SK12199A3 - Use of an oligosaccharide as an immunomodulator in a dermato-cosmetic composition - Google Patents

Abstract

The invention relates to the use of at least one oligosaccharide comprising from 2 to 6 oside residues, and having a galactose residue in the non-reducing terminal position, or of a derivative of such an oligosaccharide substituted by a hydrophobic residue, for the preparation of an immunomodulating medicament. It also relates to a dermato-cosmetic composition and a cosmetic treatment of hyperreactive skins.

Description

USE OF OLIGOSACCHARIDES AS IMMUNOMODULATORS

IN DERMATOCOSMETIC PRODUCTS

TECHNICAL FIELD

I

The present invention describes compositions useful in the field of immunology, in particular for the treatment of hypersensitivity reactions responsible for allergies and various manifestations of intolerance.

BACKGROUND OF THE INVENTION

The immune response against infectious agents, toxins or malignancies is based on recognition of the foreign nature of the antigen (or allergen) and activation of cellular or humoral mediators designed to destroy the foreign antigen. Nevertheless, this mechanism can sometimes be undesirable if it is too strong or is directed against antigens that are not substantially harmful, especially in cases of organ transplants or tissue grafts.

Immunological reactions leading to allergy are divided into 4 categories:

Type I: Mast cells bind IgE class antibodies to their Fc receptors, then antigen binding triggers mast cell degranulation and shedding of mediators (histamine, SRS-A, ECF-A).

Type II: Specific antibodies (IgG and IgM) react with antigen on the surface of the target cell, causing cytolysis either by direct activation of the killing cells or by activation of complement.

Type III: Antibodies (IgG and IgM) form immunocomplexes with antigen and complement, which settle in the tissue and activate the production of chemotactic factors for polynuclear neutrophils, resulting in local inflammation.

Type IV: T-lymphocytes sensitive to a particular antigen start to form lymphokines after reaction with it. Lymphokines induce an inflammatory response and cause macrophage accumulation. Thus, lymphocytes are key cells responsible for both cellular and humoral responses of the immune system, in particular for

I secretion of antibodies. '

Thus, allergic predispositions may be due to one or more types of immune responses. These reactions can in principle be reactions of intolerance to hygiene and care products, observed in the skin and called "hyperreactive." This manifestation due to environmental factors is due to genetic factors and acquired hypersensitivity.

SUMMARY OF THE INVENTION

It has now been found that the manifestation of hypersensitivity can be controlled by oligosaccharides containing from 2 to 6 osidic groups with a galactose residue at a non-reducing terminal position, or derivatives of these saccharides substituted with hydrophobic residues.

The present invention describes the use of oligosaccharides containing from 2 to 6 osid moieties with a galactose residue at a non-reducing terminal position, or derivatives of these saccharides for the preparation of immunomodulatory compositions, in particular for the treatment or prevention of hypersensitivity reactions.

Particularly suitable oligosaccharides may be derived from melibiosis, lactose and derivatives thereof obtained by the addition of hydrophobic groups.

Hydrophobic substituents are in particular linear or branched C 1 -C ₈ alkyls, C 1 -C ₈ alkylamines, linear or branched, even substituted C 1 -C ₁₈ carboxylic acids, linear or branched, primary, secondary or tertiary

C 1 -C 18 amides and C 1 -C 18 arylalkyls. Oligosaccharide derivatives adapted for use in the present invention should fall into one of the categories mentioned below, wherein the general formula of oligosaccharides should correspond to the following formula

Galactose - _n (oc or β) - (Hex) _p where: n represents the 1,2,3,4 or 6 position

Hex represents α- or β-linked hexose or pentose p is a number between 1 and 5

(a) - glycosides corresponding to the formula:

(I) an oligosaccharide of 1-O-R, wherein R is a linear or branched alkyl residue of 1 to 18 carbon atoms; (II) an oligosaccharide of 1-O-R-O-1-oligosaccharide, wherein:

R = (CH ₂ ) m when m is between 2 and 10

(b) - osslamine alkylated according to one of the following formulas,

While the preferred oligosaccharide is lactose, melibiosis or stachiosis:

- alkylated oslamine corresponding to one of the following formulas:

(III) an oligosaccharide of 1-NH-CO-R, wherein R is a C 2 -C 18 alkyl residue containing 0,1 or 2 double bonds; (IV) an oligosaccharide of 1-NH-CO-R-CO-NH-1- an oligosaccharide wherein R = (CH ₂ ) _m where m is between 2 and 8

(c) - an alkylamine acylated with aldonic acid, obtained by oxidation of an oligosaccharide • (V) an oligosaccharide of CO-NH-R, where R is the same as formula (III) • (VI) an oligosaccharide of CO-NH-R-NH-CO-oligosaccharide R is the same as formula (III)

(d) - or a Schiff base reduction product consisting of oligosaccharides with aliphatic mono- or diamines and corresponding to the following formula:

(VII) Gal- (Hex) nX-HN-R (VIII) Gal- (Hex) nX-HN-R-NH-X- (Hex) _n -Gal, wherein: Hex is hexose or pentose n = 0 , 1 or 2 and X = NH _l-2-hexitol

R is the same as formula (III).

According to a particular most preferred method, the oligosaccharides or derivatives thereof as defined above will be used to prepare compositions containing, in addition, a pharmaceutically acceptable carrier unsuitable for administration by the usual external route.

In fact, it has been found that the reactions involved in manifestations associated with hypersensitivity may correlate with the fixation of peptides leading to the degranulation of elastin, particularly κ-elastin, to a specific lymphocyte receptor.

Activation of these receptors triggers the ‘secretion of lytic enzymes, β-glucuronidase, elastase, and free radicals, such as superoxide ion or hydrogen peroxide, which promotes the formation of hydroxyl radicals. These products can affect the degradation of intercellular mass macromolecules, fibronectin, collagen and hyaluronan. Thus, binding of elastin peptides stimulates lymphocyte proliferation.

This phenomenon plays an important role in immunoallergic reactions of atopic origin, in particular atopic dermatitis or anaphylactic reactions, urticaria and allergic contact dermatitis. Individuals whose skin is hypersensitive or hyperactive are more and more frequent in the population. Their skin reacts easily with redness and pain and their increase in reactivity is lower due to healthy skin. The individual then experiences discomfort associated with erythema and light itching.

All of these symptoms can be alleviated or even suppressed using the oligosaccharide-containing formulation of the present invention. Melibiosis, lactose or derivatives thereof partially or completely suppress cell proliferation, even in the case of previously stimulated lymphocytes, may even inhibit the potentialization and secretion of lytic enzymes.

The composition should be formulated on a basis that does not contain any perfuming or allergenic agent. The composition may be in the form of a solution, gel, oil, cream, v / 0 or 0 / V emulsion or several emulsions in liposomal form. It can be treated by one skilled in the art, in particular using plasticizers or weak surfactants.

In particular, the composition is adapted to treat or prevent intolerance and / or allergic reactions in the skin and / or mucous membranes. In particular, the composition of the present invention is intended to prevent or reduce the formation and formation of free radicals.

Oligosaccharides or derivatives thereof may be associated with other substances that are capable of protecting and / or hydrating the skin, such as hyaluronic acid, vitamin E, glycol extract of G. biloba, sorbitol, glucosaminoglycans, alginates, etc. They may also be associated with skin-nourishing vegetable oils and / or active emollients such as wild grape extract, althaey, oats, linden, chamomile, sweet clover, Ruscus plants, procyanidol, abisabolol, coconut oil, 18-p acid -glycyrretínová.

According to another aspect of the present invention, oligosaccharides and derivatives thereof as defined above are used to prepare a composition further comprising a pharmaceutically acceptable carrier, for administration by the parenteral or enteral route. The efficacy of these oligosaccharides of the present invention is based on their surprising effect on the immune system of the cell-mediated response.

The compositions prepared according to the present invention are particularly useful for treating or preventing lymphocyte mediated hypersensitivity reactions. Specifically, most atopy, polymorphic erythema, xerodermatitis, lupus erythematosus, pemphigus, dermatitis, psoriasis and eczema are intended to treat or prevent the symptoms of these diseases.

Oligosaccharides with galactose groups at the non-reducing terminal position and derivatives thereof substituted with hydrophobic groups can be used as adjuvants limiting hypersensitivity reactions that are induced by another active procedure.

The invention thus provides a method of cosmetically treating hyperreactive skin characterized in that the composition comprises at least one oligosaccharide containing from 2 to 6 osidic groups with a galactose moiety in a non-reducing terminal position, or derivatives of these oligosaccharides substituted with hydrophobic groups and administered in a cosmetically acceptable carrier.

Particularly preferred substances suitable for implementation are melibiosis, lactose and derivatives thereof obtained by the addition of hydrophobic groups.

BRIEF DESCRIPTION OF THE DRAWINGS

Examples, invention. In the picture.

Fig. 1: The stimuli that follow are intended only to illustrate these examples and are references to the following

Inhibition of K-elastin proliferative activity by lactose and melibiosis lymphocytes.

The concentrations of lactose and melibiosis are 1 gg / ml, 10 µg / ml, 100 gg / ml, 1 mg / ml, 2 mg / ml.

Fig. 2: Inhibition of expression of elastic activity in lymphocytes stimulated with 2 gg / ml κ-elastin by lactose and melibiosis. Activity is measured in culture.

Fig. 3: Inhibition of cathepsin G expression in 2 gg / ml κ-elastin stimulated lymphocytes. Activity is measured in culture.

EXAMPLES OF THE INVENTION

Example 1

1. Methodology

Isolation of lymphocytes

Lymphocytes used for these experiments were obtained from peripheral human blood and / or tonsillectomy human tonsilectomy. Peripheral lymphocyte isolation proceeded as follows: 5 ml of blood was layered (in 15 ml centrifuge tubes) on Ficoll-pague plus (Pharmacy) so as not to mix the two phases, then centrifuged at 3000 rpm (about 600 g) for 40 minutes and at room temperature (20 ° C). The layer containing lymphocytes and monocytes was collected and mixed with 10 ml RPMI medium and centrifuged at 2200 rpm (about 400 g) for 10 minutes at 20 ° C. The sediment was resuspended in 0.5 ml RPMI, then another 4.5 ml RPMI was added and centrifuged again at 1500 rpm (about 400 g) for 10 minutes at 20 ° C. The final sediment was resuspended in 10 ml RPMI and cells were counted. Monocytes and macrophages were eliminated by adhesion to a plastic surface (incubation for 2 hours in a CO ₂ / O ₂ incubator).

Separation of polynuclear cells (PMN)

After the first centrifugation at 300 rpm (about 600 g), the residue containing red particles and PMN was resuspended in 1% polyvinyl alcohol (PVA) in Dulbecco's modified phosphatebuffered saline (DPBS), two volumes per volume of sediment, and then the solution was allowed to sediment for 20 minutes. at room temperature. The supernatant was then centrifuged for 5 minutes at 400 g and 4 ° C, the sediment was washed very carefully with DPBS to remove PVA but not activated by PMN, followed by centrifugation at 400 g and 4 ° C. The supernatant was discarded. Red blood cells were lysed by osmotic shock and then DPBS was added to regain osmotic equilibrium. After centrifugation at 400 g for 5 minutes at 4 ° C, the sediment containing PMN was resuspended in a small volume of RPMI. Cells were counted and could be lysed to release lytic enzymes (elastase and cathepsin) by adding 0.1% tritone X-100 in 1M NaCl at 0 ° C for 20 minutes, followed by centrifugation at 0 ° C for 20 minutes. minutes. The supernatant contained enzymes.

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Preparation of lymphocytes from human tonsils

Freshly removed almonds are cut into small pieces under sterile conditions in 10 ml RPMI. The tissue suspension is filtered through a coarse filter to remove larger tissue debris. The suspension was transferred to a tube and allowed to sediment for 15 minutes at room temperature. The supernatant was transferred to Ficoll-pague plus and centrifuged as described for blood-derived lymphocytes. Monocytes are also eliminated as in the case of lymphocytes isolated from blood. Lymphocyte-containing sediment was resuspended and cells counted after two centrifugations as described above. Lymphocytes obtained from almond isolation are approximately 50% of Ta-type and 50% Type B. Lymphocytes obtained from blood are approximately 80% of Ta-type and 20% Type B

Lymphocyte culture

Lymphocytes isolated as described above were incubated in a Costar 24 well plate, 500 μΐ cell suspension per well (5x10 ⁵ cells per ml or 2.5 x 10 ⁵ cells) in RPMI 1640 (eurobio) containing 10% fetal bovine serum. (ATGC), 2 mM glutamine (Gibco), penicillin and streptomycin (500

U / ml, 0.25 mg / ml), and phytohemagglutinin (Sigma) 5 gg / ml. After incubation for four days under cell culture conditions (37 ° C, CO2 / O2 incubator), the cell suspension was removed with tweezers, centrifuged at 400 g for 10 minutes and 20 ° C and the cell sediment was resuspended in 0.5 ml RPMI (without FCS) was mixed by shaking and after a further addition of 10 ml of RPMI without FCS, it was centrifuged again at 1500 rpm (400 g) for 10 minutes at 20 ° C. After two additional washes as described above, the sediment was resuspended in 10 ml of RPMI without FCS and the cells were re-transferred to culture on Costar 24 well plates.

(

Incubation with elastin peptides

Sterile κ-elastin solution (75 kD, Solabia) was added to the cells in the culture at a particular concentration (e.g., 2 µg / ml) and incubated in FCS-free medium for 2.5 hours at 37 ° C. After incubation, the plates were mixed gently, the cells collected by pipette and counted. The suspension was centrifuged for 10 minutes at 4 ° C at 1500 rpm (about 400 g), and the supernatant was dispensed into 1 ml eppendorf tubes into 0.5 ml aliquots and frozen at -40 ° C until used for immediate determination. enzymatic activity. The cell sediment was resuspended in extraction buffer (0.1% triton X-100, 1 M)

NaCl, 0.02% NaN ₃ , 0.01% Brij 35, pH 8), 1 ml per 10 ⁶ cells, and centrifuged for 15 minutes at 1600 g at 0 ° C and as described above, the supernatant obtained was added to the tubes. eppendorf and left at -40 ° C until enzymatic activity is determined.

Determination of enzymatic activity of elastase-like enzymes in leukocytes

The synthetic substrate used to determine the elastase-type enzymatic activity is Me-O-Suc-Ala-Ala-Pro-Val-pNA. 50 μΐ of medium or 20 μΐ of cell 'lysate was mixed with buffer (100 mM tris-HCl, 0.05% CaCl ₂ , 0.02% NaN ₃ , 0.01% Brij 35, pH 8) to 190 μΐ, then added 10 μΐ of 85 mM substrate solution (in N-methylpyrrolidone) to make up the total volume of 200 μΐ. Optical pressure is measured immediately at 410 nm and then after 4, 24, 48 and 72 hours incubation at 37 ° C. Elastase activity is determined in nM substrate hydrolyzed to 10 ⁶ cells per hour.

Determination of cathepsin G enzymatic activity G mg of Me-O-Suc-Ala-Ala-Pro-Val-pNA substrate in 1 ml of N-methylpyrrolidone (40 nM) was used under the same conditions as described above. .

2. Conclusions

a) Effect of elastin peptides on lymphocyte proliferation

The increase in calcium levels induced by the addition of elastin peptides to white blood cells can be considered as evidence of triggering signaling for a variety of cellular functions. One of these is the entry of cells into the proliferation cycle. This has been shown to occur in the case of skin fibroblasts in the presence of elastin peptides (Ghuisen-Itart et all, C. R. Acad. Sci., 1992, 315: 473-478). The conclusions presented in Table 1 show that stimulation corresponding to lymphocyte proliferation has been demonstrated.

Table 1

concentration κ-elastin Number of experiments Percentage of stimulation mean ± standard variation 2 pg / ml 6 62, 67 ± 37, 90 10 g / ml 13 35, 76 ± 29, 50

Maximum stimulation was observed at 2 μς / πιΐ elastin peptides and slightly weaker stimulation was demonstrated at a concentration of 10 gg / ml elastin peptides.

b) Increasing proteolytic activity by elastin peptides

When polymorphonuclear cells or lymphocytes are incubated in culture in the presence of 2 gg / ml κ-elastin, an increasing increase in elastase and cathepsin activity is observed in the cell extract and lysate. This increase is more visible in cells obtained from older arteriosclerotic individuals. An increase in this activity is not observed in the absence of kelastin. Addition of elastin peptides will increase proteolytic activity both in cell culture in the medium (salted enzymes) and in cell extract (cell-bound enzymes), both for elastase and cathepsin G, although this effect may be explained by an increase in enzymatic activity in the supernatant cell culture.

c) Effect of elastin peptides on cell survival

This experiment was performed under cell culture conditions as described above in the presence of phytohemagglutinin (PHA). 5 ml aliquots of lymphocyte suspension (2.5 x 10 ⁶ cells in 5 ml) obtained by isolation from (Human tonsils were divided into test tubes together with increasing κ-elastin concentration (BPM, PM <10 kDa): 1 gg / ml, 2 µg / ml, 10 gg / ml, 500 gg / ml, 1 mg / ml, 2 mg / ml.

After four days of incubation under standard culture conditions, cells were harvested and counted. Table 2 shows the cell loss as a function of the concentration of added elastin peptides.

Table 2

Concentration of elastin peptides in gg / ml Percentage of cell loss determined counting in Trypan blue 0 5.3 1 5, 0 2 2.5 10 17.4 100 19, 6 500 28.9 1000 39.3 2000 39.1

Elastin peptides have been shown to have higher cytotoxic activity at higher concentrations (thousands of times higher than the concentration stimulating cell proliferation and as salted lytic enzymes).

EXAMPLE 2

Effect of lactose and melibiosis on elastin receptor mediated lymphocyte reactions

1) Inhibition of cell proliferation

As shown in FIG. 1, increasing concentrations of lactose and melibiosis progressively suppress the growth stimulation induced by elastin peptides, followed by inhibition of proliferation at the highest concentrations tested (1 and 2 mg / ml), corresponding to a 5.84 x 10 ^-3 mM solution. The strongest inhibition of proliferation ranges from 16 to 30% for lactose and from 26 to 58% for melibiosis.

2) Effect of lactose and melibiosis on salted proteolytic enzymes triggered by elastin receptor

As shown above, an increase of 40 to 120% in the activity of proteolytic enzymes is observed in the cell medium as well as in the cell lysate of human lymphocytes cultured in the presence of 2 μg / ml κ-elastin. Stimulation of the increase in lytic enzymatic activity is inhibited effectively by melibiosis from as low as 1 gg / ml. This inhibition can be demonstrated for both elastase activity and cathepsin G activity. Likewise, lactose is effective in this case, but with less effect than melibiosis (Figs. 2 and 3).

EXAMPLE 3

Quantification of elastin receptors in human lymphocyte subpopulations

methodologies

Used antibodies:

primary antibody: anti 67 kD receptor for elastin / laminin antibody (bovine), IgM class, produced in mice (Elastin Products Company).

secondary antibody: mouse anti-IgG + IgM (H + L) antibody produced on goats, labeled with rhodamine (TRITC) (Jackson Immunoresearch Laboratories), for fluorescence microscopy

secondary antibody: mouse anti-IgM antibody F (ab ') ₂ / produced on goats, labeled with r-phycoerythrin (Chemicon), for flow cytometry.

Protocol:

(a) Fluorescence microscopy

Human lymphocytes isolated from human tonsils (as described in Example 1) are cultured in the presence of 5 µg / ml phytohemagglutinin (to be activated) and part of them are cultured with 2 gg / ml high molecular weight κ-elastin ( 75 kD) in RPMI 1640 medium with 10% fetal bovine serum (FCS). After incubation for 48 to 120 h at 37 ° C, the lymphocytes were washed 3 times in RPMI medium containing 2% FCS and then centrifuged at 400 g, 10 minutes at 4 ° C. Cells were then counted and divided to a concentration of 10 ⁷ cells / ml in RPMI medium containing 2% FCS.

A portion (100 μΐ) of the human lymphocyte cell suspension (about 10 ⁶ cells) was incubated with 10 μ 10 of the first antibody solution (diluted 1: 100) for 30 minutes in an ice bath. Cells were washed with 1.5 ml ice-cold RPMI with 2% FCS and centrifuged in a pre-cooled centrifuge at 4 ° C (400 g, 10 minutes).

A secondary antibody solution (conjugated to TRITC, resolved in 1: 200 ice RPMI) was added to the pellet. After gentle mixing, the suspension was incubated for 30 minutes at 4 ° C. Then 1.5 ml of ice-cold RPMI with 2% FCS was added and the suspension was centrifuged at 400g and 4 ° C for 10 minutes.

After the final wash, lymphocytes were resuspended in residual medium after the supernatant was removed by decantation. Thereafter, smears were made on the slides and allowed to air dry. After fixation in pure ethanol for 5 minutes, the samples were rehydrated inversely in several PBS baths and glycerol-PBS-buffered (9 volumes of glycerol per volume of PBS). This latter procedure increases the clarity of immunofluorescence. To identify labeled cells, preparations were observed alternately under visible light and under UV.

(b) Fluorimetry

After the last wash, lymphocytes were resuspended in 1 ml PBS and cells analyzed by cytofluorometry.

The following characterized cell populations were observed • Total T lymphocytes (CD3 +) • Helper T lymphocytes, T helpers (CD4 +) • Suppressor T lymphocytes (CD8 +) • B lymphocytes (CD20 +) • Activated T lymphocytes (CD25 +) • Memory T lymphocytes (CDA + / CD45RO +) • Granulocytes (CD15 +)

Description of used characters:

CD3

The CD3 complex is expressed by all mature human T cells. Its molecular weight is between 16 and 28 kD and is composed of five chains (γ, σ, ε, ξ, η) associated with the T cell receptor of non-covalent interactions. It is connected in the activation signal transmission.

CD4

The CD4 (T4) molecule recognizes MHC molecules II. class during the interaction of CD4 + cells with antigen presenting cells or cannon cells. It is a 59 kD glycoprotein belonging to the immunoglobulin superfamily. It is found in a subpopulation of T cells belonging to the helper / inducer group, which is about 45% of peripheral blood lymphocytes.

CD8

The CD8 molecule (T8, 30/32 kD) is a glycoprotein formed by two peptide chains. It is found in a population of cytotoxic / suppressor T lymphocytes, which is 20 to 35% of peripheral blood lymphocytes. It is also present on NK cells and 30% of the so-called. null cells in peripheral blood.

I t

CD15

CD15 antigen (3FAL, X-hapten, SSEA) is lacto-N-fucopentosis III (200 to 185 kD) at least 5 major antigens CD15 is present on the surface of polynuclear cells (which is about 90% of human granulocytes in circulation) and on circulating portions monocytes (30 to 60%). This antigen is not found on the surface of normal lymphocytes.

CD20

The CD20 antigen is a phosphoprotein with a molecular weight of 35-37 kD. It is present on all normal B cells in peripheral blood, nodules and bone marrow.

CD25

The CD25 molecule corresponds to a low affinity receptor for interleukin 2. It is a 55 kD glycoprotein expressed by activated lymphocytes (T and B) but also by activated macrophages.

CD45RO

It is a transmembrane glycoprotein with a molecular weight of 180 kD (lower molecular weight isoforms are called common leukocyte antigen, LCA). It is present on the surface of T lymphocytes, thymocytes, granulocytes, monocytes (but not present on the surface of macrophages) and in a small population of B lymphocytes. CD45RO expressing T cells are memory T cells (or induced T cells), which is about 45% of peripheral blood T cells.

CD4 + / CD45RO + cells produce so-called. auxiliary signals. In particular, IL-2 and IFN-γ production is frequent.

For labeling with antiCDx antibodies (all Sigma, except antiCD25 antibody from Serotec), 100 μΐ of lymphocyte suspension (either labeled or not) was taken together with 67 kD anti-elastin receptor antibody with a cell density of 10 ⁷ cells / ml. 10 μ susp of antiCDx antibody was added to the suspension and incubated for 30 minutes at 20 ° C (only for antiCD25 antibody incubated at 0 ° C). Then 2 ml PBS was added. After two washes (two centrifugations at 400 g at 20 ° C for 10 minutes), the cell pellet was collected, resuspended in 0.5 ml PBS and analyzed by cytofluorometry. The specificity of the label was always verified using isotype-like mouse immunoglobin (non-specific myeloma protein).

Conclusions:

(a) Fluorescence microscopy

Demonstration of the presence of elastin receptor on activated leukocytes

Part of the lymphocytes analyzed, those that express 67 kD

elastin / laminin receptor. show specific immunofluorescence under experimental conditions described above (positive) cells). percentage representation positive cells in culture on the fourth day incubation, as it was fixed fluorescence microscopy:

- cells cultured in the presence of 2 μg / ml κ-elastin:

28.52 ± 12.6%

- cells cultured without κ-elastin 28.09 ± 10.91%

In this case, there was no significant difference between the two groups, activation of the cells by PHA is probably a sufficient signal for the expression of the elastin receptor.

Percentage of positive cells in culture on day 5 of incubation

- cells cultured in the presence of 2 μς / πιΐ κ-elastin:

77.2 ± 6.7% t

Influencing cells after washing with lactose or melibiosis:

- cells cultured in the presence of 2 gg / ml κ-elastin and finally washed in culture with one μς / πιΐ lactose:

26.6 ± 6.7% (p 0.001).

Prior to washing, 77.2% of the cells were positive

- cells incubated for five days in the presence of 2 μg / ml κ-elastin and finally washed in culture with one μς / πιΐ melibiosis:

18.3 ± 9.7% (p 0.001).

Cells incubated only with the secondary antibody in the absence of the first antibody (negative control) show no fluorescence. This demonstrates that melibiosis is more effective than lactose to saturate the 37 kD lymphocyte elastin receptor subunit: lactose saturates 66% of the receptor and melibiosis 76% under experimental conditions as described above.

(b) Cytofluorometry

The percentage of human lymphocytes expressing the elastin receptor after several incubation periods is shown in Table 3.

DO: The day the lymphocytes were separated

D2, D3, D5: Second, third and fifth day after lymphocyte separation:

Table 3% percentage of positive cells

Day Without κ- elastin Count experiments With 2 µg / ml κ-elastin P TO 1.12 ± 0.2% 6 1.15 ± 0.1% - D2 22.33 ± 5,2% 5 23.02 ± 3.2% - D3 29.7 ± 0.8% 7 32.40 ± 3.5% 0,178 D5 59, 91 . ± 4,9% 10 66.42 ± 1.19% 0, 006

This experiment suggests that incubation of cells with PHA stimulates the progressive expression of elastin receptor α. This induction is further stimulated in the presence of elastin peptides. Nevertheless, the rate of this stimulation is not clear until the beginning of the fifth day of incubation in culture. Under these conditions, approximately two-thirds of the lymphocytes (&gt; 66%) express the elastin receptor on their surface.

Double Marking Results:

Table 4

Use of double lymphocyte labeling to identify subpopulations of elastin receptor expressing lymphocytes

i cells expressing the elastin receptor (67 kD) + CDx labeling with K-elastin without κ-elastin R67kD + 59.9 66, 4 CD4 + R67kD + 26, 5 45, 7 CD8 + R67kD + 11.9 16, 1 CD15 + R67kD + 0, 0 0, 0 CD20 + R67kD + 36, 7 36, 3 CD25 + R67kD + 39, 6 46, 9 CD45RO + R67kD + 26, 6 41.1

CDx + R67kD = cells labeled with anti-CDx antibody and antibody against 67kD elastin receptor subunit at the same time. Note: The sum of% together does not give 100% because the cells simultaneously express several receptors.

In this experiment, the exact characteristics of the lymphocyte subpopulations expressing the elastin receptor were determined by double labeling in the presence or absence of elastin peptides.

Most lymphocyte subpopulations tested were found to express the elastin receptor, except for CD15 + cells, which remain negative. Thus, CD15 labeling corresponds to polymorphonucleate and monocytes.

This expression is largely stimulated by the presence of elastin peptides, in the presence of which CD4 + and CD45RO + significantly increase the expression of the elastin receptor. In this case, these are subpopulations that have been recognized as helper and memory cells. It seems that positive feedback, leading to activation of the elastin receptor to its own synthesis, could be specific for these cells.

Example 4

Protection of lymphocytes against cytotoxic effect of low molecular weight κ-elastin

1) Isolation of lymphocytes

Lymphocytes were isolated as described in Example 1. The cell suspension was diluted with RPMI medium containing 5 mM glutamine and 10% FCS to a cell concentration of 10 ⁶ cells / 2 ml.

2) Cell culture

Lymphocytes were incubated in twenty-four well Costar plates (500 μΐ cell suspension / well at 10 ⁶ cells / 2 ml) in complete RPMI 1640 medium. The cell distribution was as follows;

- 10 ml of cell suspension without κ-elastin

- 10 ml cell suspension with 2 gg / ml κ-elastin a 10 ml cell suspension i with 2 gg / ml κ-elastin (control with K- elastin, but without lactose and melibiose) - 10 ml cell suspension with 2 μς / πιΐ κ-elastin + 1 mg / ml melibiose - 10 ml cell suspension with 2 μg / ml κ-elastin + 1 mg / ml

lactose

- 10 ml cell suspension + 1 mg / ml melibiosis

- 10 ml cell suspension + 1 mg / ml lactose

3) Cell counting in the presence of Trypan blue to determine cell viability

On day 5 of culture, cell suspension was harvested, cells counted in Malassez chamber in the presence of 0.1%

Trypan blue (Sigma), only dead cells were labeled Trypan blue to blue. The counting was performed immediately after addition of Trypan blue to eliminate its toxic effect.

Conclusions:

incubation cell number / ml % of dead cells (Trypan blue) K-elastin free 5, 8 ± 0.6 9.2 ± 0.4 with κ-elastin, 2 pg / ml (control) 10.23 ± 3.5 6.6 ± 1.3 with κ-elastin, 2 mg / ml 3,6 ± 1.0 41.3 ± 3.1 lactose, 1 mg / ml 7.7 ± 1.5 14.5 ± 1.9 with melibiosis, 1 mg / ml 7.26 ± 1.3 15.4 ± 1.6 with κ-elastin, 2 mg / ml + 1 mg / ml 8.8 ± 0.7 10.6 ± 2.3 lactose T 1 1 with κ-elastin, 2 mg / ml + 1 mg / ml 8.8 ± 1.6 10.5 ± 0.9

melibiose

The table shows the percentage of dead cells in culture after incubation with elastin peptides and the protective effect of lactose and melibiosis that prevents cell death. Its efficiency is close to 100%: the difference in cell viability (compared to the control without κ-elastin) incubated in the presence of 2 mg / ml elastin peptides is 32.1%. In the presence of lactose or melibiosis, the increase in the number of dead cells is only 1.3% (96% protected).

Claims (13)

PATENT CLAIMS Use of at least one oligosaccharide comprising from 2 to 6 osidic groups and having a galactose group at a non-reducing terminal position, or derivatives of these oligosaccharides substituted with hydrophobic groups, wherein the oligosaccharides glycosides correspond to the following formula galactose - n (a or P) ) - (Hex) _p wherein: n represents the 1,2,3,4 or 6 position Hex represents α- or β-linked hexose or pentose p is a number between 1 and 5 and derivatives of these oligosaccharides belonging to one of the following categories: (a) - glycosides corresponding to the formulas: (I) an oligosaccharide of 1-O-R, wherein R is a linear or branched alkyl moiety of 1 to 18 carbon atoms; (II) an oligosaccharide of 1-ORO1-oligosaccharide, wherein R = (CH ₂ ) _m where m is between 2 and 10 (b) - osslamine alkylated according to one of the following formulas, wherein the preferred oligosaccharide is lactose, melibiosis or stachiosis: alkylated oslamine corresponds to one of the following formulas: (III) oligosaccharide 1-NH-CO-R, wherein R is an alkyl residue of 2 to 18 carbon atoms containing 0,1 or 2 double bonds (IV) oligosaccharide 1-NH-CO-R-CO-NH-1 -oligosaccharide, where R = (CH ₂ ) _m , where m lies between 2 and 8 c) - an alkylamine acylated with aldonic acid, obtained by oxidation of oligosaccharides • (V, oligosaccharide CO-NH-R, where R is the same as formula (III) • (VI) oligosaccharide CO-NH-R-NH-CO-oligosaccharide, R is the same as formula (III) (d) - or a Schiff base reduction product consisting of oligosaccharides with aliphatic mono- or diamines and corresponding to the following formula: (VII) Gal- (Hex) _n -X-HN-R (VIII) Gal- (Hex) _n -X-HN-R-NH-X- (Hex) n -Gal, wherein: Hex is hexose or pentose n = 0.1 or 2 X = 1-NH ₂ -hexitol R is the same as formula (III), for the preparation of immunomodulatory drugs affecting cellular immune responses. Use of oligosaccharides according to claim 1 for the preparation of immunomodulatory agents affecting cellular immune responses, characterized in that it provides protection of lymphocytes i against the cytotoxic effect of low molecular weight κ-elastin. Use of at least one oligosaccharide containing from 2 to 6 osidic groups and having a galactose group at the non-reducing terminal position, or derivatives of these oligosaccharides substituted with hydrophobic groups according to claim 1, characterized in that they are used for preparing a preparation for treating or preventing hypersensitivity reactions and affecting cellular immune responses. Use of oligosaccharides according to claims 1 and 3, characterized in that the oligosaccharide is melibiosis or lactose or derivatives thereof as defined in claim 1. Use of oligosaccharides according to claims 1 to 4, characterized in that the composition additionally comprises a pharmaceutically acceptable carrier adapted for external topical use. 2 ^ Use of oligosaccharides according to claims 1 to 4, characterized in that the composition additionally comprises a pharmaceutically acceptable carrier intended for administration by the parenteral or enteral route. Use of oligosaccharides according to claims 1 to 6, characterized in that they are used for the preparation of a composition intended for the treatment or prevention of lymphocyte mediated hypersensitivity reactions. Use of oligosaccharides according to claims 1 to Ί, characterized in that the preparation is intended for the treatment or prevention of intolerance and / or allergic reactions on the skin and / or mucous membranes. Use of oligosaccharides according to claims 1 to 8, characterized in that the preparation is intended to prevent or reduce the formation of free radicals. I Use of oligosaccharides according to claims 1 to 8, characterized in that the preparation is intended for the treatment or prevention of symptoms of the following diseases most atopy, pemphigus, polymorphic erythema, xerodermatitis, lupus erythematosus, psoriasis, dermatitis and eczema. 11. A dermatocosmetic composition comprising at least one active ingredient in association with an adjuvant limiting hypersensitivity reactions on an active principle and wherein the adjuvant is an oligosaccharide containing from 2 to 6 moieties and having a galactose moiety at a non-reducing terminal position; or derivatives of these oligosaccharides as defined in claim 1. A method of cosmetically treating hyperactive skin, characterized in that the composition comprises at least one oligosaccharide containing from 2 to 6 osidic groups and has a galactose group at a non-reducing terminal position, or 2Γ derivatives of these oligosaccharides substituted with hydrophobic groups as defined in claim 1, and administered topically in a cosmetically acceptable form. Cosmetic treatment method according to claim 12, characterized in that the oligosaccharide belongs to the group consisting of melibiosis or its derivatives, which can be obtained by adding hydrophobic groups and is administered topically in a cosmetically acceptable form.