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In recent years, several studies have demonstrated the existence of cytokine-like molecules in invertebrates, therefore suggesting that cytokines may have been conserved throughout evolution. In this study, we investigated the presence of immunoreactive TNF alpha (ir TNF alpha) in the gastropod mollusc Biomphalaria glabrata, the specific intermediate host for the trematode Schistosoma mansoni. Immunocytochemical study indicated the presence of ir TNF alpha in mollusc hemocytes corresponding to a 53-kDa molecule detected by western blot analysis. Using ELISA tests, we demonstrated the presence of substantial amounts of ir TNF alpha in hemolymph, that were significantly decreased during the S. mansoni infection. The possible role of ir TNF alpha in the regulation of mollusc immune functions and in the host-parasite relationship is discussed.

Summary Developmental stages (sporoblasts and spores) of five species of microsporidia ( Amblyospora bracteata, A. varians, A. weiseri, Nosema apis, Tuzetia sp.), fixed by conventional EM fixatives or by glutaraldehyde supplemented with potassium chloride, were studied by freeze-fracture using ultrathin sections as controls. It was found that the polar filament is formed by coalescence of cytoplasmic vesicles of the Golgi zone. The filament is enveloped by a single cytoplasmic membrane (“filament ensheathing membrane” = FEM) showing more intramembrane particles (IP) on the EF face (in the order of 1000 IP/μm 2 ) and less IP (in the order of 500 IP/μm 2 ) on the PF face. During maturation of the spore the IP on the FEM progressively disappear. The polaroplast is a stack of cytoplasmic membranes revealing more IP (in the order of 1500 IP/μm 2 ) on the PF face and less particles (in the order of 400 IP/μm 2 ) on the EF face. When KCl is added to the fixative the polaroplast remains unswollen and its membranes are smooth. In swollen polaroplasts the membranes are modified, probably by mechanical stretching. The polaroplast membranes are not direct outgrowths of the FEM. The posterior vacuole is limited by a single cytoplasmic membrane. No evidence was found supporting the idea that the filament ends in the posterior vacuole.

In this study we describe the site and moment of histospecific differentiation in developmental stages of the annelid Platynereis dumerilii by use of biochemical markers. The monoclonal antibody (mab) OI7 and uncloned hybridoma supernatants (pAb's) OI8, OI10, OI46 and OI69 recognize neural antigens that appear asynchronously during development. By an enzymatic test, acetylcholinesterase (AChE) was found specific for nervous tissue as well. The patterns of neural structures labelled by antibodies differ, however, from those revealed by AChE staining. Experimental inhibition of transcription (with actinomycin D) and of translation (using puromycin) demonstrate that the expression of histospecific neural markers depends on both zygotic transcription and subsequent translation. The mAb OI64 labels epidermal (and neural) gland cells. The antibody 4D9, raised against the engrailed protein of Drosophila, labels single rows of ciliated cells at the posterior border of segments.

The coccidian, Coelotropha durchoni, manages to develop in its host, the polycheate annelid, Nereis diversicolor, because of its ability to circumvent the host's internal defence system. First, it avoids phagocytes by penetrating other cells, principally eleocytes and muscular cells, where it undergoes a phase of intracellular development. After becoming extracellular, a thick coat protects it from being attacked by granulocytes. This coat then breaks and is shed from its surface to permit fertilization. Parasites that have lost their coats and remain unfertilized are surrounded with granulocytes and destroyed by encapsulation. A strict hormonal correlation exists between the biological cycles of the parasite and its host. Thus, the mature spores of the coccidian parasite are disseminated when the worm lays its gametes by rupture of teguments. C. durchoni and N. diversicolor have established a biological equilibrium that permits mutual survival of both partners and constitutes a simple model for the study of the host-parasite relationship.

Coelomocytes of Nereis diversicolor synthesize and secrete vitellogenin in vitro. Using a monoclonal antibody which specifically recognized vitellogenin, we showed by immunocytochemistry that among the coelomocytes only a subpopulation, called eleocytes, countained vitellogenin. These results assert that eleocytes are the vitellogenin producing cells in nereids.

We attempted to identify the nature and origin of the pigment produced by the marine worm Nereis diversicolor in order to isolate, in inert brown capsules, foreign objects introduced into its body cavity. This brown pigment, characterized by cytochemical techniques, could be a melanin. The activity of the enzyme phenoloxidase responsible for melanin biosynthesis was detected by enzyme cytochemistry techniques in vacuoles and the Golgi apparatus of coelomocytes activated by the presence of foreign bodies. Morphological techniques combined with a monoclonal immunological probe enabled us to establish that the "G2" granulocytes contain both the precursor of the pigment in dense bodies and the capacity for phenoloxidase synthesis when activated to encapsulate foreign bodies. The "G2" granulocyte may therefore be compared to a melanocyte in which melanin is not stored as in mammals, but immediately extruded following synthesis in the form of a thick fluid.

Two peptides (B1 and B2) have been isolated from the coelomic fluid ofPerinereis cultrifera. These peptides are absent in sexually undifferentiated animals. They appear and become abundant during the oocyte submaturity stage. When B1 and B2 are simultaneously injected into very young females, they stimulate an important biosynthesis of oocyte glycoconjugates (certical alveoli). Injected separately, B1 or B2 leads to an oocyte structure similar to that of an anhormonal state. The modes of these actions were discussed.

RESUME Le mode de contamination par Aggregata a eteetudie experimentalement in vivo chez le Crabe, in vitro sur cultures cellulaires de Mollusques, puis suivi en microscopie electronique. Quelques heures apres le repas infestant, les spores s'ouvrent dans l'estomac du Crabe et les sporozoites gagnent l'intestin moyen. La ils traversent l'epithelium dans les espaces intercellulaires. La perforation de l'epaisse basale est liee a l'apparition d'une substance dense, de nature probablement enzymatique. Les lesions causees aux tissus du Crabe sont minimes. Les merozoites sont capables de penetrer en culture dans des cellules provenant de divers Mollusques; la membrane de la cellule-hǒte n'est pas rompue. Un debut de developpement est observe avec l'apparition d'un nucleole dans le noyau. ABSTRACT The mechanism of host invasion by Aggregata has been experimentally investigated both in vivo (in the crab), and in vitro (in mollusc cell cultures) and followed by electron microscopy. A few hours after the infesting meal, sporozoites escaped from sporocysts in the crab stomach: then they reached the small intestine. There, they pussed through the epithelium, making their way between the cells. Perforation of the thick basal lamina was induced by means of the elaboration of a dense product, probably an enzyme. Sporozoites stretched themselves passing through the basal lamina and entered the connective tissue surrounding the digestive tract. Damages inflicted on the host were minimal. In vitro, merozoites were able to enter cells from several molluscs by penetrating the cell's plasmalemma. A nucleolus appeared in the nuclei of intracellular merozoites.

The presence in the marine worm Nereis diversicolor of a low molecular mass protein with the capacity to bind cadmium has been previously demonstrated [5]. Poly(A) + -mRNA were extracted from coelomocytes of Nereis diversicolor and were translated either in vitro , using a rabbit reticulocyte lysate, or in vivo into Xenopus laevis oocytes. Analysis of synthesized polypeptides by enzyme-linked immunosorbent assay (ELISA) and by Western blotting, using a specific monoclonal anti-MP II antibody, showed that this metalloprotein was translated both in in vitro and in vivo translation systems, with an apparent molecular mass of 11–13 kDa. Two other products, with 26.5 and 28 kDa molecular mass, cross-reacted with the monoclonal anti-MP II antibodies. The present work confirms that coelomocytes are sites of important synthesis of MP II-mRNA.

L'etude en cryofracture du merozoite d'Aggregata eberthi confirme l'unite de structure de la paroi du germe infectieux des Coccidies (membrane plasmique polarisee par la presence d'une rosette apicale de 8+1 particules intramembranaires (P.I.M.), complexe membranaire interne forme d'un pavage de vesicules plates et d'une cape apicale). Des differences de detail specifiques existent cependant dans la disposition des P.I.M. de la cape, et dans l'arrangement des vesicules entre elles.

The growing stages of Aggregata eberthi, in the cuttlefish on the one hand (sexual generation), and in the crab on the other hand (asexual generation), are investigated.

This study considers the earlier growth stages of Coccidium Coelotropha durchoni in its host, Nereis diversicolor. Before evolving into free trophozoites and gamontes in coeliac fluid, the parasites remain in muscular and coeliac cells in microscopic intracellular form. Electron microscope reveals that these stages show an intermediary fine structure between that of a sporozoite — from which they keep some typical characteristics such as the conoid, the fibers and the involuted tubuli — and that of the future free trophozoites. The wall consisting in two clear membrans is provided with one or several micropores. The classical cytoplasmic organites clearly stand out: dictyosomes show constant relationship with ergastoplasm, the mitochondria contain short inner tubuli. Besides the paraglycogen granules and lipoid vacuoles, at least three types of vacuoles may be observed. Peculiar topographic relationship connects mitochondria and paraglycogen granules probably in formation. In the nucleus with classical membrane and heterogeneous structure, a rather voluminous nucleolus may be seen.

termined using Student's t-test. Ascaris suum larval extract damaged peritoneal leucocytes at a concentration range of 1 to 100 mg protein/1 x 106 cells (Table I). Leucocyte suspensions were assayed for lysophospholipase activity by a modification of the method of Ottolenghi (1964, J. Lipid Res. 5: 532537). Tubes containing 0.1 ml of each cell mixture, after incubation with extract, were mixed with 0.5 ml of 12.5% glycerol medium and equilibrated in a 37 C water bath for 4 min. An 0.3ml lysophosphatidylcholine solution (2 x 10-2 M) prewarmed to 37 C, was added to each test tube and allowed to react for 60 min. The reactions were stopped by adding 1.0 ml of 2 N sulfuric acid, 1.0 ml of isopropyl alcohol, and 0.4 ml of water to each test tube. Each sample then received 2.0 ml of heptane, was stirred, the heptane decanted, and free fatty acids in the heptane were quantitated by titration with 0.01 N sodium hydroxide. The amount of lysophospholipase activity in the leucocyte mixtures was directly related to the e t's t-test. Ascaris suum laramount of A. suum larval extract used to stimulate the cell mixture (Table I). Stimulation of lysophospholipase activity was lost when concentrations less than 1 x 10-5 mg A. suum protein were used as stimuli (P > 0.01). Nonviable peritoneal cells also had minimal lysophospholipase activity suggesting that cell viability has an effect on lysophospholipase activity. Mechanisms associated with in vitro lysophospholipase activity have not previously been studied using helminth extracts as stimulators of enzyme activity. The results of this study showed that leucocyte lysophospholipase activation was affected by the concentration of the stimulating agent and the viability of leucocytes. It is not certain if the i:icrease in lysophospholipase activity was the result of leucocyte plasma membrane alterations or to lysosomal degranulation, or if the enzymatic activity was specific for the A. suum extract. This investigation was supported by Grant #81 04-008 from the American Osteopathic Foundation.

International audience; Incubation of mouse thymocytes with 10 μM monensin for 1 hour induces morphological alterations characterized by the extensive dilatation and vacuolization of the Golgi complex. This effect is used to study the transport and utilization of labelled sugar nucleotides into intracellular vesicles by using thymocytes whose plasma membrane has been permeabilized by ammonium chloride treatment. It is demonstrated that monensin stimulates the incorporation of labelled sialyl, fucosyl, galactosyl, and N-acetylglucosaminyl residues. This enhanced incorporation is not due to a direct effect of monensin on glycosyltransferase activities themselves but is a consequence of a higher entry and accumulation of labelled sugar nucleotides in the dilated vesicles.

The main cellular sources of interleukin-5 (IL-5) are T lymphocytes and mast cells. Recently, IL-5 mRNA has been identified in eosinophils from patients with celiac disease, eosinophilic heart diseases, and asthma. In an attempt to determine whether IL-5 is generated by eosinophils in atopic dermatitis we have used i) in situ hybridization with 35S-labeled IL-5 RNA probe combined with immunohistochemistry using a monoclonal antibody (MoAb) (EG2) directed against the activated form of Eosinophil Cationic Protein (ECP) and ii) double-immunostaining with anti-IL-5 MoAb and polyclonal anti-ECP antibody. We found that dermal eosinophils from lesional atopic dermatitis skin express IL-5 mRNA and protein. Moreover, highly purified blood eosinophils were also labeled with anti-IL-5 antibodies. The expression of IL-5 by eosinophils in atopic dermatitis might suggest an autocrine pathway of eosinophil differentiation and activation.