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.

Purification, characterization and activities of two hemolytic and antibacterial proteins from coelomic fluid of the annelid Eisenia fetida andrei.

The coelomic fluid of the earthworm Eisenia fetida andrei exhibits antibacterial, hemolytic and hemagglutinating activities. These activities are mainly mediated by two proteins, named fetidins, of apparent molecular mass 40 kDa and 45 kDa, respectively. For the first time, the two proteins have been purified to homogeneity from dialysed coelomic fluid by means of anion-exchange chromatography. Three peaks had hemolytic activity. The first fraction was found to correspond to the 40 kDa fetidin, the second to mixed 40 and 45 kDa fetidins, the last one to the 45 kDa fetidin. Both purified proteins still exhibited their hemolytic and antibacterial activities as dialysed coelomic fluid did. In this study, the amino-acid sequence of purified proteins is compared to the amino-acid sequence predicted by cDNA. This cDNA was isolated by screening an expression cDNA library from earthworm total tissues (unpublished data).

In vitro generation of reactive oxygen species by free coelomic cells of the annelid Eisenia fetida andrei: an analysis by chemiluminescence and nitro blue tetrazolium reduction.

Coelomocytes of the earthworm Eisenia fetida andrei were activated in vitro with various stimulants in order to investigate their capacity to produce reactive oxygen species. Analysis by luminol-enhanced chemiluminescence and nitro blue tetrazolium reduction suggests the production in vitro of reactive oxygen species by both categories of free coelomocytes, leucocytes and chloragocytes, while affecting different modalities: a respiratory burst-like reaction is exhibited by leucocytes in the presence of zymosan but not bacteria; a moderate production of reactive oxygen species spontaneously occurs when chloragocytes aggregate together. A substantial reduction of reactive oxygen species production, rapidly occurring after addition of exogenous superoxide dismutase, was interpreted as indicating a prominent role of the superoxide anion, O2.-. The factors modulating the coelomocyte generation of reactive oxygen species, as well as the presence of lipofuscin in brown bodies, support the opinion that production of reactive oxygen species may be an immune defense process occurring under in vivo conditions.

Antibacterial activity of Eisenia fetida andrei coelomic fluid: III--Relationship within the polymorphic hemolysins.

The antibacterial activity exhibited by 10 different hemolytic, genetic families was established by measuring the inhibition of spontaneous in vitro growth by cell-free coelomic fluid toward 2 bacteria which are pathogenic for the earthworm: Bacillus megaterium (Gram +) and Aeromonas hydrophila (Gram -). Only two families (B and K) displayed potent inhibitory activities. This finding is consistent with the fact that the B family occurs most frequently in both natural as well as in industrial breedings. Nevertheless, evidence of a poor antibacterial defense in some frequent families suggests the existence of alternative antibacterial mechanisms.

Formation of brown bodies in the coelomic cavity of the earthworm Eisenia fetida andrei and attendant changes in shape and adhesive capacity of constitutive cells.

The formation of brown bodies in the coelomic cavity may result from an aggregation of coelomocytes around offending foreign cells such as bacteria, gregarines, incompatible graft fragments, and altered self structures such as setae or necrotic muscle cells. The initial nodule rapidly increases in volume by aggregation of new coelomocytes and various waste particles. When a brown body has reached a diameter of 1-2 mm, its external cells flatten and lose their adhesiveness toward free coelomocytes or waste particles and its pigment rapidly darkens. Brown bodies play an important role in homeostasis.

Protein analysis of earthworm coelomic fluid. III. Isolation and characterization of several bacteriostatic molecules from Eisenia fetida andrei.

A bacteriostatic activity in Eisenia fetida andrei cell free coelomic fluid is described. This activity is detected by growth inhibition of a bacteria Bacillus megaterium. Gel filtration analysis revealed eleven coelomic fluid protein fractions designated A, B,..J. Antibacterial activity was mainly found within fractions B and C. Chromatofocusing resolved fractions B-C into five different peaks named alpha BC, beta BC,... epsilon BC. Antibacterial activity appeared mediated by three different proteins characterized by their molecular weights (20,000, 40,000 and 45,000) and their isoelectric points (4.9, 5.75 and 6.0). These bacteriostatic proteins possess either hemolysis or hemagglutination activities. The polymorphic aspect of this humoral antibacterial defense is discussed.

[Traumatic rupture of the diaphragm. Clinical experience (author's transl)]. / Traumatische rupturen van het diaphragma. Klinische ervaringen

The increased number of automobile casualties increases the frequency of traumatic rupture of the diaphragm. Because of associated serious lesions, diagnosis is easily overlooked. The authors report on 11 cases and give an appraisal of the literature. In 6 patients, diagnosis was made late. The authors discuss the physiopathology, symptoms, clinical data and means of diagnosis. Roentgenography is very important. Surgical cure is always recommended because of the danger of delayed strangulation of the hernia.

[In vitro study of the cytotoxic activity of sipunculid leukocytes towards allogeneic and xenogenic erythrocytes]. / Etude in vitro de l'activité cytotoxique des leucocytes de siponcles à l'encontre d'érythrocytes allogéniques et xénogéniques

A cell mediated cytotoxic reaction analogous to that known in Vertebrates is induced in vitro by leukocytes of sipunculid worms (coelomata invertebrates). Xenogenic or allogenic erythrocytes are employed as target cells. A cytotoxic effect always occurs when killer leukocytes react against xenogenic cells. A cytotoxic activity against allogenic erythrocytes occurs in all cases when donors of leukocytes and donors of erythrocytes arise from stations far apart (Roscoff and Arcachon). Between stations near each other (4 miles apart) a cytotoxic effect is noticed only in one case out of three. No reaction is noticed between Sipunculus nudus from the same station.

[Evidence for polypeptide synthesis during cytotoxic reactions in sipunculid worms]. / Mise en évidence d'une synthèse polypeptidique au cours de la réaction de cytotoxicité chez les Siponcles.

The occurence of a polypeptidic synthesis during the in vitro cytotoxic reaction of sipunculid worns is demonstrated. Some drugs such as actinomycin D, cycloheximide, cytochalasin B, Colchicine, vinblastine and iodoacetate have been tested. Hypothesis are expressed on the use of the products synthetized by leukocytes.

Fine structure and functions of haemerythrocytes and leucocytes of Sipunculus nudus.

The marine sipunculan worm, Sipunculus nudus Linné, possesses a voluminous coelomic cavity filled with a fluid containing different types of free cells. Most of them (more than 90%) are nucleated haemerythrocytes. They arise from free, small haemerythroblastic cells which are described in this report. Different kinds of leucocytes are also found in the coelomic fluid, particularly granulocytes which are phagocytic cells showing some similarities with vertebrate macrophages. These cells seem to arise from small hyalocytes, which are cells having morphological and physiological resemblances with vertebrate lymphocytes. It appears that the older granulocytes are progressively degranulated and give rise to large hyalocytes. Ciliary cells arising from small cells, very similar to small hyalocytes, have also been observed and it is supposed that they give rise to urn cells. Lastly, some of the biological functions of the free coelomic cells of the sipunculan worms are described.

Invertebrate immunity: another viewpoint.

All vertebrates and invertebrates manifest self/non-self recognition. Any attempt to answer the question of adaptive significance of recognition must take into account the universality of receptor-mediated responses. These may take two forms: (1) rearranging, clonally distributed antigen-specific receptors that distinguish in the broadest sense between self and non-self, and non-self A from non-self B, latecomers on the evolutionary scene; (2) pattern recognition receptors, the earliest to evolve and still around, necessitating the requirement for induced second signals in T- and B-cell activation. Either strategy need not force upon invertebrates the organization, structure and adaptive functions of vertebrate immune systems. Thus, we can freely delve into the unique aspects of the primitive immune mechanisms of invertebrates. In contrast, using the opposite strategy which is still problematic, i.e. linking invertebrate and vertebrate defence, seems to give us an approach to universality that might eventually reveal homologous kinship.

Sequence and expression of an Eisenia-fetida-derived cDNA clone that encodes the 40-kDa fetidin antibacterial protein.

Fetidins are 40-kDa and 45-kDa hemolytic and antibacterial glycoproteins present in the coelomic fluid of the earthworm Eisenia fetida andrei. By screening a cDNA library with a polyclonal antifetidin serum, we have cloned a cDNA that encoded the 40-kDa fetidin. The clone contains an insert of 1.44 kb encoding a protein of 34 kDa, which corresponds to the size of deglycosylated fetidins. The recombinant protein inhibits Bacillus megaterium growth. Restriction fragment polymorphisms were observed on Southern blots and correspond to a known protein polymorphism. The sequence of the cDNA contains a peroxidase signature and fetidins from earthworm coelomic fluid have peroxidase activity. The 40-kDa and 45-kDa fetidins therefore represent two related polymorphic defence factors in invertebrates.

Treatment of achalasia with pneumatic dilatations.

During the last 13 years 264 unselected patients with achalasia have been treated solely by the technique of pneumatic dilatation. The principle of the method was to continue the series of consecutive dilatations until the result was satisfactory. This paper reports the late results in 138 patients who were treated more than three years ago (3-13 years; mean 6.6 years). The results were evaluated by somebody who had not before been involved in the treatment of achalasia. The evaluation was based on a personal interview, a standardized radiological examination, and intraluminal pressure measurements. These late results were classified as excellent in 45%, good in 32%, moderate in 17%, and poor in 6%. The best results were obtained in patients with a history of five to twenty years and a moderately dilated oesophagus (50-80 mm.). Comparison of the clinical results with the manometric data obtained before, immediately after, and late after treatment indicates that intraluminal pressure measurements may be a useful guide for the treatment of achalasia by pneumatic dilatations.