Classification and characterization of hemocytes from two Asian horseshoe crab species Tachypleus tridentatus and Carcinoscorpius rotundicauda.

In present study, transmission electron microscopy and flow cytometry were utilized to investigate the classification, characterization and immune functions of hemocytes from horseshoe crab, Tachypleus tridentatus and Carcinoscorpius rotundicauda. Three types of hemocytes were distinguished respectively: the granular cell, the semi-granular cell and the hyaline cell by transmission electron microscopy, while three hemocyte subpopulations (Gate 1 cell, Gate 2 cell, Gate 3 cell) were classified by flow cytometry. Hyaline cell was the major cell type with the highest nuclear-cytoplasmic ratio and granular cell and semi-granular cell showed lower ratios. Immune parameters of hemocytes in horseshoe crabs were investigated by flow cytometry. Different hemocyte subpopulations respond for diverse functions. Lysosomal contents and hemocyte mortality in Gate 3 cell subpopulation were higher than that in other subpopulations, while reactive oxygen species, phagocytosis and non-specific esterase, in Gate 1 cell subpopulation, were higher than those in other subpopulations. The hemocyte types between the two species had no significant differences in staining or morphology.

Detection of liposomal cholesterol and monophosphoryl lipid A by QS-21 saponin and Limulus polyphemus amebocyte lysate.

Liposomes containing cholesterol (Chol) have long been used as an important membrane system for modeling the complex interactions of Chol with adjacent phospholipids or other lipids in a membrane environment. In this study we utilize a probe composed of QS-21, a saponin molecule that recognizes liposomal Chol and causes hemolysis of erythrocytes. The interaction of QS-21 with liposomal Chol results in a stable formulation which, after injection into the tissues of an animal, lacks toxic effects of QS-21 on neighboring cells that contain Chol, such as erythrocytes. Here we have used liposomes containing different saturated phospholipid fatty acyl groups and Chol, with or without monophosphoryl lipid A (MPLA), as model membranes. QS-21 is then employed as a probe to study the interactions of liposomal lipids on the visibility of membrane Chol. We demonstrate that changes either in the mole fraction of Chol in liposomes, or with different chain lengths of phospholipid fatty acyl groups, can have a substantial impact on the detection of Chol by the QS-21. We further show that liposomal MPLA can partially inhibit detection of the liposomal Chol by QS-21. The Limulus amebocyte lysate assay is used for binding to and detection of MPLA. Previous work has demonstrated that sequestration of MPLA into the liposomal lipid bilayer can block detection by the Limulus assay, but the binding site on the MPLA to which the Limulus protein binds is unknown. Changes in liposomal Chol concentration and phospholipid fatty acyl chain length influenced the detection of the liposome-embedded MPLA.

Limulus amebocyte lysate testing: adapting it for determination of bacterial endotoxin in 99mTc-labeled radiopharmaceuticals at a hospital radiopharmacy.

UNLABELLED: A bacterial endotoxin test (BET) is required to detect or quantify bacterial endotoxin that may be present in radiopharmaceutical preparations. The test uses Limulus amebocyte lysate, which, in the presence of bacterial endotoxin and divalent calcium ions, causes the formation of a coagulin gel. (99m)Tc-labeled radiopharmaceuticals have chelating ligands such as diethylene triamine pentaacetic acid (DTPA), ethylene dicysteine (EC), L,L-ethyl cysteinate dimer (ECD), N-[2,4,6-trimethyl-3 bromoacetanilid] iminodiacetic acid (mebrofenin), dimercapto succinic acid-III (DMSA-III), dimercapto succinic acid-V (DMSA-V), and several others, which form a coordination complex with Na-(99m)Tc-O4 in the presence of reducing agents. During BET by the gel-clot method, the free sulfhydryl (-SH) and carboxyl (-COOH) in some of the chelating agents in the final (99m)Tc-labeled radiopharmaceuticals decrease the free divalent calcium ion concentration, which in turn inhibits coagulin gel formation. This study was designed using the premise that addition of calcium chloride solution to the reaction mixture would nullify this effect. METHODS: We present here the data obtained from BET assay analysis of (99m)Tc-labeled radiopharmaceuticals and the cold kits from which they are made (EC, ECD, methoxyisobutylisonitrile, DTPA, mebrofenin, methylene diphosphonic acid [MDP], DMSA-III, and DMSA-V) using 2 different dilutions, maximum valid dilution (MVD) and half maximum valid dilution (MVD/2), with and without the addition of calcium chloride at a final concentration of 300 µM. RESULTS: It was observed that at MVD and MVD/2 all of the (99m)Tc-labeled kits exhibited interference in coagulin gel formation with the exception of (99m)Tc-methoxyisobutylisonitrile, (99m)Tc-MDP, (99m)Tc-mebrofenin, and (99m)Tc-ECD. However, only the cold kits of methoxyisobutylisonitrile and MDP did not show inhibition. An addition of calcium chloride solution nullified this interference at both MVD and MVD/2 in all of the (99m)Tc-labeled radiopharmaceuticals in which interference was observed. CONCLUSION: In practice, Limulus amoebocyte lysate testing is not a method of choice for (99m)Tc-labeled radiopharmaceuticals because these radiopharmaceuticals exhibit interference. However, our study proves the hypothesis that the addition of calcium chloride can circumvent this problem. The addition of calcium chloride provides an enhanced biologic quality control testing option for the final formulation of (99m)Tc-labeled radiopharmaceuticals at the hospital radiopharmacy end.

Phagocytic activity of Limulus polyphemus amebocytes in vitro.

Phagocytosis of invading microorganisms is a fundamental component of innate immunity. The Atlantic horseshoe crab, Limulus polyphemus, possesses a single immune cell type, the granular amebocyte. Amebocytes release a repertoire of potent immune effectors in the presence of pathogens, and function in hemostasis. In contrast to other arthropod immunocytes, the properties of amebocyte phagocytosis remain poorly characterised, restricted by the technical challenges associated with handling these labile cells. We have addressed these challenges and observed the internalisation of microbial and synthetic targets by amebocytes in vitro. Confirmation of target internalisation was achieved using a combination of fluorescent quenching and lipophilic membrane probes: R18 and FM 1-43. Viability, morphological integrity and functionality of extracted amebocytes appeared to be retained in vitro. The phagocytic properties of L. polyphemus amebocytes described here, in the absence of endotoxin, are similar to those observed for arthropod immunocytes and mammalian neutrophils.

Pre-sampling contamination of filters used in measurements of airborne (1 → 3)-ß-D-glucan based on glucan-specific Limulus amebocyte lysate assay.

Air sampling for (1 → 3)-ß-D-glucan may be a good method for assessing inhalation exposure to airborne fungi. Pre-sampling contamination of filter media used for sampling (1 → 3)-ß-D-glucan may lead to substantial exposure measurement errors. Using the Limulus amebocyte lysate assay, we tested for pre-sampling levels of (1 → 3)-ß-D-glucan on three types of filters-mixed cellulose ester (MCE)[1 brand], glass fiber (GF)[1 brand], and polycarbonate (PC)[5 brands]. Levels of (1 → 3)-ß-D-glucan on MCE filters exceeded 4586.1 pg per filter. Levels on GF filters averaged 135.3 (± 28.9) pg per filter (range = 94.8-160.4 pg per filter) and levels on PC filters averaged 152.4 (± 236.1) pg per filter (range = non-detectable-1760.7 pg per filter). Efforts to clean MCE and GF filters were unfeasible or unsuccessful. Sonicating PC filters for two hours in ethanol, followed by a wash in pyrogen-free water, effectively eliminated measured levels of (1 → 3)-ß-D-glucan on four brands of PC filters, as compared to untreated PC filters. This pretreatment process did not appear to physically damage the PC filters. Air sampling results highlighted the potentially problematic contamination of untreated PC filters. Ensuring that sampling media are free of (1 → 3)-ß-D-glucan before sampling is crucial to accurately measure levels of (1 → 3)-ß-D-glucan exposure, especially in environments where levels of (1 → 3)-ß-D-glucan are low.

Endotoxin detection--from limulus amebocyte lysate to recombinant factor C.

Gram negative bacterial endotoxin is a biological pyrogen that causes fever when introduced intravenously. The endotoxin, also known as lipopolysaccharide (LPS), is found in the outer membrane of Gram-negative bacteria. During Gram-negative sepsis, endotoxin stimulates host macrophages to release inflammatory cytokines. However, excessive inflammation causes multiple organ failure and death. Endotoxins, which are ubiquitous pathogenic molecules, are a bane to the pharmaceutical industry and healthcare community. Thus early and sensitive detection of endotoxin is crucial to prevent endotoxaemia. The limulus amebocyte lysate (LAL) has been widely used for ~30 years for the detection of endotoxin in the quality assurance of injectable drugs and medical devices. The LAL constitutes a cascade of serine proteases which are triggered by trace levels of endotoxin, culminating in a gel clot at the end of the reaction. The Factor C, which normally exists as a zymogen, is the primer of this coagulation cascade. In vivo, Factor C is the perfect biosensor, which alerts the horseshoe crab of the presence of a Gram-negative invader. The hemostatic end-point entraps the invader, killing it and limiting further infection. However, as an in vitro endotoxin detection tool, variations in the sensitivity and specificity of LAL to endotoxin, and the dwindling supply of horseshoe crabs are posing increasing challenges to the biotechnology industry. This has necessitated the innovation of an alternative test for endotoxin. Thus, Factor C became the obvious, albeit tricky target for the recombinant technology effort. This chapter documents the backwater of mining the natural blood lysate of the endangered species to the monumental effort of genetic engineering, to produce recombinant Factor C (rFC). The rFC is a 132 kDa molecule, which was produced as a proenzyme inducible by the presence of trace levels of endotoxin. The rFC forms the basis of the "PyroGene" kit, which is a novel micro-enzymatic endotoxin diagnostic assay for high-throughput screens of endotoxin. Using the rFC, Lonza Inc. has spawned the "PyroSense" which serves as checkpoints of the biotechnology production line. Thus, from cloning to commercial applications, the rFC has initiated a new era in endotoxin-testing for the quality assurance of biomedical products and for the healthcare industry, whilst sparing the endangered horseshoe crabs.

Testing the role of calmodulin in the excitation of Limulus photoreceptors.

The phototransduction cascade in Limulus ventral photoreceptors involves multiple second messengers, including Ca(2+) and cGMP. Light-induced Ca(2+) release from intracellular stores is an intermediate step, but the subsequent Ca(2+)-activated reaction remains to be determined. The possibility that Ca(2+)/calmodulin (Ca(2+)/CaM) might be involved is suggested by the high calmodulin content of the transducing lobe. To test whether CaM can excite the transduction cascade we injected a 25 microM Ca(2+)/CaM solution. This produced a rapid, brief depolarization similar to that produced by light, suggesting a role for CaM in the cascade. However, an important caveat is that Ca(2+) dissociating from the Ca(2+)/CaM complex might excite this process. Several control experiments argue against, but do not entirely eliminate this possibility. To test whether endogenous CaM has a function in excitation, trifluoperazine was pressure injected into the rhabdomeric region. The response to brief flashes was not affected, but the response to steady illumination was transiently attenuated by each injection. We conclude that calmodulin should be considered a candidate to couple intermediate and late stages of the transduction cascade.

Response of the blood clotting system of the American horseshoe crab, Limulus polyphemus, to a novel form of lipopolysaccharide from a green alga.

Lipopolysaccharide (LPS, endotoxin) is a component of Gram-negative bacteria and is the principal indicator to the innate immune systems of higher animals of a Gram-negative bacterial invasion. LPS activates the blood clotting system of the American horseshoe crab, Limulus polyphemus. By stimulating blood cell degranulation, LPS triggers the release of the proteins of the clotting system from the cells, and by activating a protease cascade that converts coagulogen, a soluble zymogen, to coagulin, the structural protein of the clot, LPS triggers the production of the fibrillar coagulin blood clot. Although originally thought to be restricted to the Gram-negative bacteria and the cyanobacteria, LPS, or a very similar molecule, has recently been described from a eukaryotic green alga, Chlorella. Here we show that, like LPS from Gram-negative bacteria, the algal molecule stimulates exocytosis of the Limulus blood cell and the clotting of coagulin. The coagulin clot efficiently entraps the cells of Chlorella in a network of fibrils. Invasion and erosion of the carapace by green algae is an important cause of mortality of Limulus, and it is suggested that the cellular response to aLPS may contribute to defense against this pathogen.

Selection of a standard culture medium for primary culture of limulus polyphemus amebocytes.

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. Amebocyte 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.

Structure of the acrosomal bundle.

In the unactivated Limulus sperm, a 60- micro m-long bundle of actin filaments crosslinked by the protein scruin is bent and twisted into a coil around the base of the nucleus. At fertilization, the bundle uncoils and fully extends in five seconds to support a finger of membrane known as the acrosomal process. This biological spring is powered by stored elastic energy and does not require the action of motor proteins or actin polymerization. In a 9.5-A electron cryomicroscopic structure of the extended bundle, we show that twist, tilt and rotation of actin-scruin subunits deviate widely from a 'standard' F-actin filament. This variability in structural organization allows filaments to pack into a highly ordered and rigid bundle in the extended state and suggests a mechanism for storing and releasing energy between coiled and extended states without disassembly.

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.

Long-term in vitro generation of amoebocytes from the Indian horseshoe crab Tachypleus gigas (Müller).

Amoebocyte is the single type of cell circulating in the horseshoe crab hemolymph, which plays a major role in the defense system of the animal. Granules present in these cells are sensitive to nanogram quantities of bacterial endotoxins, which form the basis of the Limulus amoebocyte lysate (LAL) test. Normally, amoebocytes for the production of the LAL are collected by cardiac puncture; hence, development of the in vitro culture system for amoebocytes will reduce the variability of the lysate and help to conserve the 400 million-yr-old living fossil. In the present investigation we have attempted organ culture of gill flaps that have been shown to be the source of amoebocytes. The gill flaps were cultured at 28 degrees C on a rocker platform in a modified L-15 medium supplemented with 10% v/v horseshoe crab serum. This led to the release of amoebocytes outside the gill flaps for a period of 6-8 wk with a more or less steady number of amoebocytes during the weekly harvest. No significant difference was seen in the yield of amoebocytes from male and female horseshoe crabs. Confocal laser microscopy studies revealed significant difference in the size of amoebocytes released in vitro as compared with those obtained in vivo. Thus, we have optimized the culture conditions for the long-term generation of amoebocytes in vitro from the Indian horseshoe crab Tachypleus gigas by reducing the incidence of contamination, simulating in vivo conditions for the organ culture of gill flaps, and improvising the nutritional status using the modified L-15 medium, providing the desired osmolarity and pH.

Visual arrestin in Limulus is phosphorylated at multiple sites in the light and in the dark.

Arrestins participate in the termination of phototransduction in both vertebrates and invertebrates. However, the visual arrestins of invertebrates and vertebrates differ significantly from one another in that the invertebrate visual arrestins become phosphorylated rapidly in response to light while those in the photoreceptors of vertebrates do not. In an effort to understand the functional relevance of arrestin phosphorylation, we examined this process in the photoreceptors of the horseshoe crab Limulus polyphemus. We report that Limulus visual arrestin can be phosphorylated at three sites near its C-terminus and show that arrestin molecules phosphorylated on one, two, and three sites are normally present in both light- and dark-adapted photoreceptors. Light adaptation increases the amount of arrestin phosphorylated at three sites.

Phenotypic variation in Actinobacillus actinomycetemcomitans during laboratory growth: implications for virulence.

This study examined alteration of specific virulence traits associated with phenotypic changes seen when a low-passage disease-associated and well maintained parent strain of Actinobacillus actinomycetemcomitans was compared to a laboratory-grown spontaneous variant/mutant. Clinical isolates of A. actinomycetemcomitans recovered from periodontitis patients typically grow as rough, adherent colonies on primary culture but undergo transformation to smooth, non-adherent colonies following repeated passage in vitro. The relationship of these phenotypic changes to the virulence of the organism or to the processes that underlie this transformation are not understood. A fresh clinical isolate, designated strain CU1000, was obtained from the first molar site of a patient with classical signs of localized juvenile periodontitis and used as the parent strain to study virulence-related phenotypes. Following several passages of CU1000 on selective agar, a spontaneous variant that demonstrated smooth, opaque, non-adherent colonies was isolated and designated strain CU1060. This study compared the properties of these two strains with respect to colony morphology, autoaggregation, surface appendages, adherence to saliva-coated hydroxyapatite (SHA), LPS chemotype and activity, induction of fibroblast proteinase activity and antigenic properties. CU1000 demonstrated rough, raised, star-positive colonies which upon electron microscopic examination revealed the presence of large, flexible, bundled fibrils. In addition, CU1000 showed adherence to SHA, several unique protein antigens and elevated endotoxin and fibroblast proteinase activity. CU1060, on the other hand, showed minimal adherence to SHA and fewer reactive proteins compared to the fresh clinical isolates. This strain formed smooth, opaque colonies on agar, showed minimal fibril formation and limited endotoxin and fibroblast-proteinase-inducing activity. These findings demonstrate that clinical isolates of A. actinomycetemcomitans undergo significant virulence-reducing phenotypic alterations during in vitro passage and support the need to study this organism in its clinical form.

Signal transduction during exocytosis in Limulus polyphemus granulocytes.

Bacterial lipopolysaccharide (LPS)-induced exocytosis is one of the primary immune responses of the Limulus granulocyte (GR). Exocytosis can be mediated by guanine nucleotide-binding protein (G-protein)-linked surface receptors that activate phospholipase C (PLC) to produce inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG). IP3 mobilizes intracellular Ca2+ ([Ca2+]i), which can lead to exocytosis. We used activators and inhibitors of known signal transduction pathways to investigate the signaling pathway responsible for LPS-induced exocytosis in the GR. These compounds have been shown to similarly effect pathways in vertebrate and invertebrate systems and this assumption is made here. Pretreatment of GRs with cholera and pertussis toxins, which modulate G-proteins, and U73122, which inhibits PLC, inhibited LPS-induced exocytosis, but pretreatment with the tyrosine kinase inhibitor herbimycin did not. In contrast, exocytosis was induced with fluoride (a G-protein activator) and thapsigargin with Mg2+ (an inhibitor of endomembranous Ca(2+)-ATPase). Exocytosis was not induced by phorbol ester, which mimics DAG to activate protein kinase C (PKC) and it was not effected by ethanol or chelerythrine, which inhibit phospholipase D and PKC, respectively. Microinjection of GRs with different concentrations of IP3, an IP3 analog (DL-2,3,6,trideoxy-myo-inositol 1,4,5-triphosphate), Mg2+, or Ca2+ induced different percentages of exocytosis in individual cells, while HEPES buffer did not. Microfluorometric analysis of intracellular Mg2+ ([Mg2+]i) and [Ca2+]i, using the dyes Mag Fura-2AM and Calcium Green 5N, respectively, revealed [Mg2+]i and [Ca2+]i fluxes during LPS-induced exocytosis. This study suggests that LPS induces exocytosis in the Limulus GR through activation of G-protein-coupled receptors, which stimulate the IP3 signaling pathway to induce both [Ca2+]i and [Mg2+]i fluxes to facilitate vesicular and plasma membrane fusion. This is the first demonstration of the signal transduction pathway responsible for the primary immune response of the GR.

The role of hemolymph coagulation in innate immunity.

Invertebrate animals, which lack adaptive immune systems, have developed defense systems that respond to common antigens on the surface of potential pathogens. Hemolymph coagulation is one such defense system in innate immunity. The discovery of lipopolysaccharide-sensitive and (1-->3)-beta-D-glucan-sensitive serine protease zymogens in horseshoe crab (limulus) hemocytes, both of which trigger the coagulation cascade, has exemplified how the animals detect and respond to foreign materials.

A cysteine protease inhibitor stored in the large granules of horseshoe crab hemocytes: purification, characterization, cDNA cloning and tissue localization.

A cysteine protease inhibitor with an apparent Mr = 12,600, designated limulus (L)-cystatin, was isolated from hemocyte lysates of the Japanese horseshoe crab (Tachypleus tridentatus), using two steps of chromatography, including dextran sulfate-agarose, and carboxymethylated papain-agarose. L-cystatin inhibits amidolytic activity of papain by forming a noncovalent 1:1 complex with an equilibrium constant (Ki) of 0.08 nM. It also inhibits cathepsin L (Ki = 0.17 nM) and ficin (Ki = 0.52 nM), but not argingipain (a bacterial cysteine protease) and calpains. A cDNA for L-cystatin was isolated and the open reading frame coded for a mature protein of 114 amino acids, of which 99 residues were confirmed by peptide sequencing. L-cystatin shows significant sequence identities to members of the family 2 cystatin, such as bovine colostrum cystatin (33%) and human cystatin S (31%). Northern blotting revealed expression of the mRNA in hemocytes and slightly in heart but expression was negligible in hepatopancreas, intestine, stomach, and muscle. Immunoblotting revealed the localization to be in the large granules of hemocytes. Furthermore, L-cystatin has an antimicrobial activity against Gram-negative bacteria, which is much stronger than that of chicken egg white cystatin. These data suggest that the large granule-derived L-cystatin serves synergistically to accomplish an effective defense against invading microbes, together with other defense molecules that are released in response to external stimuli.

Movement of axoplasmic organelles on actin filaments assembled on acrosomal processes: evidence for a barbed-end-directed organelle motor.

The directionality of the actin-dependent motors on squid axoplasmic organelles was determined using actin filaments assembled on the barbed ends of acrosomal processes. Acrosomal processes were isolated from Limulus polyphemus sperm and incubated in monomeric actin under conditions that promoted barbed end assembly only. Newly assembled actin was stabilized and stained with rhodamine-phalloidin and the presence of filaments at the barbed ends of the acrosomal processes was verified by fluorescence microscopy and negative contrast electron microscopy. Axoplasmic organelles that dissociated from extruded axoplasm were observed by video microscopy to move along the newly assembled actin filaments at an average velocity of 1.1 +/- 0.3 microns/second. All organelles moved in the direction away from the acrosomal fragment and towards the tip of the actin filaments. Therefore, the actin-dependent organelle motor on axoplasmic organelles is a barbed-end-directed motor like other myosins analyzed. These findings support the conclusions that axoplasmic organelles are driven by a myosin-like motor along actin filaments and that these filaments as well as microtubules function in fast axonal transport.

Morphological changes of Carcinoscorpius rotundicauda amoebocyte and Escherichia coli during their interaction.

Interaction between Carcinoscorpius rotundicauda amoebocytes and Escherichia coli or Staphylococcus aureus has shown that whilst E. coli was observed to lyse, S. aureus remained intact. The heavily granulated amoebocytes were activated by the lipopolysaccharide of E. coli to undergo dramatic morphological changes with time, leading to degranulation which caused lysis of the E. coli. Degranulation of the amoebocytes involved maturation of the larger electron dense granules (0.7-2.0 micron) which were apparently mobilised towards the cell membrane whilst undergoing a loss in electron density. Some of these larger granules were expelled at 15-30 min while other intracellular granules underwent further degranulation, where parallel filaments appearing like microtubules were evident, giving the granules a striated and less dense form. By 60 min, there was complete depolymerisation of the microtubular filaments giving rise to undefined, fused and homogeneously packed striations and particulate material in the cytoplasm; the smaller electron dense granules (0.3-0.7 micron) remained unchanged. This asynchronous degranulation and preferential expulsion of granules from only some amoebocytes while maintaining others intact, implies that the horseshoe crab has safeguards in its defence mechanism, hence ensuring its continued survival as a 'living fossil'.

Culture of amebocytes in a nutrient mist bioreactor.

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 crab Limulus polyphemus. NMB culture was tried in an attempt to increase production of amebocytes, which are the source of the Limulus 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.