Studies on resistance in snails. 6. Escape of Echinostoma lindoense sporocysts from encapsulation in the snail heart and subsequent loss of the host's ability to resist infection by the same parasite.

Formation of amebocyte aggregates in the ventricular cavity of Biomphalaria glabrata, induced by developing sporocysts of Echinostoma lindoense, does not always result in destruction of the parasites, as the sporocysts occasionally escape encapsulation in the heart. When this occurs, a remarkable loss of protective capacity follows and the host snails become highly susceptible to reinfection with the same species--even more so than in control susceptible snails exposed for the first time. Although the amebocyte-producing organ is considerably enlarged after a first infection and shows numerous mitoses, the amebocytes produced by snails harboring an "escaped" infection in the heart appear unable to attack the parasites of the first or of the second exposure. Instead, the amebocytes produced accumulate in the loose connective tissues between the liver lobuli, where early developmental stages of the parasites do not occur. These amebocytes apparently have lost their ability to recognize the parasites as foreign.     

Studies on resistance in snails. 3. Tissue reactions to Echinostoma lindoense sporocysts in sensitized and resensitized Biomphalaria glabrata.

The resistance of Biomphalaria glabrata snails that have been sensitized by various levels of irradiated or nonirradiated Echinostoma lindoense miracidia increased after a second challenge infection with nonirradiated miracidia of the same species. This was demonstrated by increased suppression of migrating capacity of invading sporocysts, an accelerated host tissue reaction, and a greater tendency of snail amebocytes to flatten while attacking the parasite. Three methods of elimination of invading sporocysts were observed: (1) encapsulation by amebocytes followed by destruction of the sporocysts; (2) expulsion of the sporocyst through the host epithelium after its encapsulation in the subepithelial tissues; (3) blockade of the parasite's entry into subepithelial tissues by a localized amebocyte aggregation. The basic mechanism of host snail response to a single or a repeated challenge infection appears to be similar, though an anamnestic reaction is evident in the accelerated response following a second challenge exposure.     

An ultrastructural study on ventricular encapsulation reactions in Biomphalaria glabrata exposed to irradiated echinostome parasites

Jeong K. H., Lie K. J. and Heyneman D. 1984. An ultrastructural study on ventricular encapsulation reactions in Biomphalaria glabrata exposed to irradiated echinostome parasites. International Journal for Parasitology14: 127–133. Encapsulation reactions around sporocysts of Echinostoma lindoense and E. paraensei have been studied in the ventricle of Biomphalaria glabrata using ultrastructural procedures. Amebocytes and ameboblasts migrate from the amebocyte-producing organ to the ventricle and there surround irradiated echinostome larvae. Specialized junction sites were found that linked amebocytes in the three phases of capsule formation, migratory, flattening and phagocytic. Active engulfing of portions of the tegument by amebocytes was followed by phagocytosis of parasite internal cells and formation in amebocytes of residual bodies of indigestible material. Regression of encapsulation reactions in the ventricle was frequently associated with formation of sear tissue. Comparisons made between types of encapsulation reactions observed around echinostomes and schistosomes in B. glabrata reveal differences in speed of response, number of cells involved and size of amebocyte pseudopodia.     

Studies on resistance in snails. 5. Tissue reactions to Echinostoma lincloense in naturally resistant Biomphalaria glabrata.

Laboratory-raised juvenile albino Biomphalaria glabrata snails show a wide range of natural resistance to a single infection with 50 or 100 miracidia of Echinostoma lindoense. In the most resistant snails all sporocysts are destroyed in peripheral tissues soon after miracidial penetration. In less resistant snails some sporocysts reach the heart where they are encapsulated. In fully susceptible snails, all sporocysts rapidly migrate to the heart, where they mature and continue to develop. The greater part of our B. glabrata colony consists of snails in which sporocysts reaching the heart will survive, but in which a varying number of sporocysts will be destroyed in the tissues. These snails are usually considered susceptible, as they do become infected. Tissue reactions induced by sporocysts following a single infection in naturally resistant snails are similar to reactions in snails with an acquired resistance. In fully susceptible snails, the amebocyte-producing organ remains small and inactive. It is slightly to moderately stimulated in partially resistant snails in which destruction of sporocysts occurs in the tissues and surviving larvae are found in the ventricle. In snails in which amebocyte aggregates or capsules develop in the ventricle, the organ becomes markedly enlarged. Migration of sporocysts in the snail appears not to be continuous, as periodic rests seem to occur. Migration follows intrusion of the sporocyst through the tissues, induced by bodily distension and contraction, and then proceeds within the arteries against the blood flow, passing from one endothelial attachment site to another, possibly aided by negative pressure during ventricular diastole.     

Cytoskeletal organization of limulus amebocytes pre- and post-activation: comparative aspects

One of the major functions of circulating Limulus amebocytes is to effect blood coagulation upon receipt of appropriate signals. However, the hypothesis that Limulus amebocytes are fundamentally similar to vertebrate thrombocytes and platelets has not been tested sufficiently in previous studies of their cytoskeletal organization. Whereas the earlier data were derived from transmission electron microscopy (TEM) of thin sections of a limited number of cells, improved fluorescence labeling methods that retain cell morphology have now enabled us to survey F-actin and microtubule organization in intact individual amebocytes and in large amebocyte populations pre- and post-activation. Anti-tubulin immunofluorescence showed the marginal band (MB) of microtubules to be ellipsoidal in most unactivated cells, with essentially no other microtubules present. However, minor subpopulations of cells with discoidal or pointed shape, containing corresponding arrangements of microtubules suggestive of morphogenetic intermediates, were also observed. Texas-red phalloidin labeled an F-actin-rich cortex in unactivated amebocytes, accounting for MB and granule separation from the plasma membrane as visualized in TEM thin sections, and supporting earlier models for MB maintenance of flattened amebocyte morphology by pressure against a cortical layer. Shape transformation after activation by bacterial lipopolysaccharide was attributable principally to spiky and spreading F-actin in outer cell regions, with the MB changing to twisted, nuclei-associated forms and eventually becoming unrecognizable. These major pre- and post-activation cytoskeletal features resemble those of platelets and non-mammalian vertebrate thrombocytes, supporting recognition of the Limulus amebocyte as a representative evolutionary precursor of more specialized clotting cell types.     

Isolation and studies of the granules of the amebocytes of Limulus polyphemus,the horseshoe crab

Granules were isolated from the cytoplasm of the amebocytes of Limulus polyphemus, the horseshoe crab, by disruption of cells obtained from blood which had been drawn into 2 mM propranolol. The granules subsequently were purified by centrifugation through a sucrose gradient that contained heparin. Extracts of the granules were prepared by freezing and thawing the granule preparations in distilled water. Transmission and scanning electron microscopy of the granules revealed round or ovoid particles. However, only one type of granule appeared to be present. The ultraviolet spectrum of the extract of amebocyte granules demonstrated a peak at 277 nm at pH 7.4, and a shift into two peaks of 281 nm and 290 nm at alkaline pH. Analytical ultracentrifugation revealed a pattern similar to that observed with lysates prepared from intact amebocytes. Polyacrylamide gel electrophoresis, in the presence of urea at pH 4.5, demonstrated patterns similar to those observed with amebocyte lysate. Extracts of the granules were gelled by bacterial endotoxin. The blood of the horseshoe crab contains only one type of cell, the amebocyte. Previous studies have shown that the blood coagulation mechanism of Limulus is contained entirely within amebocytes. The current studies suggest that the granules, which pack the cytoplasm of these cells, contain all of the factors required for the coagulation of blood, including the clottable protein. The intracellularly localized coagulation system is released from amebocytes when their granules rupture during cell aggregation.     

Endotoxin-induced degranulation of the Limulus amebocyte

Exocytosis and gelation of the granule contents of the amebocyte of Limulus polyphemus have been studied in a perfusion chamber observed with Nomarski differential interference contrast microscopy. Degranulation in response to bacterial endotoxin or the ionophore A23187 was significantly inhibited by the anion channel blocking agents suramin, SITS, DNDS and sodium isethionate. db-cAMP, PGI₂ and theophylline also succeeded in imparing degranulation of the amebocytes. All of the agents tested produced inhibition of degranulation which was readily reversed by washing the system free of the inhibitor and rechallenging the amebocytes with either endotoxin or the ionophore. After isolation in vitro, amebocytes underwent spontaneous degranulation in the absence of endotoxin at a rate 1–2 orders of magnitude slower than in the presence of endotoxin. Gelation of the clottable protein released from the amebocyte granules could occur in the absence of endotoxin. This is the first demonstration of gelation under endotoxin-free conditions.     

The fine structure of oyster agranular amebocytes from regenerating mantle wounds in the Pacific oyster, Crassostrea gigas

A study of the fine structure of agranular amebocytes from regenerating mantle wounds in the Pacific oyster, Crassostrea gigas, indicated that the unspecialized monocyte-type agranular amebocytes observed in amebocyte plugs from early wounds (72-hr-old wounds) may have differentiated into the fibroblasts and myoblasts typical of late wounds (144- and 240-hr-old wounds).     

Selection of a standard culture medium for primary culture of <Emphasis Type="Italic">Limulus polyphemus</Emphasis> Amebocytes

Summary This study provides information relevant to future research aimed at producing Limulus Amebocyte Lysate (LAL) in vitro, which would potentially reduce the need to harvest and bleed horseshoe crabs as in the current methods of LAL production. To address the need for primary culture of horseshoe crab amebocytes, this study tested the effects of a variety of standard insect cell culture media on amebocyte morphology and viability after 7 d of maintenance. Amerbocyte morphology was least altered from in vivo form in Grace’s Modified Insect Medium, with no observed degranulation of cells, as compared to the other media tested. There were significant differences in amebocyte viability among the six insect cell culture media tested. Grace’s Modified Insect Medium sustained viability of 77.2±5.1% (mean ± standard deviation) of amebocytes, followed distantly by Grace’s Insect Medium with 35.1±8.7% amebocyte viability. Results indicate that Grace’s Modified Insect Medium with horseshoe crab serum supplementation was the best candidate of the six media tested for future medium optimization for Limulus amebocyte requirements.     

Phylogeny of natural cytotoxicity: cytotoxic activity of coelomocytes of the purple sea urchin, Arbacia punctulata.

Coelomocyte-mediated nonspecific cell cytotoxic activity against human and murine target cells by the purple sea urchin Arbacia punctulata was investigated in vitro. Cytotoxic activity toward target cells was shown to be mediated by different coelomocyte populations isolated by discontinuous density gradient centrifugation. The population of phagocytic amebocytes showed the strongest cytotoxic activity and the highest binding to human NK markers by cytometry analysis. Our immunophenotypic studies showed that A. punctulata phagocytic amebocytes are CD14(+), CD56(+), CD158b(+), CD3(-), CD4(-), CD8(-), and CD16(-). The cytotoxic activity was independent of experimental incubation temperatures, required viable effector cells, and required cell-cell contact between the effector and target cells. Sodium azide significantly decreased coelomocyte cytotoxicity, indicating that cytotoxicity is metabolically dependent, and EDTA reduction of cytotoxic activity is consistent with the involvement of divalent cations in the cytotoxic process. These data describe a population of sea urchin coelomocytes (the phagocytic amebocyte) that are CD14(+), CD56(+), and CD158b(+), with cytotoxic activities.     

Extracellular matrix formation by amebocytes during epithelial regeneration in the pearl oysterPinctada fucata

Summary To identify the cells which produce the extracellular matrix during bivalve wound healing, we observed epithelial regeneration inPinctada fucata and evaluated the ability of amebocytes to produce the matrix in vitro. Between days 1 and 3 after an ovary was implanted with abiotic material (a shell ball) via an incision, agranular amebocytes formed a sheath, consisting of 10–20 cell layers, between the implant and incised ovarian tissue. Extracellular matrix was deposited in the spaces between the amebocytes in the sheath. At the incised follicle, gonadal epithelial cells were attached to the newly formed matrix. When a mantle allograft (2 mm square) was implanted with abiotic material to bring them into close contact, epithelial cells emigrated from the allograft along the surface of the abiotic material where they attached to the newly formed matrix at the sheath of amebocytes. In vitro, agranular amebocytes formed a matrix composed of fibrils with a diameter of 20 nm during a 6-day culture period. Pepsin-digested extract of the cell layer forming the matrix gave protein bands with electrophoretic mobilities identical to - and -sized components of a collagen purified from this animal. The matrix exhibited immunoreaction to antiserum raised against the collagen and was stained by alcian bluc. Thus, the agranular amebocyte apparently has the ability to produce an extracellular matrix containing collagen and possibly proteoglycan(s).     

Determination of bacterial number and biomass in the marine environment.

Three techniques for the measurement of bacterial numbers and biomass in the marine environment are described. Two are direct methods for counting bacteria. The first employs an epifluorescence microscope to view bacteria that have been concentrated on membrane filters and stained with acridine orange. The second uses a transmission electron microscope for observing replicas of bacteria that are concentrated on membrane filters. The other technique uses Limulus amebocyte lysate, an aqueous extract from the amebocytes of the horseshoe crab, Limulus polyphemus, to quantitate lipopolysaccharide (LPS) in seawater samples. The biomass of gram-negative (LPS containing) bacteria was shown to be related to the LPS content of the samples. A factor of 6.35 was determined for converting LPS to bacterial carbon.     

Determination of bacterial number and biomass in the marine environment.

Three techniques for the measurement of bacterial numbers and biomass in the marine environment are described. Two are direct methods for counting bacteria. The first employs an epifluorescence microscope to view bacteria that have been concentrated on membrane filters and stained with acridine orange. The second uses a transmission electron microscope for observing replicas of bacteria that are concentrated on membrane filters. The other technique uses Limulus amebocyte lysate, an aqueous extract from the amebocytes of the horseshoe crab, Limulus polyphemus, to quantitate lipopolysaccharide (LPS) in seawater samples. The biomass of gram-negative (LPS containing) bacteria was shown to be related to the LPS content of the samples. A factor of 6.35 was determined for converting LPS to bacterial carbon.     

Limulus amebocyte clotting cascade: Roles of endotoxin and adenylate cyclase

Endotoxin, the lipopolysaccharide from the cell wall of Gram-negative bacteria, causes blood clotting in the horseshoe crab,Limulus polyphemus. Minute amounts of endotoxin stimulate the amebocytes in the blood to undergo exocytosis, which release the contents of their secretory granules to form a clot. An endotoxin-binding protein that possesses calmodulin-like activity has been isolated from the amebocyte plasma membrane. This endotoxin-binding protein can activate adenylate cyclase fromBordetella pertussis to the same extent as rat testes calmodulin. The effect of endotoxin and the endotoxin-binding protein on cyclic AMP synthesis inLimulus amebocytes was examined. Amebocytes exposed to endotoxin have increased levels of intracellular cyclic AMP. Amebocyte membranes contain an adenylate cyclase which is stimulated by NaF, guanosine (β,r-imido)triphosphate, and for skolin. This adenylate cyclase is also stimulated by the endotoxin-binding protein and calcium. Exposure of amebocytes to forskolin or dibutyryl cyclic AMP are stimulated to secrete clot components. Activation of adenylate cyclasein vivo by endotoxin via the endotoxin-binding protein may be one of the ways in which endotoxin stimulates secretion. It is suggested that endotoxin may have two actions in theLimulus system: (1) binding of endotoxin to the endotoxin-binding protein activates adenylate cyclase, promoting secretion by the amebocytes; and (2) endotoxin catalyzes a reaction on the secreted material to form a blood clot. This latter reaction is not elicited by forskolin or dibutyryl cyclic AMP.     

Culture of amebocytes in a nutrient mist bioreactor

Summary We report the first use of nutrient mist bioreactor (NMB) technology to culture animal cells. The nutrient mist approximated the amebocyte stem tissue’s natural environment, which is a thin layer of fluid in the gill leaflets of the horseshoe crabLimulus polyphemus. NMB culture was tried in an attempt to increase production of amebocytes, which are the source of theLimulus Amebocyte Lysate (LAL), the basis for a sensitive and commercially valuable endotoxin assay. Amebocyte growth in the nutrient mist bioreactor is comparable to growth in liquid medium. However, the current design of the bioreactor presents problems for primary cultures such as ours where a pyrogen-free environment is necessary and fungal decontamination is difficult.     

Limulus amebocyte clotting cascade: Roles of endotoxin and adenylate cyclase

Endotoxin, the lipopolysaccharide from the cell wall of Gram-negative bacteria, causes blood clotting in the horseshoe crab,Limulus polyphemus. Minute amounts of endotoxin stimulate the amebocytes in the blood to undergo exocytosis, which release the contents of their secretory granules to form a clot. An endotoxin-binding protein that possesses calmodulin-like activity has been isolated from the amebocyte plasma membrane. This endotoxin-binding protein can activate adenylate cyclase fromBordetella pertussis to the same extent as rat testes calmodulin. The effect of endotoxin and the endotoxin-binding protein on cyclic AMP synthesis inLimulus amebocytes was examined. Amebocytes exposed to endotoxin have increased levels of intracellular cyclic AMP. Amebocyte membranes contain an adenylate cyclase which is stimulated by NaF, guanosine (,r-imido)triphosphate, and for skolin. This adenylate cyclase is also stimulated by the endotoxin-binding protein and calcium. Exposure of amebocytes to forskolin or dibutyryl cyclic AMP are stimulated to secrete clot components. Activation of adenylate cyclasein vivo by endotoxin via the endotoxin-binding protein may be one of the ways in which endotoxin stimulates secretion. It is suggested that endotoxin may have two actions in theLimulus system: (1) binding of endotoxin to the endotoxin-binding protein activates adenylate cyclase, promoting secretion by the amebocytes; and (2) endotoxin catalyzes a reaction on the secreted material to form a blood clot. This latter reaction is not elicited by forskolin or dibutyryl cyclic AMP.A preliminary report of this work has been presented elsewhere (Liu and Liang, 1984).     

Inhibition of the development of the reproductive tract in parasitized snails

Summary

Myoblasts, muscle cells with the capacity to divide, have been detected in “Anlagen” of the male copulation organ of Lymnaea stagnalis. They only occur in the apical part of the penis. Here they could be found throughout life. Mitotic activity of these cells can be demonstrated by using an antiserum to a S-phase specific cell cycle marker, PCNA [see, e.g., Baserga (1991)]. The number/percentage of PCNA positive myoblasts is a good parameter for growth of this male copulation organ and hence also for inhibition of its growth and development as occurs in parasitized snails. In transplantation experiments, “Anlagen” of the copulation organ were used from snails 7–9 weeks after being parasitized as they can be excised in this stage and transplanted into either parasitized or nonparasitized snails. These experiments have indicated that humoral, parasitic excretory/secretory factors can be responsible for the inhibition of growth and differentiation of the copulation organ in parasitized snails as reflected by a relatively low number of PCNA positive myoblasts compared to the controls. Data obtained in in vitro experiments showed a significant decrease of the number of myoblasts in “Anlagen” cultured in the presence of parasitic E/S products. The fact that no significant effect was found on the relative low number of PCNA positive myoblasts is discussed. The effect of parasitic E/S products on these myoblasts appeared to be exerted in a direct way, not mediated by CNS-derived factors or by factors from cells in the connective tissue sheath around the CNS. Although it appears possible to use transplantation and/or in vitro culturing of these “Anlagen” as a bioassay for identification of the parasitic factor(s) responsible for the inhibitory effects on myoblasts, the methods are very laborious and do not seem very appropriate for testing many fractions of E/S products.     

Effect of miracidial dose on adoptively transferred resistance to Schistosoma mansoni in the snail intermediate host, Biomphalaria glabrata

Adoptively transferred resistance to Schistosoma mansoni in the snail intermediate host Biomphalaria glabrata was measured as a function of miracidial challenge dose. Schistosome-susceptible snails implanted with the amebocyte-producing organ (APO) from resistant donors showed 29 and 39% prevalences of infection after challenge with 5 and 10 miracidia, respectively, but 68-83% prevalences when exposed to 25-200 miracidia. Prevalences in control (untampered) susceptible snails ranged from 97 to 100% at the different miracidial doses. Higher infection prevalences at elevated doses suggest that a range of transferred resistance occurs and possibly that low levels of APO-derived plasma factors or hemocytes in some recipients can be overwhelmed by larger numbers of parasites.