CH642682A5 - Peptide substance obtained from a trematode and its use as a medicinal product - Google Patents

Description

The present invention relates to a new peptide obtained from schistosome incubates.

It specifically relates to a peptide originating from Schistosoma monsoni, of molecular weight ranging between 500 and 1000, soluble in water, thermostable, not precipitable by the reagents of proteins, dialyzing through a membrane of cross-linked dextran of the type Amicon X- 100.

According to the invention, this peptide is obtained by a process characterized in that adult Schistosoma monsoni is incubated in water, separates the bodies of the nematodes and the particulate debris from the liquid medium, subjects the liquid medium to a screw dialysis -with water, collects the dialyzed fraction and lyophilizes said fraction.

According to an advantageous embodiment of the method, the peptide is obtained by incubating Schistosoma monsoni in double-distilled water, decanting then centrifuging, then the supernatant is filtered on a Millipore filter and put into dialysis in a dialysis cell equipped with '' an Amicon X-100 filter; finally, the dialysis liquid is lyophilized, then concentrated 10 times.

This protein has very interesting pharmacological properties which are manifested by inhibiting the degranulation of the mas-tocytes of rats. This inhibition of degranulation suggests that this biological substance can inhibit the release of histamine and catecholamines contained in mast cells and thus avoid the phenomena of bronchospasm, manifestations of anaphylaxis or the risks of syncope in the states of allergy or shock, hives and digestive allergies.

Activation of mast cells by allergenic factors, such as antibodies or chemicals, leads to the release of vasoactive biological amines and can therefore be made responsible for immediate hypersensitivity or bronchoconstriction phenomena specific to allergy. The peptide substance according to the invention makes it possible to envisage the treatment of allergy manifestations by depression of the immunity reactions of vasomotor substances associated with the appearance of allergy phenomena.

The peptide substance according to the invention can be administered to humans in the form of pharmaceutical compositions in combination or in mixture with an inert, non-toxic, pharmaceutically acceptable excipient or vehicle. The peptide substance can be administered parenterally, perlingually, permucosally or rectally.

Schistosome incubation products

Ten thousand adult worms obtained by infusion of hamsters infected from 40 d are incubated in 10 ml of double-distilled water at 30 ° C for 3 h. After decantation, the supernatant obtained constitutes the incubate. This is centrifuged for 10 min at 1000 g to remove particulate debris, filtered on a 0.45 jim Millipore filter and put into dialysis for 24 h at 4 ° C. The dialysis liquid and the non-dialyzable fraction are lyophilized and concentrated 10 times when in use.

Pharmacological study of the new peptide Passive skin anaphylaxis techniques (PCA)

A - Principle

This technique consists in passively sensitizing the animal by intradermal injection of the serum. The simultaneous injection of Ag and dye (after a variable delay taking into account the tissue-milk half-life of IgG and IgE) makes it possible to appreciate at the level of the intradermal injection point the importance of the anaphylactic reaction by the appearance of a colored spot, of area proportional to the dose of Ag injected [Ovary, “Progr. Allergy ”, (1958), 459]. The PCA titer of a serum corresponds to the greatest dilution giving a blue spot of at least 5 mm. We used this technique, on the one hand, to evaluate the reagin titer of antiovalbumin sera, on the other hand, to study the inhibitory effect of the schistosome incubation products on this reaction.

B - Titration of sera

These reactions were carried out in male Wistar rats (180-200 g). 0.1 ml of the various dilutions of the serum to be assayed is injected intradermally into the shaved back of the recipient rat. After a latency period of 48 to 72 h, 5 mg of ovalbumin with 4 mg of Evans blue in solution in 0.5 ml of physiological water are injected into the caudal vein. 30 min later, the rats are sacrificed and the reactions are examined on the internal side of the skin. C - Inhibition of the passive cutaneous anaphylaxis reaction

The dialysable fraction of the schistosome incubation products (100 µl) is injected intradermally 15 min before the intravenous injection of Evans blue. The controls consist of an injection of physiological water.

1. Action of chemical compounds

Substances having a degranulating action on mast cells in vitro can also induce a positive reaction in vivo. Two substances were used: compound 48/80 (condensation product of p-methoxyphenylethylmethylamine with formaldehyde) at a concentration of 1 and 0.1 Hg / ml, and polymyxin-B-sulfate at a concentration of 10 | ig / ml. These substances (100 nl) with immediate action must be injected intradermally at the same time as the intravenous injection of 4 mg of Evans blue in solution in 0.5 ml of physiological water.

2. Inhibition of degranulation induced by an antibody-antigen system

100 μl of reaginic serum (antiovalbumin or rat serum infected with S. monsoni) are injected intradermally and the injection points are carefully identified. 48 h later, 100 μl of dialysate or physiological water are injected at the same sensitization points. Then, 15 min later, we inject Evans blue (4 mg) and ovalbumin (5 mg) or the water-soluble Ag of 5. 1% monsoni (2 mg) dissolved in 5001 * 1 of water physiological. (Ag: water-soluble Schistosoma antigen.)

Cell culture techniques A - Culture of lymphocytes

1. Recovery of cells

The inhibiting action of the dialysate has been tested against lymphocy5

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your female CBA mouse spleen (CNRS, Orléans). Mice are sacrificed by cervical dislocation. The spleen is removed sterile and dilated by means of curved forceps in RPMI medium supplemented extemporaneously with glutamine (2 nmol) and tripeptide Gly-His-Lys acetate (Eurobio) (20 | ig / ml) [Pickart et al., « Biochem. Biophys. Res. Commun. ”, 54 (1973), 562], In order to obtain better stimulation, the adherent cells are eliminated. The cells are placed in petri dishes at 37 ° C for 2 h so as to separate the adherent cells. The supernatant is removed and centrifuged. The cells are counted in a Thomas hemocytometer by an exclusion test of Trypan blue in 0.02% solution in Veronal buffer pH 8.7. The suspension is adjusted to a concentration of 107 lymphocytes / ml.

2. Mitogens

The mitogens used to induce lymphocyte stimulation are concanavalin A extracted from Concanavalia ensiformis at doses of 0.5 and 1 pg / 100 ni and phytohemaglutinin extracted from Phaseolus vulgaris (Wellcome) diluted to 1: 100.

3. Culture

The culture is carried out in microtitration plates (Nunclon, Denmark). The cells are distributed in each well at the rate of 0.5-106 cells / well. The dialyzable fraction of the adult worms incubate is added in a volume of 10 µl. The mitogens are also added, the final volume being in each well of 200 μl.

48 h later, 1 nCi of 3H tritiated Thymidine is added to each well. The incorporation of thymidine is measured after a night of contact. The cells are washed, then lysed with IN sodium hydroxide solution for 10 min at 56 ° C. The acid-precipitable material is obtained after the addition of 2 ml of cold trichloroacetic acid. After incubation for 15 min at 4 ° C and centrifugation, the precipitate is dissolved in IN sodium hydroxide solution and the samples are placed in counting flasks. The scintillating liquid used is Picofluor15 (Hewlett-Packard). The samples are counted in a Nuclear Chicago scintillation spectometer (Isocap 300).

B - Cultures of cells in continuous line

The lines used for the cytotoxicity tests are: a human monocytic line J111 (American culture type collection) (CCL 24) and a Hela line. The cells are cultured in Petri dishes in MEM (Minimum essential medium) added with 15% of fetal calf serum inactivated by heating. After 24 h of culture in the presence of dialysate, the cells are resuspended by the action of versene-trypsin and centrifuged. The pellet is taken up in MEM and counted. Cell viability is also measured by exclusion test for Trypan blue (0.02%).

The incorporation of thymidine is measured in the same way as for lymphocyte cultures.

Mast-eosinophil-dependent cytotoxicity

A - Preparation of schistosomules

The schistosomules are harvested in vitro after passage of the cercai-res through the abdominal skin isolated from Swiss mice.

B - Culture medium

The culture medium used is MEM: Eagle's minimum essential medium (production Pasteur Institute, Paris) to which 1% of calf serum inactivated by heating and 20 ng / ml of tripeptide glycyl / histidyl / lysine (Calbiochem., San Diego) are added. , Ca.).

C - Preparation of cells

10 ml of physiological water are injected intraperitoneally into male Fischer rats (Iffa Credo, l'Arbresle, France). 48 to 72 h later, the cells are removed by washing the peritoneal cavity with 20 ml of culture medium containing 25 IU / ml of calcium hepararate. The peritoneal cells are centrifuged at

1800 rpm and washed twice in the culture medium. This population is enriched in eosinophils by allowing the cells to adhere in Petri dishes (7-106 cells / ml in dishes 3 cm in diameter). After 2 h, non-adherent cells are removed and counted. The population obtained contains approximately 45% of eosino-philes and 10% of mast cells.

D - Purification of cells

To eliminate contamination due to mast cells, the cell population obtained is deposited on a 35% Ficoll solution (Pharmacia, Uppsala, Sweden). After centrifugation at 1000 g for 15 min, the interface cells are removed and washed in the medium. This population contains approximately 50% eosinophils and less than 1% mast cells.

The mast cell purification technique will be described later.

E - Cytotoxicity test

50 schistosomules suspended in 100 μl of culture medium are deposited in the cells of a microtiter plate (Nunclon) and are incubated overnight with 100 μl of rat serum infected with Schistosoma monsoni, inactivated by heating, at a final dilution 1:16. The schistosomules are then washed with the culture medium and resuspended in 100 µl of medium. 100 μl of the effector cell suspension are added so as to obtain a ratio of 4000-6000 effector cells for a schistosomule. The percentage of cytotoxicity is determined by microscopic observation after an incubation of 48 h.

Degranulation test

A - Obtaining rat peritoneal cells

The cells used for the test are obtained by washing the peritoneal cavity of male Wistar rats (180-200 g) (Janvier, Le Genest, 200 g) with 20 ml of Eagle medium (MEM) (production Institut Pasteur, Paris) . To this medium were added beforehand glutamine (2 mmol), calcium heparinate 50 U / ml and the tripeptide Gly-His-Lys acetate 20 ng / ml- The peritoneal exudate contains on average 10th mast cells, this which represents 2 to 5% of the total cells. The cells are centrifuged for 5 min at 1000 g. The mast cells are stained with toluidine blue and counted in a Fuchs Rosenthal hemocytometer.

B - Incorporation of 3H serotonin

A solution of 5-hydroxy- [3H] -tryptaminecreatininesulfate (10 Ci / mmol) (Amersham) was used for labeling the cells. The peritoneal exudate is incubated for 30 min at 37 ° C. with 2 nCi of 3H serotonin per 106 mast cells. The cell suspension is then washed 4 times in 5 ml of medium to remove the excess of labeled serotonin.

C - Measurement of serotonin release

The reaction is carried out in microtiter plates with flat bottom (Linbro, Flow Lab.). The different activating or inhibiting tested substances are brought into contact with 50 μl of the cell suspension at 106 mast cells / ml. After a variable incubation time depending on the case studied, the plate is centrifuged for 5 min at 1000 g. 50 µl of supernatant are transferred to a counting flask. Treatment of the cells with 50 μl of digestin (Merck) makes it possible to measure the total amount of radioactivity incorporated into the cells.

D - Measurement of radioactivity

10 ml of scintillating liquid (toluene Triton 1: 1 (v / v) butyl PBD 7 g / 1) are added to each counting flask. Radioactivity is measured in a scintillation spectrometer (Isocap 300,

Nuclear Chicago, Illinois) with an efficiency of 50.1% for 3H. Fluorescence quenching (quenching) is corrected by the method

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channel reports, from standard curves obtained with samples of constant activity.

E - Products used

Compound 48/80, polymyxin-B-sulfate, ATP, a-chymo-trypsin, La-lysophosphatidylcholine, dicoumarol and papa-verine, sodium cromoglycate and ovalbumin are commercial products .

F - Morphological control of degranulation

50 dj.1 of 0.1% toluidine blue (I.C.N. Laboratories, Cleveland) are added to the activated cells. The percentage of degranulated mast cells is determined by microscopic observation with a magnification of 40 × 10 (Reichert model, Austria). For each determination, at least three hundred cells were observed.

G - Ultrastructural study

The cells are fixed with a 2.5% glutaraldehyde solution in 0.1 M sodium cacodylate buffer, pH 7.2 at 4 ° C., then washed in cacodylate buffer. The cells are postfixed with 1% osmium tetroxide in distilled water for 1 h. Dehydration with acetone followed by inclusion in Araldite is then carried out. The observation is made with a Hitachi HU 12 electron microscope.

H - Purification of mast cells

The technique used has been described by Sullivan et al. in "J. Immunol. ”, 114 (1975), 1480. The cells are deposited on a solution of bovine serum albumin (BSA) 38% (weight / volume).

After 30 min at room temperature, the cells are centrifuged for 20 min at 450 g. The mast cells are then in the SAB layer. The supernatant and the interface are removed and the lower layer is washed several times with the culture medium. The mast cell preparation obtained is 99% pure with a yield of approximately 75%. Microscopic observation confirms the structural integrity of the purified mast cells.

Results and discussion

I— Development of the degranulation test A - Incorporation of serotonin by the cell suspension

1. Localization of marked serotonin in mast cells

Only mast cells and platelets have the property of incorporating exogenous serotonin. Platelets in small proportion in the peritoneal exudate are eliminated by the first washing of the cells, this being confirmed during microscopic observation. However, to demonstrate the localization of labeled serotonin in the mast cells, a preparation of purified mast cells was compared to the total peritoneal population. The peritoneal exudate is marked with tritiated serotonin, then an aliquot is taken for purification. At an equal mast cell concentration, the total amount of radioactivity incorporated is the same in the preparation containing 5% mast cells and in that containing 95% mast cells. The serotonin release curve under the action of compound 48/80 is strictly identical for the two preparations (FIG. 1). These experimental facts demonstrate the selective incorporation of serotonin in the mast cells. This reinforces the interest of the method by allowing the use of unpurified populations.

2. Structural integrity after marking

Microscopic observation as well as study by electron microscopy show that labeling with tritiated serotonin does not affect the structural integrity of membranes and mast cells.

B - Action of compound 48/80

This compound was chosen for its ability to degranulate mast cells with a large amplitude, which facilitates morphological observation and optical determination of the percentage of degranulated mast cells.

The cells are incubated for 15 min at 37 ° C. with concentrations of compound 48/80 ranging from 1 mg to 0.1 (μg / ml. On the one hand, the release of tritiated serotonin is measured, on the other hand, the percentage of degranulated mast cells obtained by microscopic observation.

The spontaneous release of tritiated serotonin represents 1 to 5% of the total radioactivity incorporated into mast cells. Each concentration is tested in triplicate and the percentage of serotonin release is calculated according to the following formula:

% = ^ 5 | xl00

X: total radioactivity of the sample S: radioactivity released spontaneously

T: total incorporated radioactivity (released after digestin action)

Compound 48/80 exhibits activity from a concentration of 1 µg / ml. The dose-response curve has a sigmoid appearance. The maximum activity corresponding to 75-86% of serotonin released is obtained for a concentration of 100 ng / ml of compound 48/80.

A close parallelism is established between the results obtained by the isotopic method and microscopic observation. The correlation study gives a coefficient r = 0.9935 (p <0.001).

C - Action of other chemical activators

Several compounds having the property of inducing mast cell degranulation were tested under the same conditions as compound 48/80. Polymyxin-B-sulfate, a-chymotrypsin, L-a-lysophosphatidylcholine and ATP induce a release of serotonin. The dose-response curves are consistent with those observed by conventional methods. The doses necessary to induce degranulation are greater than with compound 48/80. Polymyxin-B-sulfate induces release of serotonin from 10 Hg / ml, chymotrypsin 100 ng / ml and ATP 500 | ig / ml. With ATP, the percentage of serotonin released remains limited to 20% in the range of concentrations studied.

D - Inhibition of degranulation by chemical agents

Two types of degranulation inhibitors have been described. The first type of inhibitor inhibits the membrane activation step. These are substances which intervene at the level of cyclic nucleotides by causing an increase in intracellular cAMP by inhibition of phosphodiesterase. It is by this mechanism that act for example dicoumarol or sodium cromoglycate.

The second type of inhibitor acts directly at the level of intracellular metabolism. This is the case, for example, with papaverine.

We tested the effect of dicoumarol and papaverine on a de-granulation induced by polymyxin-B. The cells are first incubated for 30 min at 37 ° C. with the inhibitors; degranulation is induced by action of polymyxin-B-sulfate (100 (ig / ml) 15 min at 37 ° C. With a dose of 100 | imol / l of papaverine, the inhibition is 75%; with 10 p. mol / 1 of dicoumarol, it is 89%, so these compounds are active on mast cell degranulation and the release of tritiated serotonin.

E - Degranulation induced by an antigen-antibody reaction

The interaction of mast cells with an allergen and the corresponding reactin Ac induces degranulation, but cellular alterations are much less important than under the action of chemical compounds, which reinforces the interest of a more precise method than microscopic observation. We therefore sought the optimal conditions for degranulation induced by an anti-albumin reagent serum and the corresponding antigen.

1. Finding the optimal awareness time

The binding of IgE to the surface of the mast cell requires a contact time before degranulation can be induced by addi-

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tion of the antigen. We therefore sought the optimal time necessary to obtain mast cell sensitization.

The labeled mast cells are incubated with the antiovalbumin antiserum for variable periods of 15 min to 2 h, then brought into contact with the antigen for 15 min. Neither the antigen nor the antiserum induces serotonin release greater than the control. When the antigen is added simultaneously to the antiserum, no degranulation is induced even if the incubation times are extended, which makes the sensitization step absolutely necessary. However, it can be short-lived since the maximum release of serotonin is obtained with a contact time of 15 min.

2. Influence of the antigen concentration

We have studied a range of concentrations from 1 to 1000 (xg / ml, ie in final concentrations of 0.05 to 50 µg. The cells are brought into contact for 15 min with the different concentrations of antigen. In the range of concentrations tested, the release of serotonin increases with the antigen concentration, so for all the experiments, we used the highest concentration, 50 µg per well.

3. Sensitivity of the method

The sera of Hooded-Lister rats immunized against ovalbu-mine have PCA titers varying from 64 to 512. A serum of title 128 was tested at different dilutions. We can induce a release of serotonin with this serum up to a dilution of 54, but in this case only 5% of the incorporated serotonin is found in the supernatant. Pure serum induces the release of 20% of serotonin, which remains low and requires the use of undiluted serum for all experiments. The sera used for passive sensitization must have a PCA titer greater than 128. This limit is not due to the test itself, but to the difficulty of passively sensitizing mast cells in vitro.

4. Characteristics of anaphylactic degranulation

Two properties of IgE were used to verify that the release of serotonin follows the interaction of IgE with mast cell receptors: these are the thermolability of IgE and the high affinity of IgE for the mast cell receptor.

Thermolability: the PCA 128 anti-albumin serum is heated for 3 h at 56 ° C. This pure serum is incubated for 15 min at 37 ° C. with labeled mast cells under the conditions of passive sensitization. After addition of antigen, there is no release of serotonin whereas the same unheated serum induces a release of 21.9% of tritiated serotonin.

Table 1:

Influence of antigen concentration on serotonin release

Ovalbumin concentration (ng / ml) 1

% serotonin released2

1000

45.8 ± 7.3

100

14.9 + 3.8

10

5.6 ± 1.4

1

NS3

150 μl of the cell suspension are incubated for 15 min at 37 ° C. with 50 μl of an antiovalbunin serum (PCA titer 1: 512), then 50 μl of each of the solutions of ovalbumin indicated are added. The release of serotonin is measured after 15 min of incubation at 37 ° C.

2 Average of three experiences.

3 Not significantly different from spontaneous release.

The destruction of IgE by heating completely inhibits subsequent degranulation, which confirms that it is a phenomenon dependent on IgE attached to the surface of the mast cell.

Affinity of IgE: IgE has a strong affinity for the mast cell receptor. The binding of IgE is irreversible by washing the cells. It is therefore possible, after passive sensitization, to wash the cells and induce degranulation with the antigen.

The labeled cells are incubated with an PCV 128 anti-albumin serum for 15 min at 37 ° C. and are then washed 3 times in the culture medium. 50 µg of antigen are then added. The release of serotonin is measured after 15 min of incubation (Table 3). Without washing step, this serum induces a release of 23.7%. After 3 washes between sensitization with IgE and the addition of the antigen, 20.2% of the serotonin is released; the sensitization of the mast cells is therefore preserved by washing, which confirms the interaction of IgE with the mast cell.

F - Discussion

This method makes it possible to appreciate quantitatively the importance of degranulation induced, on the one hand, by chemical activators, on the other hand, by an anaphylactic system.

The rapid incorporation of the labeled serotonin makes it possible to avoid alterations of the cellular structure. The electron microscopy study allowed us to confirm the structural integrity of the cells.

After 3 washes, all excess serotonin is eliminated. The spontaneous release of serotonin by mast cells corresponds to approximately 1-5% of the incorporated serotonin [Bach et al., “J. exp. Med. ”, 133 (1971A), 752, obtain, by assaying histamine, a spontaneous release of 0 to 5%]. The background noise measured is therefore completely normal and confirms that the experimental conditions used to mark the cells respect their integrity and do not disturb their subsequent activation.

The selective incorporation of the marker at the mast cell level makes it possible to avoid cell purification, at least in the case where interactions with other cell types do not interfere with the release of mediators. This possibility of avoiding the cell purification step represents both a saving of time and a guarantee of better state of the cells. Measuring the release of serotonin seems to be a good technique for measuring degranulation.

The destruction of IgE by heating completely abolishes the release of serotonin. On the other hand, it is maintained if the cells are washed after passive sensitization by the reagent serum. These two experimental facts confirm that it is an anaphylactic degranu-lation whose signal is the interaction of IgE with its receptor on the mast cell. The inhibition of degranulation by sodium cromoglycate confirms that the measurement of the release of serotonin is in our conditions a good method of evaluation of degranulation, both in active anaphylaxis and after passive sensitization.

II— Production by Schistosoma mansoni of a factor inhibiting mast cell degranulation

A - Inhibition of degranulation in vitro

1. Inhibition of degranulation induced by an anaphylactic system

Serotonin-labeled rat peritoneal mast cells are incubated with the dialyzable and non-dialyzable fraction of the incubate at different concentrations. After 45 min of contact, degranulation is induced by the action of an anti-albumin and ovalbumin reactin serum under the usual conditions. Doses of dialysate of 10-50 (they are tested. On the one hand, the release of serotonin in the presence of dialysate alone is measured so as to control the cytotoxic effect of this substance, on the other hand, the release of serotonin after activation by the anaphylactic system. At low concentrations (10-30 (il), the dialysable fraction of the incubate significantly inhibits mast cell degranulation. This inhibition is dose-dependent since 54% is obtained with 10 (il and 70% with 20 (il. With 30 | il the inhibition is 49%, but a spontaneous release of serotonin begins to appear at this dose, which can explain the weakest inhibition obtained with this concentration. At the highest concentrations (40 and 50 days 1), the spontaneous release of serotonin increases considerably, and is almost complete with s

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50 (il of dialysate (87%), which corresponds not to an active process, but most certainly to a cytotoxicity of the dialysate at these concentrations. The non-dialysable fraction does not exhibit any inhibiting action.

2. Inhibition of degranulation induced by chemical agents

It was interesting to see if this substance has the property of very generally inhibiting the degranulation process, or if this inhibition is specific to a degranulation induced by antigen-antibody reactions. For this, we put labeled mast cells into the incubate, then we induced degranulation by the action of compound 48/80 or polymyxin-B-sulfate.

The doses of these compounds used to degranulate mast cells are sub-optimal doses, so as to limit cell lysis which results from explosive degranulation. With compound 48/80 (5 (μg / ml), the percentage of serotonin release is 17.1%. Preincubation with 10 μl of dialyzable fraction reduces this percentage to 15.1%; with 20 μl, obtains 10.4%, the inhibition obtained with 20 | it is 39%, although statistically significant (p <0.05), it nevertheless remains limited.

With polymyxin-B-sulfate (5 (ig / ml), 29.8% is obtained. By preincubating with 10 (11 of dialysate, 19.5%, with 20 (13.5%, which corresponds respectively at 35 and 55% inhibition, it is therefore easier to inhibit degranulation induced by po-lymyxin-B- than by compound 48/80.

3. Characteristics of the inhibitory factor a) Thermostability

The dialyzable fraction of the adult worms incubate is heated for 1 hour at 100 ° C. and then brought into contact with the labeled mast cells. The inhibition of anaphylactic degranulation obtained with the heated and unheated fractions is compared. Obtained, with 10 dj.1 of dialysate, respectively 54 and 56% inhibition. It is therefore a thermostable compound.

b) Irreversible nature of inhibition by washing

It was investigated whether the inhibition of degranulation could be removed by washing the cells after they have been brought into contact with the inhibiting fraction.

The labeled cells are incubated for 45 min with 10 μl of dialysate and then washed 3 times in the culture medium. Degranulation is induced either by the action of polymyxin-B-sulfate, or by the action of an anti-albumin and ovalbumin serum.

With the anaphylactic system, 30.3% serotonin release is obtained. With 10 (11 of dialysate, 13.3% is obtained if there is no washing step and 19.5% when the cells are washed between incubation with the dialysate and passive sensitization with the serum It is therefore impossible to restore the normal response by these washes.

With polymyxin-B-sulfate (10 µg / ml), degranulation is 23.9%. By preincubating with the dialysate, 19.9% is obtained without washing; with the washing step, 22.0% is obtained for the control and 12.7% with the dialysate, which corresponds to 34 and 42% inhibition. The same result is obtained as above with the anaphylactic reaction. The dialysate appears to induce an irreversible inhibitory signal even when it is then washed away.

c) Non-cytotoxicity of the protein substance according to the invention

The action of the dialyzable fraction of the worm incubate was

studied against lymphocytes stimulated by mitogens. This fraction was shown to inhibit lymphocyte proliferation. Although the cytotoxicity tests carried out on the lymphocyte cultures were negative, it was necessary to see if this fraction has a character of cellular toxin, that is to say if it induces a cytotoxicity or cytostaticity of the cells, whatever either the line chosen.

Two cell lines in continuous culture were studied. The first is the Hela line. These are cervical tumor cells with no particular immunological competence. The second is the human J111 monocytic line. The action of the dialysate has been studied on lymphocytes stimulated by mitogens as well as on these two cell lines. Cytotoxicity is measured by a Trypan blue exclusion test accompanied by a Thomas hemocytometer count. The growth inhibition is evaluated by measuring the incorporation of 3H thymidine (Thy).

The spleen lymphocytes of CBA mice are cultured according to the technique described above in the presence of 10 μl of dialysate and mitogens PHA (dilution 1/100) and Con A (1 (ig). The incorporation of 3 H is measured. Thy after 72 h of culture. The dose of 10 (il of dialysate proves to be extremely effective with respect to lymphocytes stimulated by mitogens. The inhibition is 96%, that is to say that the mitogenic stimulation is completely abolished The basic incorporation of thymidine in the presence or absence of dialysate is the same, which suggests that it is not a cytotoxic action.

Hela cells and monocytes are cultured according to the technique described above. The Trypan blue test makes it possible to evaluate the percentage of dead cells when these are not lysed. A toxic effect could lead to cell lysis, which is why it is necessary to also do a count in order to know the number of viable cells. With Hela cells, no difference in behavior is observed between the control cells and the cells cultured in the presence of dialysate. After 72 h of culture, the cells have multiplied and form a confluent carpet; contact inhibition limits cell proliferation, which explains the lower incorporation of thymidine at the end of culture (Table 2).

Table 2:

Incorporation of thymidine by stimulated lymphocytes in the presence of dialysate

Stimulation

Incorporation of 3H Thy by Mouse Lymphocytes

Witness cpm + ET1

Dialysate 10 (it cpm1 ± ET

-

15848 + 4463

15850 ± 2421

Con A 1 (ig

237643 + 8632 (IT2 = 15)

24896 ± 9235 (IT = 16)

PHA Yioo

206224 + 58231 (IT = 13)

22469 ± 3656 (IT = 14)

1 Number of strokes per minute (cpm) ± standard deviation (SD).

2 IT: transformation index.

(Table at the top of the next page)

The same result was obtained with the J111 monocytic line. Cell multiplication is slower, but within 48 hours an increase in the number of cells is observed, this increase being identical with and without dialysate.

At the doses commonly used to demonstrate inhibitory effects, the dialyzable fraction of the incubate has no toxic action on the cells, whatever their origin.

B - In vivo inhibition of degranulation

I. PCA induced by chemical agents

Compound 48/80 and polymyxin are capable of inducing a positive reaction in vivo when injected intradermally. The reaction is instantaneous, which is why the intravenous injection of Evans blue must be done simultaneously. The limiting dilution giving a positive reaction is determined for each of these compounds on a series of control rats. For compound 48/80, this is 0.1 µg / ml for the polymyxin 10 µg / ml.

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Table 3:

Action of the dialysate on the viability and growth of Hela cells

Time

Witness

Dialysate 10 | il

3H

Numeration1 3 H Thy (cpm + ET)

4.6- 10s ± 0.5 • 10s 23114 + 9477

4.9-105 + 0.9-105 30040 ± 11177

8H

Numeration 3 H Thy (cpm ± ET)

4.1 • 105 ± 1.0-105 17847 + 1675

3.0-105 + 0.4-105 19,629 + 977

24h

3H Thy number (cpm + ET)

7.7-105 + 0.4-105 22,719 + 651

5.6-105 + 0.5-10s 21873 + 1550

72H

3H Thy count (cpm ± ET)

21.6- 10s ± 3.8-105 5,726 + 386

22.0 -105 ± 3.0 -105 6,015 + 1,764

1 Thomas hemocytometer count after exclusion test for Trypan blue.

2 Incorporation of 3 H Thy expressed in counts per minute (cpm) ± standard deviation (SD).

The dialysate is injected 10 min before the induction of degranulation at the rate of 100 μl by injection, intradermally. Simultaneously, Evans blue is injected intravenously and 48/80 or polymyxin intradermally. The positive controls consist of a first injection of physiological water then a second of 48/80 or of polymyxin. After injection of the dialysate, a marked reduction in the intensity of the reaction is observed, whether with compound 48/80 or with polymyxin.

2. PCA induced by an anaphylactic reaction a) Anti-albumin and ovalbumin serum

A passive cutaneous anaphylaxis reaction was induced using an anti-albumin serum of PCA 1: 128 titer. The animals are sensitized by reactin serum diluted 1:64. After 48 h of sensitization, the dialysate is injected at the same point, an injection of physiological water constituting the positive control. Ovalbumin is injected intravenously with Evans blue. A strong positive reaction is obtained with reactin serum. This reaction is practically completely abolished if the dialysate has been injected beforehand.

b) Infected rat serum and water-soluble antigen of S. monsoni

A second anaphylactic system was chosen: unlike in vitro experiments, it is possible to obtain a positive reaction with a rat serum infected with S. monsoni when the PCA technique is used. The infected rat serum is taken at the 45th d of the infection, that is to say at the most favorable period for obtaining specific anti-S IgE. monsoni. The serum (PCA titer 1: 8) is injected pure intradermally 48 hours before the injection of dialysate. The intravenous injection of water-soluble Ag S. monsoni and Evans blue produced a strong positive reaction for the control. This is completely abolished with the dialysate. The results obtained with the chemical compounds are confirmed, the inhibition being even more marked in the case of the anaphylactic reaction.

C - Inhibition of the dependent mast cell / eosinophil cytotoxicity system

The mechanism of this cytotoxicity reaction has been described by Capron et al. Eosinophils brought into contact with schistosomules sensitized by IgGa Ab are capable of coating and killing these. The presence of mast cells or degranulated mast cell supernatants is essential for cytotoxicity. It is likely that mast cells activated by IgE or IgGa complexes release eosinophil activation products. These are then able to destroy the schistosomules. It was therefore interesting to see if the dialyzable fraction of the adult worm incubate was capable, by blocking mast cells, of inhibiting this cytotoxicity reaction.

10 μl of dialysable fraction are deposited in contact with the sensitized schistosomules at the same time as the cells obtained after adhesion for 2 h. The percentage of schistosomules dead after 48 h of culture is determined. The presence of the dialysable fraction of the incubate drops the percentage of cytotoxicity from 63 to 17%, ie an inhibition of 74%. The dialyzable fraction most likely acts on mast cells, but it is also possible that it acts on eosinophils. This is why a second experiment 30 consisted in incubating beforehand purified mast cells with the dialysable fraction and then adding these mast cells to purified eosinophils and sensitized schistosomules. The percentage of cytotoxicity which was 14% drops to 3% when the mast cells have been preincubated with the dialyzable fraction. The same percentage of inhibition is found, although the positive control is much weaker. This suggests that the action of the dialysable fraction is located at the mast cell level.

D - Conclusion

40 The dialyzable fraction of the incubate of adult worms S. monsoni has an inhibitory action with respect to mast cell degranulation. Experiments carried out in vitro have shown that this fraction is capable of inhibiting degranulation, whether it is induced by an anaphylactic system or by chemical agents. The use 45 of the isotopic method of measuring degranulation makes it possible to very precisely assess the inhibition. When degranulation is induced by the action of an anti-albumin serum and the corresponding antigen, doses of 10 to 30 days are responsible for an increasing inhibition of the release of serotonin. With higher 50 concentrations, a cytotoxic effect appears. Cell lysis or at least alteration of the cell membrane leads to a spontaneous release of serotonin. This measurement of the spontaneous release of serotonin thus constitutes a good indicator of the possible cytotoxicity of the dialysate. It should be noted that this cytotoxicity 55 at high concentrations is not surprising and corresponds to the characteristics of a large number of biological substances. The same inhibitory effect is observed when degranulation is induced by polymyxin-B. This effect is much more difficult to demonstrate when it comes to compound 48/80. This can be explained by the fact that the degranulation induced by compound 48/80 is the consequence of a mechanism different from that of conventional activation. For example, the dependence of degranulation on the level of intracellular cyclic nucleotides could not be demonstrated when it is induced by compound 48/80 [Johnson et al., 65 "J. Immunol. ”, 112 (1974), 511; Diamond, "Acta Physiol. Scand. ", 71 (1967), 283], while it has been clearly demonstrated in the general mechanism of degranulation [Kaliner and Austen," J. Immunol. ”, 112 (1974), 664]. The possibility of inhibiting a degra

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Cancellation induced by a chemical agent such as polymyxin-B suggests that the dialysate acts by inhibition of an intracellular activation signal rather than by competition phenomena at the level of IgE receptors.

A characteristic of the inhibitory substance is that it is thermostable (1 h-100 ° C), which is specific to substances with a molecular weight <10,000. The inhibition is irreversible by washing the cells. This result suggests that the dialysate induces an intracellular signal at the cell level which prevents further activation, for example by blocking a stage of metabolism necessary for the process of degranulation. To explain this irreversible phenomenon, however, we cannot exclude the possibility of a strong affinity bond between the mast cell and the inhibiting factor.

Tests carried out on Hela cells and human monocytes in continuous culture have shown that the dialysate has no cytotoxic or cytostatic activity on these cells, at the doses used to demonstrate an inhibitory effect on lymphocytes or mast cells. The dialysate under the conditions used is not a cellular toxin, but has a specific action vis-à-vis certain cell types including lymphocytes and mast cells.

The use of the PCA technique has made it possible to confirm in vivo the inhibitory role played by the dialysate. The chemical compounds 48/80 and polymyxin-B have the advantage of inducing an immediate positive reaction without an awareness period, which facilitated the experiments at first. The same number of injections is made for each point (physiological water or dialysate), which eliminates the hypothesis of an inhibition due to the trauma of the intradermal injection. The dialysate alone does not induce a positive reaction. If the action of the dialysate consisted of a tissue injury

sular at the injection site, it is likely that this would result in mast cell lysis with release of mediators and vasodilation, therefore a positive reaction with the dialysate alone. The absence of a reaction with the dialysate makes it possible to say that it is not a simple cytotoxic reaction. The presence of the dialysate leads to a strong attenuation of the reaction caused by 48/80 or polymyxin. Almost total inhibition is observed in the anaphylactic system, whether it is an antiovalbumin serum or a rat serum infected with S. monsoni. The fact that the dialysate is injected after or io by chemical agents suggests an intervention at the level of cellular metabolism in one of the stages of activation leading to degranulation. One might think that the action of the dialysate is not located at the mast cell level, but acts by destruction of the released mediators. The use of measurement of released tritiated serotonin rules out this assumption, since radioactivity is anyway measured, even if the released serotonin has been further degraded.

The purification of this substance provides important information on its nature, its mechanism of action and its identification in vivo. On the other hand, it makes it possible to determine the pharmacological interest of the product of the invention in the treatment of allergic phenomena.

Finally, it should be noted that schistosomiasis is rife in the regions of the Yangtze valley which are a very important site of cirrhogenic hepatitis endemic leading to one of the world foci of liver cancer. There is relative protection for populations infested with Schistosoma. It can therefore be inferred from the existence in Schistosoma extracts of a chemical factor protecting the host against the responsible virus.

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Claims (4)

642 682 1. Peptide originating from Schistosoma monsoni, of molecular weight ranging between 500 and 1000, soluble in water, thermostable, not precipitable by the reagents of proteins, dialyzing through a membrane of cross-linked dextran of the type Amicon X-100. 2. Method for obtaining the peptide according to claim 1, characterized in that adult Schistosoma monsoni is incubated in water, separates the bodies of the nematodes and the particulate debris from the liquid medium, subjects the liquid medium to dialysis vis-à-vis water, collects the dialyzed fraction and lyophilizes said fraction. 2 3. Method according to claim 2, characterized in that adult Schistosoma monsoni is incubated in water for 3 h, separates the bodies of the nematodes, subjects the filtrate to centrifugation, separates the supernatant, the filter on a Millipore filter, subjects the filtrate to dialysis against distilled water in a dialysis cell equipped with an Amicon X-100 filter for 24 h, collects the dialyzed fraction which is lyophilized. 4. Pharmaceutical compositions containing as active ingredient the peptide of claim 1 in association or in mixture with an excipient or an inert non-toxic carrier pharmaceutically acceptable.