Agent inducing maturing of dendritic cells

FIELD: medicine.

SUBSTANCE: invention concerns medicine, be more specific to oncology, and concerns the substances stimulating maturing of dendritic cells (DC). Application of fucoidan from Fucus evanescens or polysaccharide composition from Fucus evanescens consisting of fucoidan in amount of 60-80% and poly-mannuronic acid in amount of 20-40%, as an agent possessing ability to induce maturing of dendritic cells is offered. The declared fucoidan preparations have a standardised composition and, hence, possess direct biological effect. They keep the properties for long time (3 years).

EFFECT: increase of DC functional activity under the influence of the declared substances.

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The invention relates to medicine, specifically to Oncology, and applies to substances that stimulate the maturation of dendritic cells (DC).

Studies have shown that the use of dendritic cells in cancer therapy has promising results. Dendritic cells - cells of the immune protection is a potential antigen presenting cells (APC) and play a major role in the regulation of the immune response [Banchereau J., et al. Immunobiology of dendritic cells. Annu. Rev. Immunol. 2000. 18. 767-811].

Dendritic cells there are at least two functionally and phenotypically different stages of development and maturation. At the first stage of immature dendritic cells (iD) circulate in the blood, and then penetrate into the peripheral tissues, where they learn the ability to capture and pressure antigens. In further Mature dendritic cells (mDC) migrate to the lymph nodes, presenterat peptide fragments of antigens with molecules of T lymphocytes inducyruya immune response. The ability of DC to stimulate T cells depends on their stage of maturation.

When dendritic cells are fully Mature, they are a source of information about the content of cancer cells. In many types of cancer dendritic cells in the cancerous area are immature and not able to effectively convey information about cancer cells balances the immune system, particularly T-lymphocytes. To t the th to information about cancer was reliable, DC cells to Mature - this is the main point. So getting a large number of Mature DC cells from the circulating blood of patients cancer patients appears to be an important moment in the immunotherapy of cancer.

Monocytes cultured with cytokines (interferons, tumor necrosis factor (TNF), a number of interleukins, colony-stimulating factor (CSF)) differentiate into immature dendritic cells. The DC maturation occurs after the processing of their substances, inducing maturation of dendritic cells, such as tumor necrosis factor (TNF-α), γ-interferon (IFN-γ), or CD40L (the family of tumor necrosis factor) [Reis e Sousa C: Dendritic cells in a mature age. Nat. Rev. Immunol. 2006. 6(6). 476-483].

Known use as an agent for the maturation of dendritic cells bacilli Calmette-guérin (BCG), compounds of imidazoquinolines, artificial donateware of polyribonucleotide, and lipopolysaccharide [EN 2005121256 A, 2006.02.10].

It is known that fucoidan from Fucus vesiculosus (Sigma) inhibits the binding T epithelial cells to CD11b/CD18, purified from neuropile, and also increases the viability of DC production of IL-12 TNF-alpha, MHC I and II, CD54 and CD86. Also this fucoidan reduces absorption of antigen and increases the growth of allogeneic splenocytes, shows immunostimulating effect and affect the maturation of DC on the way, including NF-kappa-B activation [Kim, M.H., and Joo, H.G. Immunostimulatory effects of fucoidan on bne marrow-derived dendritic cells. Immunol. Lett. (2008), 115, 138-143].

The objective of the invention is the expansion of the means of inducing the maturation of dendritic cells.

The problem is solved by the use of fucoidan from Fucus evanescens or polysaccharide composition of Fucus evanescens, consisting of fucoidan in the amount of 60-80% and polymannuronic acid in the amount of 20-40%, as a means of inducing the maturation of dendritic cells.

Technical result provided by the invention, is expressed in inducing the activity of fucoidan from Fucus evanescens or polysaccharide composition of Fucus evanescens on the maturation of dendritic cells.

Fucoidan from Fucus evanescens, denoted hereinafter "Fe", patented by the applicant as a means of having anticoagulant and immunotropic effect [EN 2247574 C2, 10.03.2005].

Polysaccharide composition of Fucus evanescens, consisting of fucoidan in the amount of 60-80% and polymannuronic acid in the amount of 20-40%, indicated hereinafter "Fc", patented by the applicant as a biologically active product, which is an additional source of immunoactive polysaccharides and soluble dietary fibers, which increase nonspecific resistance of the organism [EN 2315487 C1, 2008.01.27].

The claimed technical solution is new, as available in the patent and scientific and medical literature is not detected description application product Fe or Fc in image quality is as funds inducing the maturation of dendritic cells.

For the specialist is not obvious from the prior art, the possibility of using Fe or Fc as a means of inducing the maturation of dendritic cells.

The fucoidans is a family vysokotarifitsirovannyh Homo - and heteropolysaccharides brown algae, having L-fucose as the main structural units, but differ from each other physico-chemical properties, monosaccharide composition, molecular mass and other characteristics. This family can be added new connections, obtained from various species of brown algae, with their intrinsic physico-chemical and other characteristics, and with certain biologically active properties.

It is established that the manifestation of biological activity in any specific effects of certain types of algae does not imply that this biological activity will occur in fucoidans isolated from other species of algae. This is confirmed by, for example, research on the linkages between the structure of fucoidans from various sources and their biological activity [Cumashi, A., Ushakova, N. A. et. al. (2007) A comparative study of the anti-inflammatory, anticoagulant, antiangiogenic, and antiadhesive activities of nine different fucoidans from brown seaweeds. Glycobiology 17, 541-552].

The authors of the above is the Uta article shows which of the nine investigated fucoidans only five show inhibition of thrombin activity. It was also shown that the fucoidans decrease of leukocytes with peritonitis in rats. The possible mechanism of this action is inhibition of P-selectin binding activity. The activity of fucoidans were decreased in the next row: L. saccharina, L. digitata, F. evanescens, F. serratus, F. distichus, F. spiralis, A. nodosum. The fucoidans from Cladosiphon okamuranus and Fucus vesiculosus showed no inhibition of P-selectin binding activity.

Thus, the proposed solution meets the criterion of "inventive step".

Fucoidan from Fucus vesiculosus (Sigma), denoted hereinafter "Fv"is a non-homogeneous product. It contains a mixture of fucoidans and uronic acids, as well as the impurity neugeborne nature [Fitton, J.H. Fucoidans: healthful saccharides from the sea. GlycoScience&Nutrition (2005), 6, 1-6]. This circumstance does not allow an unambiguous conclusion about what fucoidan, and not another component of this mixture, induces the maturation of dendritic cells.

Preparations Fe and Fc are standardized composition and, therefore, have directed the biological effect. They do not contain polysaccharides another nature, lipids and retain their properties for a long time (3 years). All these advantages open the possibility for application of fucoidan from Fucus evanescens or Polish IGNOU songs from Fucus evanescens as a means of inducing the maturation of dendritic cells.

The invention is illustrated by the following drawings:

Figure 1 shows the induction of maturation of dendritic cells under the influence of drugs Fe and Fc. Fv - drug comparison; lipopolysaccharide (LPS) - positive control.

Figure 2 presents the dose-dependent effect of Fc on the maturation of dendritic cells.

Figure 3 is a diagram of the induction of markers of dendritic cells under the action of Fc and LPS.

Figure 4 is a diagram of the induction of markers of dendritic cells using the methods of electron microscopy with the use of PI-conjugated CD 80 antibody and FITC-conjugated MHC-II antibodies.

Figure 5 presents the phagocytic activity of dendritic cells under the action of Fc and LPS.

Figure 6 presents the expression of cytokines in dendritic cells treated with Fc and LPS.

Figure 7 presents the expression of IFN-γ in the mixed lymphocyte culture under the action of Fc and LPS.

On Fig presents the growth stimulation of T cells under the action of dendritic cells treated with Fc and LPS.

The invention is illustrated in the example.

Abbreviations

TNF-αThe tumor necrosis factor
IFN-gγ-interferon
CD40LThe family of tumor necrosis factor
iDCImmature dendritic cells
mDCMature dendritic cells
BCGBacillus Calmette-guérin
LPSLipopolysaccharide
CD1Cluster of differentiation 1
CD3Cluster of differentiation 3
CD14Cluster of differentiation 14
CD40Cluster of differentiation
CD80Cluster of differentiation
CD86Cluster of differentiation
GM-CSFGranulocyte-macrophage colony-stimulating factor
BSAAlbumin
FITCFluoresceinisothiocyanate
DPhycoerythrin
MHC-IIThe major histocompatibility complex
CCR7Chemokinesis receptor 7
IL-4Interleukin-4
IL-10Interleukin 10
IL-12Interleukin 12
PBSPhosphate buffer

Obtaining dendritic cells

Monocytes were purified from peripheral blood cells selection using super-paramagnetic media MACS (Miltenyi Biotec., Bergisch Gladbach, Germany)conjugated with antibodies to CD 14 (cluster of differentiation 14). The purity of monocytes, certain flow cytometer, more than 95%. Monocytes were cultured in medium containing recombinant GM-CSF (800 u/ml) (Novartis, Munich, Germany) and IL-4 (125 u/ml) (Strathmann Biothec., Hamburg, Germany) for 6 days to generate DC. Differentiation of monocytes was tested by expression of CD1 and CD 14.

Analysis of the phenotype of dendritic cells

Cells (1×106) were collected, washed with phosphate buffer (PBS)containing bovine serum albumin (BSA)and labeled with FITC (fluoresceinisothiocyanate) and RE (phycoerythrin)-conjugated antibodies by incubation on ice for 30 min followed by washing in PBS. Viable cells would and analyzed on a FACSCalibur flow cytometer (Beckton Dickinson, Franklin lakes, NJ). Appropriate antibodies were used as a control for nonspecific staining. Drugs Fv and Fc increased the level of expression of cluster of differentiation of CD40, CD80, CD83 in dendritic cells like LPS. However, the Fe had a lesser effect on the expression of markers of dendritic cells than Fv and Fc. The expression of MHC II (major histocompatibility complex) did not increase under the effect of fucoidans (Figure 1). The expression of CD80, CD83 iDC increased depending on the doses Fc (Figure 2). In addition, Fc increased the expression of CD80, CD83, CD86 and CCR7 (chemokine receptor 7) in dendritic cells (Figure 3).

Confocal microscopy

Cells were fixed for 15 min in 3% solution of paraformaldehyde in PBS, incubated 10 min in 0.1% solution of Triton X-100 in PBS. Slides were incubated in blocking buffer at 25°C for one hour. After washing (3 times with PBS), the cells were incubated at 4°C for one hour with PE-conjugated antibodies against CD-89 or MHC-II and again washed with PBS. The samples were analyzed using microscopy (Carl Zeiss LSM 510). Treatment of dendritic cells with Fc drug for two days induces typical morphological changes during maturation of dendritic cells (Figure 4). Immunohistochemical analysis showed that the immunoreactivity CD86 and MHC-II significantly increased after two days of cultivation with fucoidan F and lipopolysaccharide.

Phagocytic activity

For analysis of endocytosis 1×105cells were incubated with 1 mg/ml FITC-dextran (42 kDa) at 37°C for one hour. Cells were washed twice with cold HBSS, incubated, and analyzed on a flow cytometer. A parallel experiment was conducted at 4°C.

FITC-dextran was tested for its ability to penetrate into cells. Phagocytic activity of DC treated with Fc, was higher than in control (Figure 5).

Analysis of cytokines

Cells were incubated in medium RPMI-1640 containing Fc (50 μg/ml) or LPS (1 μg/ml) for 24 h in the presence or in the absence of T cells. Profiles of cytokine secretion of IL-10, IL-12, TNF-a, IFN-g in solutions were measured in three independent experiments using ELISA kits (R&D System), the sensitivity of ≥ 5 PCG/ml Standard cytokines were used as controls. The expression level of the immunosuppressant IL-10 compared with control was increased in cells treated with Fc or LPS. However, the expression of IL-12 (p70) in cells treated with Fc was lower than during stimulation with lipopolysaccharide (Fig.6). Fc stimulated the ability of DCs to Express TNF-α, and the expression level comparable to the level obtained by treatment of LPS. The expression of IFN-γ in T cells increased when dendritic cells with T cells at a ratio of 1:25 were treated with Fc or LPS (Fig.7).

The definition of the functionality is through the activity of dendritic cells (mixed lymphocyte culture)

T cells were purified from peripheral blood of healthy volunteers, and the purity was tested by staining with FITC-conjugated CD3 antibodies. Fc or LPS-treated dendritic cells were treated with 50 μg/ml of mitomycin C for 1 h and then 1×105T cells were added to 96 well plates. Cells were cultivated for 5 days at 37°C, 5% CO2and then add 1 µci of [3H]thymidine (NEN-DuPont, Boston, MA). The radioactivity of the collected cells was measured after 18 hours Testing dendritic cells for their ability to support the activation of T-cells showed that they stimulated the proliferation of T cells, when grown for 5 days in the ratio (T:DC=1:25) (Fig).

The use of fucoidan from Fucus evanescens or polysaccharide composition of Fucus evanescens, consisting of fucoidan in the amount of 60-80% and polymannuronic acid in the amount of 20-40%, as a means of inducing the maturation of dendritic cells.



 

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