JP6529835B2 - Dendritic cell activator - Google Patents

Description

  The present invention relates to dendritic cell activators useful for dendritic cell activation.

  Dendritic cells are cells that play a central role in the immune response, which detects and eliminates pathogenic bacteria and virus-infected cells, etc. Foreign antigens are presented on the surface of their own cells, and the information is displayed on T cells and the like. It is transmitted to other immune cells and plays a role in activating the immune response for eliminating foreign substances. In recent years, dendritic cells have found utility in dendritic cell vaccine therapy and the like, and development of an active ingredient that activates dendritic cells is desired.

  On the other hand, in the case of milk fermented products containing Lactobacillus kefiri, in particular Lactobacillus kefili P-IF strain, the present applicant has been directed to Clostridium microorganisms which are said to be bad bacteria of human intestinal microflora. It is reported that it contains a component showing a strong antibacterial effect and is excellent as an active ingredient of the intestinal environment-improving agent (patent documents 1 and 2 below).

Patent 4456160 gazette Patent No. 4519202

  Providing new active ingredients for dendritic cell activators broadens the range of treatment and treatment options. In addition, if a natural product or its primary processed product is an active ingredient, it is desirable because the risk of side effects is considered to be small.

  Therefore, an object of the present invention is to provide a dendritic cell activating agent which contains a natural product or a primary processed product thereof as an active ingredient and is useful for activating dendritic cells.

The present inventors earnestly studied in order to achieve the above-mentioned object, and came to complete the present invention. That is, the present invention is as follows.
[1] A dendritic cell activating agent comprising a fermented milk product of Lactobacillus kefiri or a processed product thereof as an active ingredient.
[2] The dendritic cell activating agent according to the above [1], which is used to activate immature dendritic cells.
[3] The dendritic cell activator according to [1], which is used to enhance the ability of dendritic cells to produce an immunomodulatory cytokine.
[4] The dendritic cell activating agent according to the above [1], which is used to be administered to cultured dendritic cells.
[5] The dendritic cell activating agent according to the above [1], which is used to be administered together with a vaccine or antigen derived from a pathogen.
[6] Any one of the above-mentioned [1] to [5], wherein the Lactobacillus kefiri (Lactobacillus kefiri) is Lactobacillus Kefiri P-IF (Accession No. FERM BP-10896) The dendritic cell activator according to claim 1.

  According to the dendritic cell activating agent of the present invention, a fermented milk product of Lactobacillus kefiri or a processed product thereof is used as an active ingredient, and therefore dendritic cells can be activated safely with less side effects. Thus, the dendritic cell activator of the present invention is useful for dendritic cell vaccine therapy and the like.

In Test Example 1, CD80, which is a costimulatory and maturation promoting molecule for dendritic cells, wherein the addition (1 μg, 10 μg) of the lactobacillus treated product (LbK-PFT) of Lactobacillus kefiri to milk cells is a costimulatory and maturation promoting molecule for dendritic cells; It is a graph which shows the result of having investigated the influence on the expression of CD86 and HLA-DR. In Test Example 1, CD80, which is a costimulatory and maturation promoting molecule for dendritic cells, wherein the addition (20 μg, 40 μg) of the lactobacillus treated product (LbK-PFT) of Lactobacillus kefiri to milk cells is a costimulatory and maturation promoting molecule for dendritic cells, It is a graph which shows the result of having investigated the influence on the expression of CD86 and HLA-DR. In Test Example 2, the addition (1 μg, 10 μg, 20 μg, 40 μg) of the fermented milk fermentation product (LbK-PFT) of Lactobacillus kefiri (LbK-PFT) to the dendritic cells was a cytokine (TNF-α, dendritic cells). It is a graph which shows the result of having investigated the influence on production of IL-6). In Test Example 3, the addition (1 μg, 10 μg) of a fermented milk processed product (LbK-PFT) of Lactobacillus kefiri to E. coli LPS-stimulated dendritic cells (1 μg, 10 μg) co-stimulates dendritic cells and It is a graph which shows the result of having investigated the influence on the expression of the maturation promoting molecules CD80, CD86, and HLA-DR. In Test Example 3, the addition (20 μg, 40 μg) of the fermented milk processed product (LbK-PFT) of Lactobacillus kefiri to E. coli LPS-stimulated dendritic cells (20 μg, 40 μg) co-stimulates dendritic cells and It is a graph which shows the result of having investigated the influence on the expression of the maturation promoting molecules CD80, CD86, and HLA-DR. In Test Example 4, addition (1 μg, 10 μg, 20 μg, 40 μg) of dendritic cells under stimulation with E. coli LPS (LgK-PFT) of a product treated with lactation of Lactobacillus kefiri (LbK-PFT) is a dendritic cell. It is a graph which shows the result of having investigated the influence on the production of the cytokine (TNF-α, IL-6, IL-10) of

  The Lactobacillus kefiri is not particularly limited as long as it is a microorganism belonging to Lactobacillus kefiri, and preferably Lactobacillus Kefiri P-IF (Accession No. FERM). Use BP-10896). Lactobacillus kefiri can be cultured by a known method or in accordance therewith, and for example, it is anaerobically kept using a commercially available MRS medium "Lactobacilli MRS broth" (trade name, a product of Difco). Mass culture is possible by culturing or the like.

  A fermented milk of Lactobacillus kefiri can be obtained by inoculating Lactobacillus kefiri as a starter with a milk raw material and exposing it to predetermined fermentation conditions. As a milk raw material for milk fermentation, for example, milk, whey, fermented milk, lactic acid bacteria beverage, skimmed milk, skimmed milk powder, whole milk powder, concentrated milk, skimmed concentrated milk, condensed milk, skimmed milk, sugared condensed milk , Sweetened skimmed milk and the like. These may be dissolved or diluted or concentrated in water or the like as appropriate, and then subjected to a process such as autoclave sterilization as necessary to be used as a raw material for milk fermentation. As a method for inoculating a microorganism for fermentation (starter), a method of adding a preculture solution in MRS medium or the like, a method of adding the remaining milk fermented material when fermentation is performed before, and the like can be mentioned. As culture conditions, a method of anaerobically standing culture at 20 to 30 ° C., more preferably 24 to 26 ° C., for 1 to 10 days, more preferably 3 to 7 days is preferably employed.

  A plurality of bacterial species may be used in combination as a starter for the above-mentioned milk fermentation. According to this, since milk fermentation is caused by the symbiotic growth of a plurality of microorganisms, a milk fermented product having good taste and appearance can be obtained. For example, at least one yeast selected from the group consisting of Kazachstania thulensis (Kazachstania turicensis), Kazatzania unispora (Kazachstania unispora), and Kluyveromyces marxianus may be used in combination. Alternatively, Lactobacillus kefiri P-IF (Lactobacillus kefiri P-B1) (Lactobacillus kefiri P-B1) (Lactobacillus kefiri P-IF) (Accession No. FERM BP-10896) as Lactobacillus kefiri P Accession No. FERM BP-11115) may be used in combination. In a particularly preferred embodiment, Lactobacillus kefiri P-IF (Accession No. FERM BP-10896), Lactobacillus kefiri P-B1 (Lactobacillus kefiri P-B1) (Accession No.) as a starter for the above-mentioned milk fermentation FERM BP-11115), Kazattania thulensis P-Y3 (Kazachstania turicensis P-Y3) (Accession No. FERM BP-11116), Kazattania unispora P-Y4 (Kazachstania unispora P-Y4) (Accession No. FERM BP-11117), and Lactobacillus kefili P-IF (Lactobacillus) in the microbial community of a fermented milk after fermentation using a complex microorganism containing at least I. m. P. Y 5 (Kluyveromyces marxianus P-Y 5) (Accession No. FERM BP-11118) It is preferable to use a fermented milk prepared so that kefiri P-IF) (Accession No. FERM BP-10896) occupies 80% or more, more preferably 90% or more.

  In the present invention, a fermented milk product of Lactobacillus kefiri obtained as described above or a processed product thereof is used as an active ingredient of the dendritic cell activating agent. Here, “processed material” includes heat sterilization, concentration and drying of fermented milk, separation of bacterial cells, removal of bacterial cells, crushing of bacterial cells, dilution with aqueous solvent or water-containing alcohol solvent, etc., aqueous solvent And those prepared by applying one or a combination of two or more types of treatment such as extraction to a water-containing alcohol solvent or the like. In addition, it includes those prepared by applying one or a combination of two or more kinds of treatments such as heat sterilization, concentration, drying, removal of solid matter, etc. to an extract extracted with an aqueous solvent or a hydroalcoholic solvent etc. .

  For example, Lactobacillus kefiri is killed by subjecting the cells to a heat treatment at 60 to 120 ° C., more preferably 95 to 120 ° C. Thus, the fermented milk can be powdered by spray drying, but it may be sterilized by the heat applied at the same time. Alternatively, the powdering of the fermented milk may be performed by lyophilization. According to this, Lactobacillus kefiri can be provided in a live state by returning the freeze-dried product of the milk fermented product with water or the like.

  In the dendritic cell activating agent of the present invention, there is no particular limitation in blending other materials in addition to the above-mentioned active ingredient, and if necessary, a pharmaceutically acceptable base material or carrier is added. Using, for example, in the form of tablets, granules, capsules, pills, powders, solutions, powders, jellies, lozenges, injections, aspirants, coatings, etc. Can.

  The dendritic cell activating agent of the present invention may be administered to humans or animals, and the administration form is not particularly limited. For example, oral administration, intravenous administration, intracerebral local administration, intraperitoneal administration, aspiration, nasal administration and the like can be mentioned. Among them, the oral administration form is preferred from the viewpoint of reducing burden on the user and ease of taking.

When the dendritic cell activating agent of the present invention is orally administered, the daily dose is 0.01 in terms of the amount of the dry matter of a fermented milk product of Lactobacillus kefiri. It is preferably (g / kg body weight) to 10 (g / kg body weight), more preferably 0.05 (g / kg body weight) to 5 (g / kg body weight), 0.1 (g / kg body weight) It is even more preferable that it is kg body weight to 1 (g / kg body weight). Alternatively, it may be 1 × 10 ¹³ (in / kg body weight) to 1 × 10 ¹⁶ (in / kg body weight) in terms of the number of kefili bacteria contained in the fermented milk of Lactobacillus kefiri. Is preferably 5 × 10 ¹³ (in / kg body weight) to 5 × 10 ¹⁵ (in / kg body weight), more preferably 1 × 10 ¹⁴ (in / kg body weight) to 1 × 10 ¹⁵ (in. Even more preferably, If the dose is lower than these ranges, a sufficient effect can not be obtained, and if the dose is higher than the range, the risk of producing any side effect is increased. In addition, the number of Kefiridium bacteria contained in the dried product of the fermented milk of Lactobacillus kefiri is 5 × 10 ¹² (cells / g) to 3 × 10 ¹⁵ (cells / g). Is preferable, and 5 × 10 ¹³ (in / g) to 2 × 10 ¹⁵ (in / g) is more preferable, and 5 × 10 ¹⁴ (in / g) to 1 × 10 ¹⁵ (in / g) It is even more preferred.

  The form of the product for administering the dendritic cell activating agent of the present invention is not particularly limited as long as the effect is not impaired. For example, it can be used in various product forms such as pharmaceuticals, quasi-drugs, health foods, functional foods, nutraceuticals, supplements, etc., or in combination with such products. Alternatively, it may be an animal product for pet animals and the like.

  Another mode of use of the dendritic cell activating agent of the present invention is administration with a vaccine or antigen derived from a pathogen. According to this, the immune function by dendritic cells can be enhanced specifically to the vaccine and the antigen. Vaccines or antigens include oncogenic tissues, oncogenic cells, oncogenic tissue / cell-derived peptides, attenuated pathogenic viruses, pathogenic virus-derived peptides, attenuated pathogenic bacteria, pathogenic bacterial-derived peptides, etc. .

  Another alternative mode of use of the dendritic cell activating agent of the present invention is administration to cultured dendritic cells. More specifically, utilization for dendritic cell vaccine therapy and the like can be mentioned. In dendritic cell vaccine therapy, monocytes such as peripheral blood mononuclear cells (PBMCs) are separated from a donor (patient) and cultured in a culture plate and granulocyte-macrophage colony stimulating factor (GM- Monocyte-derived immature dendritic cells are prepared by stimulation with CSF), interleukin 4 (IL-4) or the like. Whether or not the obtained cells acquire dendritic cellularity can be determined by the expression of a dendritic cell marker such as CD11c. The expression of the marker can be measured by flow cytometry or the like. The immature dendritic cells are brought into contact with a desired vaccine or antigen to enhance the immunocompetence for the vaccine or antigen, and then returned to the donor (patient) in a predetermined amount. Thereby, the pathogen can be eliminated from the body by its immune function.

  The dendritic cell activating agent of the present invention can promote the above-mentioned immature dendritic cells to acquire immunocompetence, and further to enhance the immunocompetence against a specific vaccine or antigen. In this case, preferably, the cultured dendritic cells are administered together with a vaccine or antigen derived from a pathogen. Vaccines or antigens include oncogenic tissues, oncogenic cells, oncogenic tissue / cell-derived peptides, attenuated pathogenic viruses, pathogenic virus-derived peptides, attenuated pathogenic bacteria, pathogenic bacterial-derived peptides, etc. . Whether or not the immature dendritic cells have acquired immunocompetence can be determined by the expression of the dendritic cell maturation marker CD80, CD86 and the like. The expression of the marker can be measured by flow cytometry or the like.

When the dendritic cell activating agent of the present invention is administered to cultured dendritic cells, the dose per number of dendritic cells is in the state of a dry matter of a milk fermentation product of Lactobacillus kefiri. The amount is preferably 0.1 (μg / 1 × 10 ⁶ ) to 1000 (μg / 1 × 10 ⁶ ), preferably 1 (μg / 1 × 10 ⁶ ) to 100 (μg / L). more preferably 1 × 10 ^6), 5 ([mu] g / 1 × 10 ⁶⁾ it is even more preferred is to 50 ([mu] g / 1 × 10 ⁶ cells). Alternatively, it is 1 × 10 ¹¹ (units / 1 × 10 ⁶ ) to 1 × 10 ¹⁵ (units / 1) in terms of the number of kefili bacteria contained in the fermented milk of Lactobacillus kefiri. It is preferable that it is × 10 ^6, more preferably 1 × 10 ¹² (unit / 1 × 10 ⁶ ) to 1 × 10 ¹⁴ (unit / 1 × 10 ⁶ ), and 5 × 10 ¹² it is even more preferred is a number / 1 × 10 ⁶ cells) to 5 × ^{10 13} (number / 1 × 10 ⁶ cells). If the dose is lower than these ranges, a sufficient effect can not be obtained, and if the dose is higher than the range, the risk of cytotoxicity is increased.

  The invention will now be described by way of example, which do not limit the scope of the invention.

[Materials and Methods]
(1) Preparation of milk-fermented product of Lactobacillus kefiri
Lactobacillus kefiri P-IF (Accession No. FERM BP-10896) is inoculated into MRS liquid medium, precultured at 25 ° C., and a part thereof is used as a defatted milk medium (ion-exchanged water 90). 10 parts by mass of skimmed milk powder was mixed with the parts by mass, inoculated into a medium prepared by autoclave sterilization and inoculated, and anaerobically incubated and fermented at 25 ° C. for 5 days. The resulting fermented culture was a yellow-brown, yogurt-like liquid. After heat sterilization, it was lyophilized. The suspension was suspended in distilled water at a concentration of 1 mg / mL, centrifuged at 1200 × g for 5 minutes, the supernatant was filter-sterilized, and this was used as “LbK-PFT” in the test. In the following tests, the concentration of “LbK-PFT” is the concentration converted by the amount of the above-mentioned lyophilizate.

(2) Preparation of Monocyte-Derived Dendritic Cells Monocyte-derived dendritic cells were prepared by a conventional method. Specifically, it was prepared as follows.
Peripheral blood mononuclear cells (PBMCs) obtained from healthy donors (approved by Charles Drew University Institutional Review Board (IRB)) were separated by Ficoll-Hypaque density gradient centrifugation. The cells were allowed to adhere to the culture plate for 2 hours and then non-adherent cells were removed. Adherent monocytes were treated with 10% FBS, 1 mM glutamine, 100 U / mL penicillin, 100 μg / mL streptomycin, 50 ng / mL human granulocyte macrophage colony stimulating factor (GM-CSF) and a humidified atmosphere of 37 ° C. and 5% CO ₂ The cells were cultured for 6 hours in RPMI 1640 medium supplemented with 10 ng / mL recombinant human IL-4. Half of the medium was replaced with fresh medium every two days, and dendritic cells were collected after six days. According to the percentage of cells expressing the dendritic cell marker CD11c, the purity of the obtained dendritic cells was over 95%.

<Test Example 1>
Monocyte-derived dendritic cells (cell number about 0.5) under the same culture conditions as in the preparation of dendritic cells except that different concentrations of LbK-PFT (1, 10, 20, 40 μg / mL) were added. X 10 ⁶ ) was cultured for 24 hours, and the phenotype of dendritic cells after culture was examined. Specifically, the expression of cell surface markers CD80, CD86 and HLA-DR was analyzed by flow cytometry. FIGS. 1A and 1B show representative analysis results arbitrarily selected from four individual experiments.

  As shown in FIGS. 1A and 1B, the expression of CD80, CD86, and HLA-DR increased depending on the dose of LbK-PFT (indicated by arrows in the figure). These cell surface markers are costimulatory and maturation promoting molecules that enhance the antigen presentation ability of dendritic cells, and LbK-PFT has the effect of promoting the activation of immature dendritic cells and enhancing the antigen presentation ability It became clear that there was.

Test Example 2
The supernatant after culture of Test Example 1 was collected, and the cytokines (TNF-α, IL-6, IL-10) in the supernatant were each measured by a specific ELISA kit (BD Pharmingen).

  As shown in FIG. 2, addition of LbK-PFT significantly increased the production of TNF-α and IL-6 in dendritic cells. In addition, IL-10 was below the detection limit even if it added LbK-PFT.

<Test Example 3>
Monocyte-derived dendritic cells were cultured for 24 hours in the same manner as in Test Example 1 except that E. coli LPS (1 μg / mL) was added in addition to LbK-PFT, and the phenotype of dendritic cells after culture was determined Examined. FIGS. 3A and 3B show representative analysis results arbitrarily selected from four individual experiments.

  As shown in FIGS. 3A and 3B, the expression of CD80, CD86, and HLA-DR increased depending on the dose of LbK-PFT. These cell surface markers are costimulatory and maturation promoting molecules that enhance the antigen presentation ability of dendritic cells, and LbK-PFT is an antigen that further stimulates dendritic cell activation under stimulation with E. coli LPS. It became clear that there is an action effect that enhances the presentation ability.

<Test Example 4>
The supernatant after culture of Test Example 3 was collected, and the cytokines (TNF-α, IL-6, IL-10) in the supernatant were each measured by a specific ELISA kit (BD Pharmingen).

  As shown in FIG. 4, addition of LbK-PFT significantly increased the production of IL-6 and IL-10 by dendritic cells under stimulation with E. coli LPS. On the other hand, the production of TNF-α by dendritic cells under stimulation with E. coli LPS was reduced by the addition of LbK-PFT.

Claims (5)

A dendritic cell activating agent comprising a fermented milk of Lactobacillus Kefiri P-IF (Accession No. FERM BP-10896) or a solvent extract thereof as an active ingredient.   The dendritic cell activator according to claim 1, which is used to activate immature dendritic cells.   The dendritic cell activator according to claim 1, which is used to enhance the ability of dendritic cells to produce an immunomodulatory cytokine.   The dendritic cell activator according to claim 1, which is used to be administered to cultured dendritic cells. The dendritic cell activator according to claim 1, which is used to be administered together with a vaccine or antigen derived from a pathogen.