Binding properties of Paracentrotus lividus (Echinoidea) hemolysin.

1. Paracentrotus lividus hemolysin binds erythrocytes, zymosan particles, lipopolysaccharide and laminarin surfaces but not auto and allogeneic cell membranes. 2. The binding could, at least for erythrocytes, involve phospholipids and cholesterol. 3. The protease activity of the coelomic fluid is not related to hemolysis. 4. The finding that very low concentrations of Zn2+ inactivate the hemolysin suggests a possible regulative function of the ion in the hemolytic reaction. 5. Ultrastructural observations on rabbit erythrocyte membranes indicate that most likely the transmembrane pores are induced by the lytic molecules.     

Cytotoxic activity in the coelomic fluid of the annelidEisenia foetida Sav

The annelid Eisenia foetida not only causes hemolysis of red blood cells of several vertebrate species, but also has a toxic effect on a variety of cell types, such as chicken fibroblasts, guinea-pig polymorphonuclear leukocytes and insect hemocytes. However, it has no influence on the vitality of the coelomocytes of Lumbricus terrestris and other lumbricides, nor on the hemocytes of the snail Helix pomatia, the mussels Anodonta cygnea and Unio tumidus, free cells of the turbellarian Euplanaria sp. or whole Rhabditis oxycerca (nematode) and the protozoons Paramaecium caudatum and an amoeba of the Proteus-type. By electrofocussing the hemolytic activity of pooled coelomic fluid was separated into 7 hemolytic bands. Three of them are cytotoxic. The cytotoxic effect is a result of the destruction of the cell membrane, as shown by measuring the release of lactate dehydrogenase (LDH). The bacteriostatic and bacteriocidal action of the coelomic fluid of E. foetida against a broad spectrum of gram positive and gram negative bacteria was tested. An antibacterial activity could be observed only against Proteus vulgaris and Bacillus megaterium. It was noted that the so-called Eisenia foetida-factor acts on an antigenic structure at the cell surface when anti-sheep-E-antibody was used under competitive conditions. The binding between the Eisenia foetida-factor and this membrane structure is relatively strong as it cannot be removed by subsequent treatment with anti-sheep-E-antibody or 2 M KCl.     

Antibacterial activity of the hemolytic system from the earthworm Eisenia fetida andrei

The coelomic fluid and the cocoon albumen of the earthworm Eisenia fetida andrei are demonstrated to possess an antibacterial activity. The antibacterial activity and the already known hemolytic activity are due, in fact, to the same lipoproteic molecules. The antibacterial activity (bacteriostatic effect) is only directed against the highly pathogenic soil bacteria. Only these pathogenic bacteria strains possess at least one common antigen with the sheep red blood cells.     

Coelomic fluid: a noninvasive source of DNA in earthworms

To investigate whether coelomic fluid secreted by earthworms can be a noninvasive source of DNA, we amplified and sequenced DNA extracted from the coelomic fluid and muscle tissue of eight worms. The sequences obtained using DNA extracted from both sources were identical. All cytochrome c oxidase subunit I (COI) mitochondrial DNA sequences, including those retrieved from GenBank, formed a monophyletic group of Metaphire sieboldi. The results indicate that we successfully extracted total DNA from coelomic fluid secreted by earthworm.     

The flow cytometric analysis of in vitro phagocytic activity of earthworm coelomocytes (Eisenia foetida; Annelida)

We have detected in vitro phagocytic activity of earthworm coelomocytes (Eisenia foetida, Annelida) by flow cytometry using FITC-labelled synthetic 2-hydroxy-ethylmethacrylate particles (FITC-HEMA). We have observed that the phagocytic activity is increased after in vivo stimulation by a protein antigen (arsanylated human serum albumin) and after pre-incubation of particles in cell-free coelomic fluid.     

Cellular clot formation in a sipunculan worm: entrapment of foreign particles, cell death and identification of a PGRP-related protein

Clotting in animals having open or closed circulatory system comprises humoral and cellular mechanisms. Sipunculans are a small phylum of non-segmented marine worms that do not have a true circulatory system. These worms can form a cellular clot without transforming cell-free coelomic fluid into an insoluble mass. The clot may also contribute to immune response by entrapping foreign particles. We evaluated the formation of a cellular clot ex vivo in the sipunculan Themiste petricola after activation through glass contact and sea water, the ability to entrap magnetic beads and non-pathogen cyanobacteria particles within the clot, and the presence of a peptidoglycan recognition protein S (PGRP-S) antigen in cells forming the clot. Our results showed that the clot was formed by homotypic aggregation of large granular leukocytes (LGLs), a subtype of cells found in the coelomic fluid. Aggregated LGLs served to entrap magnetic beads and non-pathogen cyanobacteria particles, and PGRP-S antigen was detected both in non-activated LGLs and in activated homotypic aggregates through immunofluorescence, Western blot and flow cytometry. Cellular clots were found to be positive to Annexin V-FITC labelling. Complete disintegration of cytoplasm with shedding of microparticles, nuclear isolation and degradation were also observed by light microscopy and flow cytometry. In conclusion, cellular clot formation in Themiste petricola may serve both haemostatic and immune functions entailing rapid activation changes in LGLs, entrapment of foreign particles within a homotypic aggregate, and further cell death.     

Identité immunologique et métabolique entre le liquide coelomique,les coelomocytes et les ovocytes de Perinereis cultrifera (Annélide Polychète)

Immunological study of Perinereis cultrifera reveals the existence of an identical antigen in the coelomic fluid of females (oocyte diameter > 140 (μm), in oocytes, and in coelomocytes. This factor is not found in the body fluid of males or young females.

The elution patterns obtained after Sephadex chromatography shows a similar glycoprotein fraction (fraction I) in the coelomic fluid, in coelomocytes, and in soluble oocyte extracts. This fraction includes the main part of the antigenic components of the coelomic fluid.

Gas chromatography reveals that identical monosaccharides are present, albeit in varying proportions, in samples of fraction I obtained from the different sources.

The metabolic interrelationships of coelomocytes, coelomic fluid and oocytes is discussed. Glycoprotein synthesized by coelomocytes may be discharged into the coelomic fluid and contribute to the development of the cortical alveoli of the oocytes. No evidence of an involvement of this material in yolk synthesis has been obtained.     

Haemostatic and immune role of cellular clotting in the sipunculan Themiste petricola

Sipunculans, a small phylum of coelomated marine worms closely related to polychaete annelids, lack a true circulatory system. We have previously shown that the sipunculan Themiste petricola can form a cellular clot, without congealing, of cell-free coelomic fluid. The clot is formed by the aggregation of large granular leukocytes (LGLs) and may serve not only haemostatic but immune functions, since dissimilar particles may become entrapped within it. We have now evaluated the capacity of a massive clot, induced in vitro by sea water contact, to stop coelomic fluid flow. We have further studied smaller clots induced on glass-slides either with or without the presence of bacteria placed for entrapment within the clot. The fate of clotting LGLs is cell death while forming a cohesive mass, although cytoplasmic and nuclear remnants are shed from the clot. These remnants and any bacteria that avoid clot entrapment or are detached from the clot are engulfed by non-clotting cells that include small granular leukocytes (SGLs) and large hyaline amebocytes (LHAs). Both cell types can be found other than in the clot but SGLs also occur around the clot edges heavily loaded with engulfed material. The cytoskeletal arrangement of SGLs evaluated with phalloidin-rhodamine correspond to motile cells and contrast with that of clotting LGLs that form a massive network of F-actin. Thus, the complementary roles between clotting LGLs and non-clotting SGLs and LHAs act a central immune strategy of Themiste petricola to deal with body wall injury and pathogen intrusion into the coelomic cavity.     

CHANGES IN CALCIUM CONCENTRATIONS OF SERUM AND COELOMIC FLUID OF THE BULLFROG, RANA CATESBEIANA, DURING LARVAL DEVELOPMENT AND METAMORPHOSIS

The serum calcium levels of bullfrog tadpoles (stage 26 to 33) and adults are higher than those of the coelomic fluid. The serum levels increase gradually from stage 26 (7.6 mg/100 ml) to stage 30 (8.4 mg/100 ml), and then sharply to stage 33 (10.5 mg/100 ml), while the coelomic fluid levels increase from 7.1 to 8.7 mg/100 ml during this period. Only minor differences are found in serum and coelomic fluid sodium levels among larval stages with the exception of a temporary decrease during metamorphic climax.
These results suggest that the adult type of regulation of serum calcium concentrations is established during larval development and is fully achieved after the completion of metamorphosis. The control mechanism for serum calcium may be different from that for coelomic fluid.     

Recognition of sphingomyelin by lysenin and lysenin-related proteins

Lysenin is a sphingomyelin (SM)-specific toxin isolated from the coelomic fluid of the earthworm Eisenia foetida. Lysenin comprises a family of proteins together with lysenin-related protein 1 (LRP-1, lysenin 2) and LRP-2 (lysenin 3). In the present study, we characterized LRP-1 and LRP-2 together with lysenin using maltose-binding-protein-tagged recombinant proteins. LRP-2 specifically bound SM and induced hemolysis like lysenin. In contrast the binding and hemolytic activities of LRP-1 were 10 times less than those of lysenin and LRP-2. Lysenin and LRP-2 share 30 common sites of aromatic amino acids. Among them, only one position, phenylalanine 210, is substituted for isoleucine in LRP-1. The activity of LRP-1 was dramatically increased by introducing a single amino acid substitution of isoleucine 210 to phenylalanine, suggesting the importance of this aromatic amino acid in biological activities of lysenin and LRPs. The importance of aromatic amino acids was further indicated by a systematic tryptophan to alanine mutation of lysenin. Lysenin contains six tryptophan residues of which five are conserved in LRP-1 and -2. We showed that the conserved tryptophans but not the nonconserved one were required both in the recognition of SM and in the hemolytic activity of lysenin. Our results suggest the importance of tryptophan in the toxin function likely due to a direct recognition of SM or in maintaining the protein structure.     

Comparative immunological analysis of echinoderm cellular and humoral defense factors

Coelomocyte are found in the fluid filling coelomic cavity of echinoderms and depending on species can be a mixture of several morphologically different types. There are among them: granular and agranular amoebocytes, morula cells, vibratile and lymphocyte-like cells. All these cells take part in cellular response to immune challenges through phagocytosis, clotting, encapsulation of foreign particles, cytotoxicity, and the production of antimicrobial agents, such as reactive oxygen and nitric oxide. The data are given on a variety of humoral factors found in the coelomic fluid, including different types of lectines, agglutinins, hemolysins, acute phase proteins and antimicrobial factors. The discussion on cooperation between cellular and humoral arms of defense reactions during inflammation reveals the crucial role of coelomocytes in immune response. It is suggested that the sea urchin complement system (that is homologous to the alternative pathway in vertebrates) is appeared initially in echinoderms as a protein cascade that points to opsonization of foreign cells and particles, augmenting their phagocytosis and subsequent destruction by coelomocytes. So the identification of a simple complement system as a part of the echinoderm immune response shows that these animals as well as all invertebrate deuterostomes share innate immune system homologies with vertebrates. Studying the simpler immune response demonstrated by echinoderms is important for understanding the ancestral deuterostome defense system and reconstructing the evolution of immune system in higher vertebrates.     

Hemagglutinin activity in Nereis coelomic fluid

1. Nereis coelomic fluid agglutinates rat, mouse, chicken, guinea pig and rhesus monkey erythrocytes (RBC). 2. Lipid fractions of the particulate matter from coelomic fluid are hemagglutinins exhibiting different activity inhibition profiles with complex polysaccharides. 3. The high mol. wt hemagglutinin from coelomic fluid supernatant is not a protein and is inhibited by bovine submaxillary mucin (BSM), thyroglobulin, transferrin and their asialo derivatives. 4. Coelomic fluid supernatant has a population of low mol. wt protein hemagglutinins inhibited by BSM, fetuin, antiserum to coelomic fluid and some mannan preparations. 5. Hemagglutination by lipids characterized by RBC specificity and specificity for inhibition by carbohydrate is noteworthy and may be significant in studies of cellular interactions and immunity in invertebrates.     

Interactions between earthworm hemolysins and sheep red blood cell membranes

The hemolytic activity exhibited by the coelomic fluid of the Annelid Eisenia fetida andrei is mediated by two lipoproteins of mass 40 and 45 kDa, each of them capable of hemolysis. Such an activity is not inhibited by zymosan, inulin or lipopolysaccharide (LPS), nor by hydrazine or methylamine, suggesting that earthworm hemolysins are not related to C3 or C3b complement components. Among the membrane lipids tested (phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, sphingomyelin and cholesterol) only sphingomyelin inhibited hemolysis. The analysis of E.f. andrei proteins bound to sphingomyelin microvesicles, as well as to sheep red blood cell (SRBC) membranes, revealed a polymerization of E.f. andrei 40 kDa and/or 45 kDa hemolysins. Consequently, sphingomyelin appears a likely candidate for hemolytic complex receptor. Electron microscopy observations suggested that the polymerization causes an open channel through the lipid bilayer. As demonstrated using metal ions, heparin, chondroitin sulfate, poly(L-lysine) and protamine chloride, the mode of action of earthworm hemolytic complex is not analogous to that of C9 or perforine.     

Echinoid phagocytes in vitro

A method is described for obtaining pure monolayers of phagocytes from the sea urchin Strongylocentrotus droebachiensis in vitro. The coelomic fluid contains four types of cells. About 67% of the cells are phagocytes, the rest is comprised of the red and white morula cells and the vibratile cells. The different cell types could be separated by centrifugation on a discontinuous gradient of sodium metrizoate. Release of granula from the vibratile cells was found to be responsible for rapid and extensive clotting of the coelomic fluid immediately after its removal from the animal. Clotting was prevented by adding a mixture of 50 mM mercaptoethanol, 3 mM caffeine and 2 mM TAME (p-tosyl- -arginine methyl ester) to the coelomic fluid. The phagocytes were isolated from other cell types by their attachment to glass, and were grown at 10 °C in a simple peptone-sea water medium. The phagocytes are very motile cells and spread rapidly on glass, accompanied by a complete change of their morphology to flattened cells with peripheral ruffling. After few hours in vitro the cells fuse to form monolayer-syncytia, and later still cell clusters and free floating balls of cells are formed. During a culture period of 10 days there was no change in the DNA content per culture, while a small increase in protein was found.     

Erythrocyte membrane structural features that are critical for the lytic reaction of Spirographis spallanzani coelomic fluid hemolysin

1. Hemolytic activity of Spirographis spallanzani coelomic fluid depends on factor(s) strongly influenced by calcium but not by sulfhydril or disulfide reagents.2. The lytic reaction was suppressed by low zinc ion concentrations but it was not influenced by the presence of proteinase inhibitors.3. These data indicate that S. spallanzani hemolysin is a non-enzymatic, calcium-dependent, zincinhibitable factor that occurs naturally in the coelomic fluid.4. In the absence of calcium, enzymatic desialization converted sheep erythrocytes into susceptible targets, suggesting the involvement of erythrocyte surface sialic acid.5. However, the inhibitory effect of the sugar on anti-rabbit lysis was partially removed by addition of calcium.6. Attempts to characterize membrane components that are critical for hemolysis were performed by inhibition experiments.7. We found that saccharides, glycoproteins, mucosubstances as well as rabbit erythrocyte soluble tryptic fragments were ineffective in inhibiting hemolysis.8. Sonicated dispersion of phosphatidyl choline, phosphatidyl glycerol, phosphatidyl ethanol, sphingomyelin and cholesterol did not influence the hemolytic reaction.9. Rabbit erythrocyte extracted from membrane lipids (chloroform phase) did not modify the lytic activity against rabbit red blood cells.10. Conversely, the methanol phase consistently reduced the lytic capacity of the fluid.11. The heat-stable, trypsin-resistant inhibitory factor was most probably a small molecule, since dialysis removed the inhibitory effect.     

Detection of two immunochemically identical forms of mannan-binding lectin in the sea urchin <Emphasis Type="Italic">Strongylocentrotus nudus</Emphasis>

This study revealed a new lectin (MBL-SN) in the coelomic fluid of the sea urchin Strongylocentrotus nudus. Based on the peculiarities of molecular structure and carbohydrate specificity, MBL-SN can be assigned to the mannan-binding lectin family. Using polyclonal monospecific rabbit antibodies against MBL-SN, the presence of MBL-SN in the sea urchin was detected in two forms: a soluble form dissolved in the coelomic fluid and an extracellular matrix-bound form. The biosynthesis site of this lectin may be one of the subpopulations of morula cells-coelomic fluid cells that perform heterosynthesis. Our results demonstrate the similarity of the sea urchin lectin MBL-SN to the previously investigated MBLs of the holothurians Cucumaria japonica and Apostichopus japonicus, and suggest a similarity to MBLs of vertebrates, which also have soluble and bound forms.     

New intrinsically radiopaque hydrophilic microspheres for embolization: synthesis and characterization

Polymeric particles currently used for embolization procedures have the disadvantage that they are radiolucent, that is, invisible on X-ray images, and consequently the interventional radiologist has to resort to angiography to (indirectly) monitor the fate of the particles. Here, we introduce intrinsically radiopaque hydrophilic microspheres. Since these microspheres can directly be visualized on X-ray images, using these microspheres for embolization purposes will allow superprecise location of the embolic material, both during and after the procedure. The microspheres, which are prepared by suspension polymerization, are based on the radiopaque monomer 2-[4-iodobenzoyl]-oxo-ethylmethacrylate and hydroxyethylmethacrylate (HEMA) and/or 1-vinyl-2-pyrrolidinone (NVP) as hydrophilic component. It has been shown that for clinically relevant X-ray visibility the spheres should contain at least 20 wt % iodine. At this iodine content, copolymerization with HEMA results in spheres that hardly imbibe water (EQ = 1.08). When HEMA is replaced by NVP, the volume swelling ratio can be significantly increased (to 1.33).     

Binding of streptavidin with biotinylated thermosensitive nanospheres based on poly(N,N-diethylacrylamide-co-2-hydroxyethyl methacrylate)

Thermosensitive polymer nanospheres based on N,N-diethylacrylamide and 2-hydroxyethyl methacrylate (HEMA) have been prepared, characterized, and conjugated with biotin. The thermosensitivity of poly(N,N-diethylacrylamide) was enhanced by the incorporation of HEMA up to about 40 mol %. Atomic force microscopic images show that these particles can be closely packed even without the surface charges as in the latex particles. Biotinylation reduces the thermosensitivity of the copolymer nanospheres. The biotinylated hydrogel nanospheres showed a reduction in size upon binding with streptavidin, indicating the formation of a less hydrophilic conjugate. No aggregation of the biotinylated particles due to the cross-linking effect of streptavidin was observed. This size change could be reversed by the addition of free biotin to the system. The interaction is specific, and no such changes were observed when streptavidin was replaced by bovine serum albumin.     

Bacterial flora of the sea urchin Echinus esculentus.

A total of 85 isolates of mesophilic, aerobic, heterotrophic bacteria were isolated from the gut, peristomial membrane, and coelomic fluid from specimens of the sea urchin Echinus esculentus from the Clyde Sea area of Scotland. These isolates were compared with 26 isolates from sand and seawater in the same locality. Overall, strains of Pseudomonas and Vibrio predominated. Gut (with an average bacterial viable count of 2 X 10(7) per 3-cm section) and coelomic fluid (which was often sterile and rarely had more than 40 bacteria per ml) had similar distributions of genera, with Vibrio predominating and Pseudomonas and Aeromonas next in abundance. In contrast, the flora of the peristomial membrane (with an average count of detachable bacteria of 2.5 X 10(5) per membrane) resembled that of sand/seawater in having Pseudomonas predominating, gram-positive forms or Vibrio next in abundance, and smaller numbers of Aeromonas, Flavobacterium, Acinetobacter, and Moraxella.