte-producing organs (APOs) of Biomphalaria glabrata were maintained in nonnutritive saline with, or without, extracts of miracidia and adults of Schistosoma mansoni, and examined histologically. The hematopoietic cells remained viable and showed measurable mitotic activity for up to 6 days, with little evidence of tissue death. APOs accumulated fluid and became swollen by as soon as 24 hr, but no cell exomigration was observed. Parasite extracts elicited an increase in the number of dividing cells in the APO, suggesting that the extract may directly stimulate a response from the hematopoietic cells by providing either nutrients or mitogenic growth factors.
Lipopolysaccharide (LPS) is a pathogen associated molecular pattern (PAMP) to which the internal ... more Lipopolysaccharide (LPS) is a pathogen associated molecular pattern (PAMP) to which the internal defense system (IDS) of both vertebrates and invertebrates responds. We measured the mitotic response of the hematopoietic tissue of the schistosome-transmitting snail, Biomphalaria glabrata, to crude LPS from Escherichia coli 0127:B8. In a dose-response study, snails were injected with a range of concentrations of crude LPS, and mitotic figures were enumerated in histological sections of amebocyte-producing organ (APO) fixed at 24 h post-injection (PI) following a 6 h treatment with 0.1% colchicine. In APOs from Salvador strain snails, which are genetically resistant to infection with Schistosoma mansoni, LPS concentrations of 0.01 mg/ml and above triggered a large increase in mitotic activity, whereas in APOs from schistosome-susceptible NIH albino snails, concentrations of 0.1 mg/ml elicited a much smaller, but statistically significant increase. A time course study, without colchicine treatment, revealed that in Salvador APOs the mitotic response to 0.1 mg/ml occurred by 18 h PI, peaked at 24 h, and returned to control levels by 72 h; NIH albino APOs showed no detectible response. When Salvador APOs were exposed to crude LPS in vitro, no increase in mitotic activity occurred, a result suggesting the possible requirement for a peripheral tissue or hemolymph factor. The increased cell proliferation induced by crude LPS represents a novel systemic response of an invertebrate IDS to one or more PAMPs from a Gram-negative bacterium.
Genetically resistant strains of the snail Biomphalaria glabrata show hyperplasia of hematopoieti... more Genetically resistant strains of the snail Biomphalaria glabrata show hyperplasia of hematopoietic tissue in the amebocyteproducing organ (APO), which is the anterior wall of the pericardial sac, following infection with miracidia of Schistosoma mansoni or injection of parasite extract [1]. At the organismal level it is not known whether the parasite stimulates the hematopoietic tissue directly, or indirectly, e.g. by eliciting production of hemocyte cytokines that are mitogenic. We recently described an in vitro technique for measuring cell division in the APO of B. glabrata and reported that parasite extracts stimulated proliferation of hematopoietic cells in APOs maintained in non-nutritive saline [2]. These results suggest that parasite molecules may be able to directly stimulate the APO. At the cellular level, very little is known about signalling mechanisms involved during hematopoiesis, and in general, intracellular signalling mechanisms in molluscs are not well understood [3]. Exposure of B. glabrata embryonic (Bge) cells to phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C (PKC), induces phosphorylation of the mitogen-activated protein kinase (MAPK) ERK1/2, and inhibitor studies suggest that both this effect and cell spreading result from PKC-and Ras-mediated phosphorylation of the mitogen-activated protein kinase kinase (MAPKK), MEK1/2 [3]. Additionally, PKC signal transduction pathways occur in molluscan hemocytes. Specifically, when stimulated with PMA, hemocytes of B. glabrata generate H 2 O 2 , and this production is greater in schistosome-resistant snails than in schistosomesusceptible snails [4]. PMA also induces superoxide anion production in hemocytes of Littorina littorea [5]. Hemocytes of Lymnaea stagnalis produce NO in response to PMA or laminarin, and this NO production is blocked by inhibitors of PKC or ERK [6]. Likewise, the ERK signalling pathway is also active in B. glabrata and L. stagnalis hemocytes during spreading and H 2 O 2 production [7], and ERK signalling is disrupted in hemocytes from schistosome-susceptible, but not schistosome-resistant, strains of B. glabrata by sporocysts of S. mansoni and their excretory-secretory products [8]. Finally, PKC and ERK inhibitors impair phagocytosis in Mytilus gallaprovincialis hemocyte subpopulations [9]. Since PKC-mediated signalling pathways apparently have been well-conserved in molluscs, we investigated whether these are also involved in regulating hematopoiesis in the APO. As in several of the above studies, we employed PMA, which is an analogue of diacylglycerol (DAG) and an activator of the DAG-sensitive PKC pathway [10]. In order to test the hypothesis that PKCmediated MAPK activation is involved in cell division in the APO, we used U0126 (1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio] butadiene). This substance is a highly selective inhibitor Developmental and Comparative Immunology 33 (2009) 725-727
Amebocyte-producing organs (APOs) of Biomphalaria glabrata were maintained in nonnutritive saline... more Amebocyte-producing organs (APOs) of Biomphalaria glabrata were maintained in nonnutritive saline with, or without, extracts of miracidia and adults of Schistosoma mansoni, and examined histologically. The hematopoietic cells remained viable and showed measurable mitotic activity for up to 6 days, with little evidence of tissue death. APOs accumulated fluid and became swollen by as soon as 24 hr, but no cell exomigration was observed. Parasite extracts elicited an increase in the number of dividing cells in the APO, suggesting that the extract may directly stimulate a response from the hematopoietic cells by providing either nutrients or mitogenic growth factors.
Lipopolysaccharide (LPS) is a pathogen associated molecular pattern (PAMP) to which the internal ... more Lipopolysaccharide (LPS) is a pathogen associated molecular pattern (PAMP) to which the internal defense system (IDS) of both vertebrates and invertebrates responds. We measured the mitotic response of the hematopoietic tissue of the schistosome-transmitting snail, Biomphalaria glabrata, to crude LPS from Escherichia coli 0127:B8. In a dose-response study, snails were injected with a range of concentrations of crude LPS, and mitotic figures were enumerated in histological sections of amebocyte-producing organ (APO) fixed at 24 h post-injection (PI) following a 6 h treatment with 0.1% colchicine. In APOs from Salvador strain snails, which are genetically resistant to infection with Schistosoma mansoni, LPS concentrations of 0.01 mg/ml and above triggered a large increase in mitotic activity, whereas in APOs from schistosome-susceptible NIH albino snails, concentrations of 0.1 mg/ml elicited a much smaller, but statistically significant increase. A time course study, without colchicine treatment, revealed that in Salvador APOs the mitotic response to 0.1 mg/ml occurred by 18 h PI, peaked at 24 h, and returned to control levels by 72 h; NIH albino APOs showed no detectible response. When Salvador APOs were exposed to crude LPS in vitro, no increase in mitotic activity occurred, a result suggesting the possible requirement for a peripheral tissue or hemolymph factor. The increased cell proliferation induced by crude LPS represents a novel systemic response of an invertebrate IDS to one or more PAMPs from a Gram-negative bacterium.
Genetically resistant strains of the snail Biomphalaria glabrata show hyperplasia of hematopoieti... more Genetically resistant strains of the snail Biomphalaria glabrata show hyperplasia of hematopoietic tissue in the amebocyteproducing organ (APO), which is the anterior wall of the pericardial sac, following infection with miracidia of Schistosoma mansoni or injection of parasite extract [1]. At the organismal level it is not known whether the parasite stimulates the hematopoietic tissue directly, or indirectly, e.g. by eliciting production of hemocyte cytokines that are mitogenic. We recently described an in vitro technique for measuring cell division in the APO of B. glabrata and reported that parasite extracts stimulated proliferation of hematopoietic cells in APOs maintained in non-nutritive saline [2]. These results suggest that parasite molecules may be able to directly stimulate the APO. At the cellular level, very little is known about signalling mechanisms involved during hematopoiesis, and in general, intracellular signalling mechanisms in molluscs are not well understood [3]. Exposure of B. glabrata embryonic (Bge) cells to phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C (PKC), induces phosphorylation of the mitogen-activated protein kinase (MAPK) ERK1/2, and inhibitor studies suggest that both this effect and cell spreading result from PKC-and Ras-mediated phosphorylation of the mitogen-activated protein kinase kinase (MAPKK), MEK1/2 [3]. Additionally, PKC signal transduction pathways occur in molluscan hemocytes. Specifically, when stimulated with PMA, hemocytes of B. glabrata generate H 2 O 2 , and this production is greater in schistosome-resistant snails than in schistosomesusceptible snails [4]. PMA also induces superoxide anion production in hemocytes of Littorina littorea [5]. Hemocytes of Lymnaea stagnalis produce NO in response to PMA or laminarin, and this NO production is blocked by inhibitors of PKC or ERK [6]. Likewise, the ERK signalling pathway is also active in B. glabrata and L. stagnalis hemocytes during spreading and H 2 O 2 production [7], and ERK signalling is disrupted in hemocytes from schistosome-susceptible, but not schistosome-resistant, strains of B. glabrata by sporocysts of S. mansoni and their excretory-secretory products [8]. Finally, PKC and ERK inhibitors impair phagocytosis in Mytilus gallaprovincialis hemocyte subpopulations [9]. Since PKC-mediated signalling pathways apparently have been well-conserved in molluscs, we investigated whether these are also involved in regulating hematopoiesis in the APO. As in several of the above studies, we employed PMA, which is an analogue of diacylglycerol (DAG) and an activator of the DAG-sensitive PKC pathway [10]. In order to test the hypothesis that PKCmediated MAPK activation is involved in cell division in the APO, we used U0126 (1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio] butadiene). This substance is a highly selective inhibitor Developmental and Comparative Immunology 33 (2009) 725-727
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