Spherule Cell Research Articles

The use of an integrative approach to identify coelomocytes in three species of the genus Holothuria (Echinodermata)

AbstractCoelomocytes in the Holothuroidea are traditionally identified according to their morphology through light, fluorescence, or electron microscopy. Former studies have typically used only one method, with few works combining two or more approaches. Studies using cytocentrifugation to study these cells are scarcer. Thus, for the first time, an integrative approach was used to compare coelomocytes in Holothuroidea. This approach consisted of living and stained cells, scanning electron microscopy (for spherule cells), and accurate morphometric analyses. Specifically, we used specimens of Holothuria grisea, Holothuria arenicola, and Holothuria tubulosa to test whether cytocentrifugation could be valuable in comparative studies with coelomocytes, whether an integrative approach could help to understand spherule cell diversity, and whether closely related species, even those having distinct geographic distributions and ecological requirements, would have a similar population of coelomocytes. Our results showed seven distinct cell types in these species, including phagocytes, fusiform cells, morula cells, acidophilic spherulocytes, spherulocytes, progenitor cells, and crystal cells. Total and differential cell counts, along with morphometric parameters, were similar among species. Morphometric analyses of spherule cells revealed consistent differences among the diameter of their cytoplasmic spherules, as well as a set of different morphotypes in acidophilic spherulocytes and spherulocytes. Cytospin preparations proved to be quite useful because they provided constant morphological and morphometric data, allowing accurate identification of the cell types and comparisons among species. Moreover, this study highlighted (1) that the spherule diameter is a good parameter to separate spherule cells and (2) a putative maturation process to acidophilic spherulocytes and spherulocytes. Lastly, we showed that the cells of these species are very similar, regardless of their geographic distribution and ecology. Thus, our work contributes to a better understanding of the coelomocytes in Holothuria, a genus with a wide geographic distribution. The present study may be useful to establish these species as important model organisms, as well as bring insights into the functions of coelomocytes.

Echinochrome A Release by Red Spherule Cells Is an Iron-Withholding Strategy of Sea Urchin Innate Immunity

Cellular immune defences in sea urchins are shared amongst the coelomocytes - a heterogeneous population of cells residing in the coelomic fluid (blood equivalent) and tissues. The most iconic coelomocyte morphotype is the red spherule cell (or amebocyte), so named due to the abundance of cytoplasmic vesicles containing the naphthoquinone pigment echinochrome A. Despite their identification over a century ago, and evidence of antiseptic properties, little progress has been made in characterising the immunocompetence of these cells. Upon exposure of red spherule cells from sea urchins, i.e., Paracentrotus lividus and Psammechinus miliaris, to microbial ligands, intact microbes, and damage signals, we observed cellular degranulation and increased detection of cell-free echinochrome in the coelomic fluid ex vivo. Treatment of the cells with ionomycin, a calcium-specific ionophore, confirmed that an increase in intracellular levels of Ca²⁺ is a trigger of echinochrome release. Incubating Gram-positive/negative bacteria as well as yeast with lysates of red spherule cells led to significant reductions in colony-forming units. Such antimicrobial properties were counteracted by the addition of ferric iron (Fe³⁺), suggesting that echinochrome acts as a primitive iron chelator in echinoid biological defences.

The haemocytes of the colonial aplousobranch ascidian Diplosoma listerianum: Structural, cytochemical and functional analyses

Diplosoma listerianum is a colonial aplousobranch ascidian of the family Didemnidae that is native to the northeast Atlantic and exhibits a cosmopolitan distribution in temperate waters. It lacks a shared colonial circulation crossing the tunic, and the zooids are connected only by the common tunic. In the present study, the haemocytes of this ascidian were analysed via light and electron microscopy. Their phagocytic and enzymatic activities, staining and immunostaining properties, and lectin affinity were examined with various classical methods reconsidered and modified for small marine invertebrates. Eight morphotypes were identified in reference to corresponding cell types described in other ascidians: undifferentiated cells (haemoblasts), storage cells for nitrogenous catabolites (nephrocytes) and immunocytes. The immunocytes are involved in immune responses, acting as (1) phagocytes, rich in hydrolases and involved in the clearance of both foreign particles and effete cells (hyaline amoebocytes and macrophage-like cells); (2) cytotoxic cells, able to degranulate and induce cytotoxicity through the release of the enzyme phenoloxidase after an immune stimulus (granular amoebocytes and morula cells); and (3) basophilic cells with an affinity for ConA and NPA that contain heparin and histamine and that show sensitivity to the compound 48/80, promoting their degranulation (mast cell-like granulocytes). In addition, a particular cell type showing exceptional development of the Golgi apparatus and large vacuoles containing a filamentous material has been recognised (spherule cell), for which a role in tunic repair and fibrogenesis has been hypothesised.

Conical Tomography of a Ribbon Synapse: Structural Evidence for Vesicle Fusion

To characterize the sites of synaptic vesicle fusion in photoreceptors, we evaluated the three-dimensional structure of rod spherules from mice exposed to steady bright light or dark-adapted for periods ranging from 3 to 180 minutes using conical electron tomography. Conical tilt series from mice retinas were reconstructed using the weighted back projection algorithm, refined by projection matching and analyzed using semiautomatic density segmentation. In the light, rod spherules contained ∼470 vesicles that were hemi-fused and ∼187 vesicles that were fully fused (omega figures) with the plasma membrane. Active zones, defined by the presence of fully fused vesicles, extended along the entire area of contact between the rod spherule and the horizontal cell ending, and included the base of the ribbon, the slope of the synaptic ridge and ribbon-free regions apposed to horizontal cell axonal endings. There were transient changes of the rod spherules during dark adaptation. At early periods in the dark (3–15 minutes), there was a) an increase in the number of fully fused synaptic vesicles, b) a decrease in rod spherule volume, and c) an increase in the surface area of the contact between the rod spherule and horizontal cell endings. These changes partially compensate for the increase in the rod spherule plasma membrane following vesicle fusion. After 30 minutes of dark-adaptation, the rod spherules returned to dimensions similar to those measured in the light. These findings show that vesicle fusion occurs at both ribbon-associated and ribbon-free regions, and that transient changes in rod spherules and horizontal cell endings occur shortly after dark onset.

Molecular cloning and characterization of Coccidioides immitis antigen 2 cDNA

Previous experiments have provided evidence that Coccidioides immitis antigen 2 (Ag2) is a major T-cell-reactive component of mycelia and spherule cell walls. Here we report the identification and cloning of the cDNA that encodes Ag2 from a lambda ZAP cDNA expression library constructed from spherule-derived RNA. DNA sequence analysis established that the 1,255-bp clone contains a 174-bp 5' untranslated region, a 582-bp open reading frame which encodes for a protein consisting of 194 amino acids, and a 375-bp 3' untranslated region, including a poly(A) tail. The recombinant Ag2 protein has a predicted molecular mass of 19.5 kDa and contains an 18-amino-acid N terminus which has been tentatively identified as a signal peptide. The Ag2 cDNA was ligated into the pGEX-4T-3 vector and expressed in Escherichia coli TG-1 cells as a glutathione S-transferase fusion protein. The recombinant fusion protein showed reactivity with sera from patients with coccidioidomycosis and elicited delayed-type footpad hypersensitivity responses in Coccidioides-immune mice. These results suggest that the Ag2 cDNA can be used for the large-scale production of this immunologically important protein.

The coelomocytes ofholothuria polii(echinodermata). I. light and electron microscopy

Abstract The ultrastructure of Holothuria polii coelomocytes is described and related to cell functions. Amebocytes, spherule cells and progenitor cells are the major circulating cellular types. Two morphological phases (bladder and filipodial) of the amebocytes are distinguished. Type I and Type II spherule cells are described on the basis of the morphological and ultrastructural characteristics of their granules. Maturative stages of these cells are also observed. Probably a third type of spherule cell is present. It could correspond to the acidophilic and Schmorl's positive cells. Progenitor cells lacking elaborate cytoplasmic specialization and with a probable staminal function are also normal circulating coelomocytes of Holothuria polii.

TheHolothuria poliibrown bodies

Abstract Holothuria polii brown bodies are associated with the defence mechanisms of the host. Naturally and experimentally induced brown bodies are histologically constituted by nodules in a granular extracellular matrix containing distinct spherule cell types. Nodules of natural brown bodies consist in flattened amoebocytes surrounding large antigens, whereas nodules from experimentally induced brown bodies originate from the aggregation of phagocyting amoebocytes. An extracellular granular matrix characterizes both types of brown bodies. It probably originates from degranulation of spherule cells. The presence of acid phosphatase and arylsulfatase in the homo‐genate of brown bodies suggests that these structures are able to digest enclosed substances via lysosomes.

Antigenic heterogeneity of an alkali-soluble, water-soluble cell wall extract of Coccidioides immitis.

The antigenic composition of an alkali-soluble, water-soluble cell wall extract of Coccidioides immitis, designated C-ASWS, was assessed by two-dimensional immunoelectrophoresis against goat antisera to C-ASWS and coccidioidin. The results established that C-ASWS from mycelia or spherule cell walls is heterogeneous in composition, containing two distinct antigenic components. One is present as a polymer that is antigenically identical to a polymeric antigen in coccidioidin, designated antigen 2. The other component detected in C-ASWS presented an unusual precipitin pattern in that a cathodal leg was demonstrable in the absence of an anodal leg. This incomplete precipitinogen was also detected in coccidioidin. In addition to the finding that C-ASWS is antigenically heterogeneous, the results provide evidence that the conformational and/or configurational structure of the C-ASWS antigen 2 (or antigen 2-like polymer) is altered during physicochemical extraction. This conclusion is based upon the finding that the immunoelectrophoretic profile of the C-ASWS polymer differs from that of coccidioidin antigen 2. The C-ASWS polymer is characterized by having a small cathodal precipitin peak connected to a large anodal peak, whereas coccidioidin antigen 2 is characterized by a predominant cathodal peak.

Haemopoiesis in Lepidoptera. III. A note on the multiplication of spherule cells and granular haemocytes

Blood films from early sixth instar larvae of Euxoa declarata (Lepidoptera: Noctuidae) stained in hematoxylin – eosin – alcian blue showed unequivocal examples of mitosis in spherule cells. The improved visibility of mitosis and the estimation of the mitotic index from counts of dividing cells per 1000 cells of each type indicated a far greater potential maximum production of spherule cells and granular haemocytes by mitosis than reported previously. Certain other methods of staining showed similar clear examples of spherule cell mitosis.

Comparison of Coccidioides immitis arthrospore, mycelium, and spherule cell walls, and influence of growth medium on mycelial cell wall composition.

Comparative lipid content, cell wall yield, neutral monosaccharide, glucosamine, and protein (amino acid) contents of arthrospores, mycelia, and spherules of Coccidioides immitis Cash were studied. Cellular lipid contents were found in the decreasing order: spherules, arthrospores, mycelia. Lipid content of mycelia did not reach the level of arthrospores or spherules even when mycelia were grown on relatively rich media. Cell wall yields of spherules were lower than for mycelia when grown on comparable media. Cell walls of arthrospores, mycelia, spherules, and spherule culture filtrate all contained 3-O-methylmannose, mannose, and glucose, but in varying amounts. Cell wall yield and cell wall glucose content increased in mycelia grown in increasingly rich media, whereas mannose content either decreased or remained constant.

Biochemical and physiological studies of certain ticks (Ixodoidea). Qualitative and quantitative studies of hemocytes.

Qualitative and quantitative studies were made of the hemocytes of 4 tick species from 2 genera representing the 2 principal tick families: Hyalomma anatolicum excavatum Koch and Hyalomma dromedarii Koch (Family Ixodidae), and Argas ( Persicargas ) persicus (Oken) and Argas ( Persicargas ) arboreus Kaiser, Hoogstraal & Kohls (Family Argasidae). The hemocytes were studied from laboratory-reared ticks representing various nutritional states and developmental stages, and both sexes of each species. Hematocrits and total hemocyte counts gave the highest values in unfed adults. Hemocyte numbers decreased during engorgement and continued to fall in the ixodids after dropping from the host; however in argasids counts were slightly increased during this period. Neither sex nor species influeneed the hematonits and total hemocyte counts. The hemocytes were classified into 3 major categories based on Giemsa-staining properties: prohemocytes, plasmatocytes, and spherule cells. The latter 2 categories were subdivided into early and late forms on the basis of extent of vacuolation and spherule density, respectively. Mitotic figure and binucleated cells were also observed. No granular hemocytes were found. Supplementary observations were made with supravital staining and with phase contrast. All cell types appeared to be structurally uniform in the 4 tick species studied. Differential hemocyte counts showed that plasmatocytes accounted for a larger portion of the total cell population than than the other 2 basic types. Values for argasid plasmatocytes were 1.5–2.0 fold greater than for counterpart ixodids. In ixodids the relative proportion of early and advanced forms shifted toward the advanced form as the tick passed through the hunger, engorgement, and preoviposition states. Prohemocyte counts were highest in unfed adult ixodids. Spherule cell numbers increased about 2 fold with engorgement of ixodids but were considerably reduced in argasids. Mitotic figures did not exceed 1.2% and binucleated cells 0.32% of the total cell population. These 2 forms were usually found together, most notably in premolt third nymphal instars and in unfed adults. Change in nutritional state had a greater influence upon the differential hemocyte count than did change in sex, species, or genus.

In vitro transformations of the insect plasmatocyte in some insects

In vitro transformation of insect plasmatocyte have been studied in physiological saline-versene, tissue culture, and the haemolymph. Transformation of a plasmatocyte into a granular cell can occur either by fragmentation of portions of the cytoplasm or by a gradual rounding-off of the fusiform plasmatocyte. The spherule cell is derived either from a plasmatocyte directly or from a granular cell. Cystocytes are formed from round plasmatocytes or from granular cells. Although gradual transformation of a plasmatocyte and a spherule cell into an adipohaemocyte was not observed, a granular cell was found changing into an adipohaemocyte. Oenocytoids are formed from fusiform plasmatocytes by cytoplasmic fragmentation. Podocytes and vermiform cells are derived from fusiform plasmatocytes by gradual transformation. All these transformations seem to be governed by specific physiological conditions. Oenocytoid- and lamellocyte-like cells are present in the haemolymph of Periplaneta americana (L.). The polymorphic plasmatocyte is the basic type of haemocyte in insects.

AN INVESTIGATION ON THE SWIMMING REACTION OF THE ANEMONE STOMPHIA COCCINEA. II. HISTOLOGICAL LOCATION OF A REACTING SUBSTANCE IN THE ASTEROID DERMASTERIAS IMBRICATA.

AbstractThe sea anemone, Stomphia coccinea, propels itself through the water by a series of whip‐like swimming motions when stimulated by the asteroid Dermasterias imbricata. It has been previously concluded that the reacting substance is located only in the coelomic fluid and in the aboral surface of this asteroid. The general histology of the body wall of Dermasterias indicates this asteroid is atypical to other asteroids. The dermis of the aboral surface is unique. This tissue has a spherule cell which is abundant, measures 28–32 μ and is not found on the lateral or oral surfaces.Histochemically the material in dermal spherules correlates well with the chemical properties of a stimulatory carbohydrate previously isolated from this tissue. It is probable that the stimulatory substance that causes Stomphia to swim is related to or identical witht his material.A prey‐predator relationship for the swimming response cannot be substantiated for the specific case of Dermasterias as the stimulatory asteroid. The response can only be considered a chemoreceptive phenomenon.