Bge Cells Research Articles

Biomphalaria glabrata embryonic cells express a protein with a domain homologous to the lectin domain of mammalian selectins.

We have cloned from Biomphalaria glabrata, the intermediate host of the helminth parasite Schistosoma mansoni, a 36-kDa apparent-molecular-mass molecule (BgSel) that shares sequence identity with selectins of the cell-adhesion-molecule superfamily. BgSel exhibited in its C-terminal part a putative C-type lectin domain similar to the selectin lectin domain. Using antibodies to the recombinant BgSel protein, we demonstrated the presence of BgSel in snail hemocytes as well as in the cell line derived from B. glabrata embryos (Bge). Anti-BgSel antibodies specifically recognized a 79-kDa component in Bge-cell-secreted products that was supposed to represent the native form of BgSel, as was confirmed after glycosidase treatment. Lectins are known to be implicated in recognition mechanisms participating in humoral and cellular immunity in molluscs. The possible role of BgSel in the interaction between sporocysts and Bge cells, particularly in the in vitro model of sporocyst development dependent on Bge cell factors, remains to be determined.

Cloning of a beta integrin subunit cDNA from an embryonic cell line derived from the freshwater mollusc, Biomphalaria glabrata.

A cDNA encoding an integrin subunit was cloned and structurally characterized from an embryonic cell line derived from Biomphalaria glabrata, snail intermediate host of the human blood fluke Schistosoma mansoni. Cells of the B. glabrata embryonic (Bge) snail cell line were initially tested for their sensitivity to the integrin-specific tetrapeptide inhibitor Arg-Gly-Asp-Ser (RGDS). Washed Bge cells when exposed to 0.5 to 2.0mM of RGDS were significantly inhibited in their ability to spread on a glass substrate. Spreading inhibition was specific, since a control peptide Arg-Gly-Glu-Ser (RGES) did not have the same effect. RT-PCR was performed using previously reported degenerate oligonucleotide primers to the ligand binding domain (LBD) of known beta integrin subunits and Bge cDNA. A 137 bp fragment was amplified, TA-cloned, sequenced, and the na and deduced aa sequences were compared with other beta integrins. Databank analysis showed that the 137 bp product shared >/=55.6% aa similarity to other beta integrin LBDs. Southern and northern blot analyses using the 137 bp sequence as a probe revealed binding to Bge genomic DNA restriction fragments and to an approximately 8 kb poly-(A)+RNA transcript, respectively. An exact 5' primer synthesized to the 137 bp product and an oligo-d(T) primer then were used to amplify from Bge cDNA, a partial beta integrin sequence of 2285 bp that contained a 1971 bp ORF. The remaining upstream coding region was obtained using 5' RACE methods. The complete ORF, consisting of 2364 bp, encoded a 788 aa sequence with shared similarity to other known beta integrins (44.6-61.5%). Sequence and structural comparisons, which include a characteristic LBD, a series of three homologous cysteine-rich repeats, membrane proximal sequence (LLTFIHD), cytoplasmic NPXY motifs, and predicted domain lengths of the molluscan protein, clearly identifies it as an integrin homologue. This report represents the first cloning of a cDNA putatively encoding an integrin subunit from molluscan cells, and establishes the Bge cell line as a model for studying cellular adhesion in molluscs at the molecular level.

Biomphalaria glabrata embryonic (Bge) cell line supports in vitro miracidial transformation and early larval development of the deer liver fluke, Fascioloides magna

A Bge cell co-culture system, previously shown to support the in vitro production of daughter sporocysts from mother sporocysts of Schistosoma mansoni and S. japonicum, has proven capable of supporting the in vitro development of intramolluscan stages of the deer liver fluke, Fascioloides magna. Miracidia commenced transforming within 4 h of incubation with Bge cells, and had completely shed their epidermal plates within 18-24 h. Redial stages were visible inside in vitro-transformed mother sporocysts after 12-16 days of co-culture with Bge cells, and emerged as fully-developed larvae starting at 14-20 days post-cultivation. Rediae survived over 60 days of in vitro culture, and reached a maximum size of 150-170 microns. Although particulate material was visible in their cecae, rediae were not observed to actively feed on Bge cells. Bge cells did not attach to or encapsulate larval stages at any time throughout the incubation period. Unlike Schistosoma spp., in which a high percentage of miracidia spontaneously shed their ciliated epidermal plates and transformed into mother sporocysts in Chernin's balanced saline solution (CBSS), transformation of F. magna was dependent on Bge cell products. Less than 5% of F. magna miracidia transformed spontaneously in either CBSS or Bge medium with 10% fetal bovine serum (complete or C-Bge). However, incubation of miracidia in either Bge cell-conditioned C-Bge medium or a greater than 30 kDa fraction concentrated from conditioned CBSS increased transformation rates to 67 and 83%, respectively. This secretory Bge cell factor(s) appeared to be protein in nature since its activity was completely abrogated by heat or proteinase K treatments. Overall, these results demonstrate that Bge cells are required for stimulating in vitro miracidial transformation and supporting early larval development of a fasciolid trematode under culture conditions. This is the first report of in vitro development of rediae from miracidia for a digenetic trematode.

Transfection and heat-inducible expression of molluscan promoter-luciferase reporter gene constructs in the Biomphalaria glabrata embryonic snail cell line.

Studies were initiated to begin developing a genetic transformation system for cells derived from the freshwater gastropod, Biomphalaria glabrata, an intermediate host of the human blood fluke Schistosoma mansoni. Using a 70-kD heat-shock protein (HSP70) cDNA probe obtained from the B. glabrata embryonic (Bge) cell line, we cloned from Bge cells a complete HSP70 gene including a 1-kb genomic DNA fragment in its 5'-flanking region containing sequences indicative of a HSP promoter. Identified in the 5'-half (416 nucleotides) of this genomic fragment were TATA and CAAT boxes, two putative transcription initiation sites, and a series of palindromic DNA repeats with shared homology to the heat-shock element consensus sequence (Bge HSP70(0.5k) promoter). The 3'-half of this upstream flanking region was comprised of a 508-base intron located immediately 5' of the ATG start codon. To determine the functionality of the putative snail promoter sequence, Bge HSP promoter/luciferase (Luc) reporter gene constructs were introduced into Bge cells by N-(1-(2,3-dioleoyloxy) propyl)-N,N,N-trimethylammonium methylsulfate (DOTAP)-mediated transfection methods, and assayed for Luc activity 48 hr following a 1.5-hr heat-shock treatment (40 degrees C). Compared with control vectors or the Bge HSP70(0.5k/1.0k) promoter constructs at 26 degrees C, a 10- to 300-fold increase in Luc expression was obtained only in the Bge HSP70 promoter/Luc-transfected cells following heat-shock. Results of transfection experiments demonstrate that the Bge HSP70(0.5k) DNA segment contains appropriate promoter sequences for driving temperature-inducible gene expression in the Bge snail cell line. This report represents the first isolation and functional characterization of an inducible promoter from a freshwater gastropod mollusc. Successful transient expression of a foreign reporter gene in Bge cells using a homologous, inducible promoter sequence now paves the way for development of methods for stable integration and expression of snail genes of interest into the Bge cell line.

Comparison of Echinostoma caproni Mother Sporocyst Development In vivo and In vitro Using Biomphalaria glabrata Snails and a B. glabrata Embryonic Cell Line

Biomphalaria glabrata embryonic (Bge) cells have previously been shown to permit a successful cocultivation of Schistosoma mansoni and Schistosoma japonicum from miracidia to mother sporocysts (MS) and then to the production of daughter sporocysts (DS). To investigate further the properties of the Bge culturing system we used Echinostoma caproni under identical in vitro conditions. In vitro-derived miracidia were used either for experimental infections of B. glabrata snails, or for in vitro cultivation with Bge cells. Histological analysis showed that the development of MS in B. glabrata was similar to the previously described development in Biomphalaria pfeifferi in terms of final site of infection, development dynamics, growth dynamics, reproduction intensity, and life spans. Only short delays in migration dynamics were observed in B. glabrata. When cultivated under in vitro conditions, E. caproni MS could live for up to 17 wk in the presence of Bge cells, as compared with 2 wk in cell-free Bge medium. The presence of Bge cells also permitted significant growth of MS and development through complete embryogenesis of the next intramolluscan stage (embryos of 100-110 cells). However, degeneration of MS consistently occurred before production of this second generation. During the entire cultivation period, no visible contact was observed between MS and Bge cells, suggesting that development of MS was only triggered by soluble factors released by Bge cells.

Characterization of an Intracellular Receptor for Activated Protein Kinase C (RACK) from the MolluscBiomphalaria glabrata, the Intermediate Host forSchistosoma mansoni

Lardans, V., Serra, E., Capron, A., and Dissous, C. 1998. Characterization of an intracellular receptor for activated protein kinase C (RACK) from the molluscBiomphalaria glabrata, the intermediate host forSchistosoma mansoni. Experimental Parasitology88, 194–199. A receptor for activated protein kinase C (RACK) was characterized from the molluscBiomphalaria glabrata, the intermediate host for the human parasiteSchistosoma mansoni. This protein was shown to possess structural and functional characteristics of other RACK proteins from various cells and organisms. Its ability to bind mammalian PKCs also confirmed the conservation of PKC and RACK interactive domains throughout evolution. Results of immunolocalization indicated the presence of Bg RACK in the cytoplasm of mollusc hemocytes andB. glabrataembryonic (Bge) cells with a more intense staining around the nucleus. These results are in agreement with the association of RACK proteins with cytoskeletal elements.

Heat-Shock Response in a Molluscan Cell Line: Characterization of the Response and Cloning of an Inducible HSP70 cDNA

Sublethal heat-shock of cells of the Bge (Biomphalaria glabrataembryonic) snail cell line resulted in increased or new expression of metabolically labeled polypeptides of approximately 21.5, 41, 70, and 74 kDa molecular mass. Regulation of this response appeared to be at the transcriptional level since a similar protein banding pattern was seen upon SDS–PAGE/fluorographic analysis of polypeptides produced byin vitrotranslation of total RNA from cells subjected to heat shock. Using a yeast (Saccharomyces cerevisiae) 70-kDa heat-shock protein (HSP70) probe to screen a cDNA library from heat-treated Bge cells, we isolated a full-length cDNA clone encoding a putative Bge HSP70. The cDNA was 2453 bp in length and contained an open reading frame of 1908 bp encoding a 636-amino-acid polypeptide with calculated molecular mass of 70,740 Da. Comparison of a conserved region of 209 amino acid residues revealed >80% identity between the deduced amino acid sequence of Bge HSP70 and that of yeast (81%), the human blood flukeSchistosoma mansoni(for whichB. glabrataserves as intermediate host) (81%),Drosophila(81%), human (84%), and the marine gastropodAplysia californica(88%, 90%). In addition to the extensive sharing of sequence homology, the identification of several eukaryotic HSP70 signature sequences and an N-linked glycosylation site characteristic of cytoplasmic HSPs strongly support the identity of the Bge cDNA as encoding an authentic HSP70. Results of a Northern blot analysis, using Bge HSP70 clone-specific probes, indicated that gene expression was heat inducible and not constitutively expressed. This is the first reported sequence of an inducible HSP70 from cells originating from a freshwater gastropod and provides a first step in the development of a genetic transformation system for molluscs of medical importance.

Schistosoma japonicum: In VitroCultivation of Miracidium to Daughter Sporocyst Using aBiomphalaria glabrataEmbryonic Cell Line

In vitrocultivation ofSchistosoma japonicummiracidia to the mother sporocyst (MS) and then to the daughter sporocyst (DS) stage was achieved using theBiomphalaria glabrataembryonic (Bge) cell line as a coculture system. When comparing the effect of Bge cell and MS density on MS development, it was apparent that Bge cell density had a highly significant effect on both MS viability and growth. Viability and growth rate of MS cultured under high cell density conditions (350 cells/mm2) were almost 2 times greater than those of MS cultured under conditions of low cell density (60 cells/mm2). Growth under high cell density conditions corresponded to a 20 to 30 times increase in MS estimated volume within the first 9 weeks of cultivation. Emergence of fully formed motile DS was first observed after 11 weeks of cocultivation. A few DS lived for 14 weeks after emergence and attained a size of 770 ± 100 μm in length and 48 ± 13 μm in width. In contrast to what was observed in Bge cell/Schistosoma mansonicocultures, Bge cells did not encapsulateS. japonicumMS. Our results show that, although the cellular interactions between Bge cells and schistosomes MS display some level of specificity, Bge cells apparently secrete soluble factors that permit excellent survival and can trigger advancedin vitrodevelopment ofS. japonicum.

Production of Schistosoma mansoni Daughter Sporocysts from Mother Sporocysts Maintained in Synxenic Culture with Biomphalaria glabrata Embryonic (BGE) Cells

In vitro production of Schistosoma mansoni daughter sporocysts (DS) from miracidium-derived mother sporocysts (MS) was achieved by synxenic larval cultivation with cells of the Biomphalaria glabrata embryonic (Bge) cell line. The in vitro growth and viability of MS cocultured with Bge cells or in Bge cell-conditioned medium were significantly extended beyond that of larvae cultured in fresh medium alone. However, complete DS development and emergence from MS were achieved only in the presence of Bge cells. Introduction of either miracidia or previously transformed MS onto Bge cell monolayers resulted in an initial attachment of cultured cells to sporocysts, followed by a gradual encapsulation of larvae by multiple layers of Bge cells. Sporocysts and their encapsulating cells eventually formed large cellular aggregates, within which MS increased 4-fold in size during the first 20 days of cultivation. The timing of in vitro DS development was somewhat variable; however, in general, early embryo formation, i.e., germ cell aggregates with surrounding primitive epithelium, was first detected at 15-20 days of culture, whereas motile, intra-MS daughter stages were seen at 25-30 days and thereafter. Mature, first generation DS, measuring 136 +/- 46 microns long by 22 +/- 6 microns wide, emerged from MS starting at approximately 30-45 days of initial cultivation. Although the basic morphology and size of emergent, in vitro-derived DS were comparable to those propagated in vivo, there was a large reduction in the in vitro reproductive capacity of the MS and a delay in DS culture development.(ABSTRACT TRUNCATED AT 250 WORDS)

Bge Snail Cell-Line Antigens: Ineffectiveness as Antischistosomal Vaccine in Mice

Mice and rabbits were immunized with antigens derived from Bge cells, Biomphalaria glabrata hemolymph, or Schistosoma mansoni. Antisera from mice given molluscan antigens did not form immunoprecipitates with soluble antigen from adult worms, but their binding to surfaces of sporocysts, cercariae, and schistosomules suggests the presence of cross-reacting determinants. In vitro, cell-mediated immune responses to Bge antigens were not demonstrable in infected nor in immunized mice. Mice immunized with Bge cell-line antigens and challenged with S. mansoni cercariae showed no reduction in worm burden when compared with control mice.

Metacercarial Cyst Formation In vitro of Echinostoma paraensei

Cercariae of Echinostoma paraensei Lie and Basch 1968 encysted normally in the presence of Biomphalaria glabrata embryo (Bge) cells in culture, partially in culture conditioned medium, and not at all in fresh culture medium alone. At the ultrastructural level the cyst is composed of 2 well defined regions. The outer cyst wall (OCW) is particulate to fibrous in nature, formed from secretory granules released from the cercarial tegument. Membranous scrolls or rodlets secreted from the subtegumental cystogenous gland cells are then added to this layer, forming the inner cyst wall (ICW). After 24 hr the cultured cyst is enclosed by a thin cellular capsule similar to that found around cysts in the snail host. The capsule also contains collagen fibers, not found elsewhere in Bge cell cultures.

Low-Temperature Preservation of the Biomphalaria glabrata Cell Line

Biomphalaria glabrata embryo (Bge) cells are tolerant of a wide range of conditions for preserva- tion at low temperature. It is recommended that the cells, suspended in complete medium with 10% DMSO, be frozen at a rate of 0.5-2°C/min. If a controlled-rate freezer is not available, successful freezing is achieved by placing the lightly insulated sealed ampoule (Fig. 1) into the freezing compartment of a refrigerator (about - 16°C) for 1 hr, then transferring it directly to an ultradeep freeze at about -86°C for another hour before removing it from the insulation and placing it into liquid nitrogen at -198°C for semipermanent storage. There is minimal lag in growth after the cells are thawed. It is apparent that, if 5 × l0 5 or more cells are used for freezing, a certain and quick recovery of the culture after thawing is assured. Using this technique, we have accumulated a bank of 38 frozen ampoules in which cells are preserved with the genetic constitution inherent to this cell line at the fifty-sixth to sixty-third passage.