WO2018084172A1 - Th1細胞を誘導する細菌 - Google Patents
Th1細胞を誘導する細菌 Download PDFInfo
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- WO2018084172A1 WO2018084172A1 PCT/JP2017/039522 JP2017039522W WO2018084172A1 WO 2018084172 A1 WO2018084172 A1 WO 2018084172A1 JP 2017039522 W JP2017039522 W JP 2017039522W WO 2018084172 A1 WO2018084172 A1 WO 2018084172A1
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- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5082—Supracellular entities, e.g. tissue, organisms
- G01N33/5088—Supracellular entities, e.g. tissue, organisms of vertebrates
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2207/00—Modified animals
- A01K2207/12—Animals modified by administration of exogenous cells
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/105—Murine
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
- A01K2267/035—Animal model for multifactorial diseases
- A01K2267/0368—Animal model for inflammation
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
- A61K2039/541—Mucosal route
- A61K2039/542—Mucosal route oral/gastrointestinal
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55588—Adjuvants of undefined constitution
- A61K2039/55594—Adjuvants of undefined constitution from bacteria
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/22—Klebsiella
Definitions
- the present invention in 2015, Japan Medical Research and Development Organization (AMED), innovative advanced research and development support project, unit type “Realization of optimal medical care based on network understanding of biostasis maintenance / transformation / failure mechanism” This was obtained as a result of a research project based on a commissioned project “Technology Creation” Research Area (Research and Development Project Name: “Development of New Treatment Methods for Intractable Diseases Based on Understanding Characteristics of Intestinal Bacterial Bacteria”).
- AMED Japan Medical Research and Development Organization
- the present invention relates to a bacterium that induces proliferation or activation of Th1 cells in the intestinal tract (hereinafter also referred to as “Th1 cell-inducing bacterium”).
- the present invention also relates to a composition for stimulating immunity or a composition for inducing the proliferation or activation of Th1 cells, comprising the Th1 cell-inducing bacterium or a physiologically active substance derived therefrom as an active ingredient.
- the present invention relates to a method for stimulating immunity or a method for inducing proliferation or activation of Th1 cells using the Th1 cell-inducing bacterium or a physiologically active substance derived from the Th1 cell-inducing bacterium. .
- the present invention provides a vaccine composition comprising the Th1 cell-inducing bacterium or an antigen specific for the bacterium as an active ingredient, or an antigen specific for the Th1 cell-inducing bacterium or the Th1 cell-inducing bacterium. And a method for inducing an immune response to the Th1 cell-inducing bacterium in the subject by ingesting the subject.
- the present invention provides a composition for suppressing immunity or a composition for suppressing the proliferation or activation of Th1 cells, which contains, as an active ingredient, a substance having antibacterial activity against the Th1 cell-inducing bacteria.
- the present invention relates to a method for suppressing immunity or a method for suppressing proliferation or activation of Th1 cells using the substance or the like.
- the present invention relates to a method for screening a Th1 cell-inducing bacterium or the like that suppresses or induces the proliferation or activation of Th1 cells in the intestinal tract, and a kit or a non-human animal used for such a screening method.
- the present invention relates to a composition for examining a disease caused by Th1 cells, comprising a substance for specifically detecting the Th1 cell-inducing bacteria.
- the resident flora plays a very important role in the physiology and health maintenance of the host.
- the constitutive abnormality of the resident flora is called Dysbiosis, and it is gradually becoming clear that it causes various diseases. If elucidation of the mucosal resident flora progresses, it is likely to lead to the development of new disease countermeasures and treatments for various diseases, but the detailed mechanism has not been sufficiently elucidated due to its complexity.
- Non-Patent Documents 1 to 6 Non-Patent Documents 1 to 6).
- the problem to be solved by the present invention is to provide a bacterium that induces the proliferation or activation of Th1 cells in the intestinal tract.
- Another object of the present invention is to identify oral bacteria that induce Crohn's disease and the like by colonizing the intestinal tract. As a result, it aims at providing the composition etc. which treat the disease, such as Crohn's disease, etc. which target the oral bacteria identified, or improve or prevent.
- IFN- ⁇ interferon gamma
- the cells were found to increase significantly (see FIGS. 1 and 2 etc.).
- Intestinal bacteria of mice in which this increase in Th1 cells was observed were cultured, and eight types of bacterial strains were successfully isolated (see FIG. 3 etc.).
- FIG. 4 etc. the mixed solution of these 8 strains was administered to a sterile mouse, it became clear that they could induce Th1 cells sufficiently (see FIG. 4 etc.).
- K. pneumoniae 2H7 strain or E. pneumoniae When E. coli strain 2B1 is established in a sterile IL-10-deficient mouse, In comparison with the colonization of 2B1 stock in E. coli. pneumoniae 2H7 strain colonization was found to cause more severe enteritis (see FIG. 17 etc.). From this, it is derived from saliva derived from a patient with Crohn's disease. Pneumoniae 2H7 strain was strongly suggested to be involved in the onset of enteritis.
- K.K. pneumoniae 2H7 strain is considered to have a gene and a structure involved in Th1 cell induction not possessed by BAA-2552 and 700721 strains.
- Th1 cells were remarkably induced in the large intestine as in the above-mentioned Crohn's disease patient. Furthermore, as a result of identifying a bacterium that induces Th1 cells, although it is a strain different from the 2H7 strain, We found 11E12 strain belonging to Klebsiella aeromobilis, a closely related species of pneumoniae.
- Table 1 shows the annotation and information (KEGG or UniProt) of genes related to carbohydrate metabolism in the genes related to the induction of the colon Th1 cells
- Table 2 shows the colon Th1 cells in the strains belonging to Klebsiella. The degree of induction and the degree of possession of the gene related to the carbohydrate metabolism are indicated.
- Table 3 shows the annotation and information (KEGG or UniProt) of genes related to membrane transport in the genes related to the induction of colonic Th1 cells
- Table 4 shows the induction level of colonic Th1 cells in strains belonging to Klebsiella. And the degree of possession of the gene related to the membrane transport.
- Table 5 shows gene annotations and information (KEGG or UniProt) related to amino acid metabolism in the genes related to the induction of the colon Th1 cells
- Table 6 shows the induction level of the colon Th1 cells in strains belonging to Klebsiella. And the degree of possession of the gene related to the amino acid metabolism.
- Table 7 shows gene annotations and information (KEGG or UniProt) related to gene regulation in the genes related to the induction of colon Th1 cells
- Table 8 shows the induction level of colon Th1 cells in strains belonging to Klebsiella. And the degree of possession of genes related to the gene regulation.
- Table 9 shows annotations and information (KEGG or UniProt) of genes other than Tables 1 to 8 in the genes related to the induction of the colon Th1 cells, and Table 10 shows colon Th1 cells in strains belonging to Klebsiella. The degree of induction level and the degree of possession of the other genes are shown.
- these genes having known functions include genes involved in the metabolism of mannose, fructose or galactose, and the present invention has been completed.
- the present invention provides the following.
- ⁇ 1> A bacterium that induces proliferation or activation of Th1 cells in the intestinal tract.
- ⁇ 2> The bacterium according to ⁇ 1>, further belonging to Klebsiella pneumoniae or Klebsiella aeromobilis.
- ⁇ 4> The bacterium according to any one of ⁇ 1> to ⁇ 3>, further comprising a gene involved in metabolism of at least one sugar of mannose, fructose, and galactose .
- a composition for stimulating immunity comprising the bacterium according to any one of ⁇ 1> to ⁇ 4> or a physiologically active substance derived from the bacterium as an active ingredient.
- the composition according to ⁇ 5> further comprising a composition for inducing proliferation or activation of Th1 cells.
- ⁇ 7> The bacterium according to any one of ⁇ 1> to ⁇ 4> or a physiologically active substance derived from the bacterium is ingested by the subject to induce proliferation or activation of Th1 cells in the subject.
- Method. ⁇ 8> A method of stimulating immunity in a subject by causing the subject to ingest the bacterium according to any one of ⁇ 1> to ⁇ 4> or a physiologically active substance derived from the bacterium.
- ⁇ 9> A vaccine composition comprising the bacterium according to any one of ⁇ 1> to ⁇ 4> or an antigen specific for the bacterium as an active ingredient.
- ⁇ 10> A method for inducing an immune response to the bacterium in the subject by causing the subject to ingest the bacterium according to any one of ⁇ 1> to ⁇ 4> or an antigen specific for the bacterium.
- ⁇ 11> A substance having antibacterial activity against the bacterium according to any one of ⁇ 1> to ⁇ 4> or the bacterium according to any one of ⁇ 1> to ⁇ 4> A composition for suppressing immunity comprising a substance that binds to a physiologically active substance as an active ingredient.
- composition according to ⁇ 11> or ⁇ 12> which is a composition for treating, ameliorating or preventing a disease caused by Th1 cells.
- ⁇ 14> A substance having antibacterial activity against the bacterium according to any one of ⁇ 1> to ⁇ 4> or the bacterium according to any one of ⁇ 1> to ⁇ 4> A method for suppressing the growth or activation of Th1 cells in a subject by causing the subject to ingest a substance that binds to the physiologically active substance.
- ⁇ 15> A substance having antibacterial activity against the bacterium according to any one of ⁇ 1> to ⁇ 4> or the bacterium according to any one of ⁇ 1> to ⁇ 4> A method for suppressing immunity in a subject by causing the subject to ingest a substance that binds to the physiologically active substance.
- ⁇ 16> A substance having antibacterial activity against the bacterium according to any one of ⁇ 1> to ⁇ 4> or the bacterium according to any one of ⁇ 1> to ⁇ 4> A method of treating a subject caused by Th1 cells in a subject to treat, ameliorate, or prevent a substance that binds to a physiologically active substance.
- ⁇ 17> A non-human animal in which the bacterium according to any one of ⁇ 1> to ⁇ 4> is established in the intestinal tract.
- the non-human animal according to ⁇ 17> which is a non-human animal model of a disease caused by Th1 cells.
- the method for producing a non-human animal according to ⁇ 17> or ⁇ 18>, A method comprising the steps of causing a non-human animal to ingest a bacterium that induces Th1 cell proliferation or activation in the intestinal tract and allowing the bacterium to settle in the intestinal tract of the animal.
- ⁇ 20> A kit for evaluating the proliferation or activation of Th1 cells, comprising the bacterium according to any one of ⁇ 1> to ⁇ 4>, intestinal epithelial cells, and peripheral blood mononuclear cells. . ⁇ 21> A kit for evaluating the proliferation or activation of Th1 cells, comprising intestinal epithelial cells and peripheral blood mononuclear cells.
- ⁇ 22> A method for screening a bacterium that induces proliferation or activation of Th1 cells in the intestinal tract, Ingesting a test sample into a non-human sterile animal; Detecting the number or activity of Th1 cells in the intestinal tract of the non-human sterile animal; Isolating bacteria from an intestinal sample of a non-human sterile animal in which Th1 cell proliferation or activation is detected in the above step.
- a method for screening a physiologically active substance that induces proliferation or activation of Th1 cells in the intestinal tract Ingesting a non-human sterile animal with a physiologically active substance derived from a bacterium that induces the proliferation or activation of Th1 cells in the intestine; Detecting the number or activity of Th1 cells in the intestinal tract of the non-human sterile animal; Determining that the physiologically active substance is a physiologically active substance that induces proliferation or activation of Th1 cells in the intestinal tract when Th1 cell proliferation or activation is detected in the step.
- a method for screening a bacterium that induces proliferation or activation of Th1 cells in the intestinal tract In a system comprising intestinal epithelial cells and peripheral blood mononuclear cells, adding a test bacterium to the intestinal epithelial cells; Detecting the number or activity of Th1 cells in the system; A step of determining that the test bacterium is a bacterium that induces the proliferation or activation of Th1 cells in the intestinal tract when Th1 cell proliferation or activation is detected in the step.
- a method for screening a physiologically active substance that induces proliferation or activation of Th1 cells in the intestinal tract In a system comprising intestinal epithelial cells and peripheral blood mononuclear cells, a step of adding to the intestinal epithelial cells a physiologically active substance derived from a bacterium that induces proliferation or activation of Th1 cells in the intestinal tract; Detecting the number or activity of Th1 cells in the system; A step of determining that the physiologically active substance is a physiologically active substance that induces proliferation or activation of Th1 cells in the intestinal tract when Th1 cell proliferation or activation is detected in the step; Including methods.
- a screening method for a substance that induces proliferation or activation of Th1 cells in the intestinal tract In a system comprising intestinal epithelial cells and peripheral blood mononuclear cells, a step of adding to the intestinal epithelial cells a bacterium that induces proliferation or activation of Th1 cells in the intestinal tract, and a test substance; Detecting the number or activity of Th1 cells in the system; When the number or activity of Th1 cells detected in the step is increased compared to that in the case where the test substance is not added, the test compound proliferates or activates Th1 cells in the intestinal tract. Determining that the substance is a substance that induces.
- ⁇ 27> A screening method for a substance that induces proliferation or activation of Th1 cells in the intestinal tract, A step of ingesting a test substance into the non-human animal according to ⁇ 17>; Detecting the number or activity of Th1 cells in the intestinal tract of the non-human animal; When the number or activity of Th1 cells detected in the step is increased from that in the case where the test substance is not ingested, the test substance proliferates or activates Th1 cells in the intestinal tract. Determining that the substance is a substance that induces.
- a composition for stimulating immunity comprising a bacterium, a physiologically active substance or a substance obtained by the screening method according to any one of ⁇ 22> to ⁇ 27> as an active ingredient.
- composition according to ⁇ 28> which is a composition for inducing the proliferation or activation of Th1 cells.
- a vaccine composition comprising a bacterium obtained by the screening method according to ⁇ 22> or ⁇ 24> or an antigen specific for the bacterium as an active ingredient.
- a method for screening a substance having an activity of inducing or exacerbating a disease caused by Th1 cells A step of ingesting a test substance into the non-human animal according to ⁇ 18>; Detecting the extent of a disease lesion caused by Th1 cells in the non-human animal; When the degree of lesion detected in the step is higher than that in the case where the test substance is not ingested, the test substance has an activity of inducing or exacerbating a disease caused by Th1 cells. And a step of determining that the substance has a substance.
- ⁇ 32> A composition for inducing or exacerbating a disease caused by Th1 cells, comprising a substance obtained by the screening method according to ⁇ 31> as an active ingredient.
- ⁇ 33> A method for screening a bacterium that suppresses the proliferation or activation of Th1 cells in the intestinal tract, Ingesting a test sample into a non-human sterile animal; Detecting the number or activity of Th1 cells in the intestinal tract of the non-human sterile animal; Isolating bacteria from an intestinal sample of a non-human sterile animal in which inhibition of Th1 cell proliferation or activation is detected in the above step.
- a method for screening a physiologically active substance that suppresses the proliferation or activation of Th1 cells in the intestinal tract A step of ingesting a non-human sterile animal with a physiologically active substance derived from a bacterium that suppresses the proliferation or activation of Th1 cells in the intestinal tract; Detecting the number or activity of Th1 cells in the intestinal tract of the non-human sterile animal; Determining that the physiologically active substance is a physiologically active substance that suppresses the proliferation or activation of Th1 cells in the intestinal tract when suppression of Th1 cell proliferation or activation is detected in the step. , Including methods.
- a method for screening a bacterium that suppresses the proliferation or activation of Th1 cells in the intestinal tract In a system comprising intestinal epithelial cells and peripheral blood mononuclear cells, adding a test bacterium to the intestinal epithelial cells; Detecting the number or activity of Th1 cells in the system; A step of determining that the test bacterium is a bacterium that suppresses the proliferation or activation of Th1 cells in the intestinal tract when suppression of the proliferation or activation of Th1 cells is detected in the step.
- ⁇ 36> A method for screening a physiologically active substance that suppresses the proliferation or activation of Th1 cells in the intestinal tract,
- a screening method for a substance that suppresses the proliferation or activation of Th1 cells in the intestinal tract In a system comprising intestinal epithelial cells and peripheral blood mononuclear cells, a step of adding to the intestinal epithelial cells a bacterium that induces proliferation or activation of Th1 cells in the intestinal tract, and a test substance; Detecting the number or activity of Th1 cells in the system; When the number or activity of Th1 cells detected in the step is lower than that in the case where the test compound is not added, the test substance proliferates or activates Th1 cells in the intestinal tract. And a step of determining that the substance is a substance that suppresses the above.
- a screening method for a substance that suppresses the proliferation or activation of Th1 cells in the intestinal tract A step of ingesting a test substance into the non-human animal according to ⁇ 17>; Detecting the number or activity of Th1 cells in the intestinal tract of the non-human animal; When the number or activity of Th1 cells detected in the step is lower than that in the case where the test substance is not taken, the test substance proliferates or activates Th1 cells in the intestinal tract. And a step of determining that the substance is a substance that suppresses the above.
- composition for suppressing immunity comprising a bacterium, a physiologically active substance or a substance obtained by the screening method according to any one of ⁇ 33> to ⁇ 38> as an active ingredient.
- composition according to ⁇ 39> further comprising a composition for inhibiting the proliferation or activation of Th1 cells.
- the composition according to ⁇ 39> or ⁇ 40> which is a composition for treating, ameliorating or preventing a disease caused by Th1 cells.
- ⁇ 42> A method for screening a substance having an activity of treating, ameliorating or preventing a disease caused by Th1 cells, A step of ingesting a test substance into the non-human animal according to ⁇ 18>; Detecting the extent of a disease lesion caused by Th1 cells in the non-human animal; When the degree of the lesion detected in the step is lower than that in the case where the test substance is not ingested, the test substance treats, improves or prevents a disease caused by Th1 cells. Determining that the substance has activity.
- ⁇ 43> A composition for treating, ameliorating or preventing a disease caused by Th1 cells, comprising a substance obtained by the screening method according to ⁇ 42> as an active ingredient.
- compositions for examining a disease caused by Th1 cells comprising an antibody that specifically recognizes the bacterium according to any one of ⁇ 1> to ⁇ 4>.
- composition for examining a disease caused by Th1 cells comprising a polynucleotide for detecting the nucleotide sequence specific for the bacterium according to any one of ⁇ 1> to ⁇ 4>.
- ⁇ 46> A physiologically active substance derived from the bacterium according to any one of ⁇ 1> to ⁇ 4>.
- ⁇ 48> An antibody that specifically recognizes the bacterium according to any one of ⁇ 1> to ⁇ 4>.
- ⁇ 49> A polynucleotide for detecting a nucleotide sequence specific to a bacterium according to any one of ⁇ 1> to ⁇ 4>.
- bacteria that induce proliferation or activation of Th1 cells in the intestinal tract such as 2H7 strain, 11E12 strain, 34E1 strain, BAA-1705 strain, 700603 strain, and 40B3 strain belonging to Klebsiella (Th1 cell-inducing bacteria) It is possible to suppress the growth or activation of Th1 cells by targeting, and suppressing or killing the growth of the bacteria. It is also possible to suppress immunity in the intestinal tract by suppressing the growth of the Th1 cell-inducing bacteria, thereby treating, improving or preventing diseases caused by Th1 cells such as Crohn's disease and ulcerative colitis. Is also possible.
- Th1 cell-inducing bacteria or physiologically active substances thereof by using such Th1 cell-inducing bacteria or physiologically active substances thereof, the proliferation or activation of Th1 cells is induced, and immunity is activated, thereby treating infectious diseases, It is also possible to enhance the anticancer effect.
- Bacterial species are shown in the figure. Also, eight isolated bacterial strains corresponding to OTUs are marked in green. Graph showing the percentage of Th1 cells in colon LPs of exGFB6 mice colonized with 8 species mixture (8-mix), Fu-21f + Ve-2E1 mixture, Kp-2H7, 7 species mixture (7-mix) or Ec-2B1 is there. In the figure, “GF” indicates data for the non-bacterial group. Error bars indicate mean ⁇ standard deviation. *** indicates P ⁇ 0.001 (based on one-way analysis of variance (ANOVA) followed by Tukey's post-hoc test).
- ANOVA analysis of variance
- the ratio (%) of IFN- ⁇ + cells (Th1 cells) in CD4 + T cells was analyzed by q-PCR in the colon LPs of GF ICI mice or GF BALB / c mice in which only Kp-2H7 was established. It is a graph which shows the result. In the figure, each point represents data for an individual mouse. Error bars indicate mean ⁇ standard deviation. * Indicates P ⁇ 0.05, ns indicates no significant difference (P> 0.05) (based on one-way analysis of variance (ANOVA) followed by Tukey's post-hoc test) ). It is the graph which analyzed the proliferation of Kp-2H7 in the presence of each antibiotic. Kp-2H7 was cultured in a 96-well plate for 24 hours at 37 ° C.
- Untreated SPF B6 mice (Cont, control) or SPF continuously treated with antibiotics (Tyl: tylosin or Amp: ampicillin) via drinking water from 4 days before administration of 2 ⁇ 10 8 CFU Kp-2H7
- “ ⁇ ” and “+ Kp-2H7” represent the Kp-2H7 non-administered group and the administered group, respectively.
- Each point represents data for an individual mouse. Error bars indicate mean ⁇ standard deviation.
- “GF WT” and “GF Il10 ⁇ / ⁇ ” indicate the results of GF wild type and GF Il10 ⁇ / ⁇ mice, respectively.
- “GF WT + Kp-2H7” and “GF Il10 ⁇ / ⁇ + Kp-2H7” The results of individuals in which only Kp-2H7 was established in wild-type and GF Il10 ⁇ / ⁇ mice are shown.
- the upper row shows a representative example of the result of analysis by H & E staining
- the lower row shows a representative example of the result of analysis by a scanning electron microscope.
- Scale bar indicates 200 ⁇ m It is a graph which shows the result of having analyzed the histological enterocolitis score about the proximal colon of GF wild type or GF Il10 ⁇ / ⁇ mouse which established only Kp-2H7 or Ec-2B1.
- “GF” indicates the result of the group not administered with bacteria.
- Each point represents data for an individual mouse. Error bars indicate mean ⁇ standard deviation. *** indicates P ⁇ 0.001 (based on one-way analysis of variance (ANOVA) followed by Tukey's post-hoc test).
- SPF B6 mice SPF WT or SPF continuously treated with antibiotics (Amp: 200 mg / L ampicillin) via drinking water from 4 days before oral administration or non-administration of 2 ⁇ 10 8 CFU Kp-2H7
- Am 200 mg / L ampicillin
- GF IL10 - / - shows the proximal colon of mice were observed through a microscope - mice, GF IL10 were established only Kp-2H7 - / - GF were established only mice and EC-2B1 IL10 - / It is a photograph. In the figure, a representative example of the result of analysis by H & E staining is shown, and the scale bar indicates 200 ⁇ m.
- GF indicates that the group was not administered with Kp-2H7.
- Each point represents data for each mouse. Error bars indicate mean ⁇ standard deviation. Ns indicates significant difference. Not (P> 0.05), *** indicates P ⁇ 0.001 (based on one-way analysis of variance (ANOVA) followed by Tukey's post-hoc test).
- K is a fluorescence micrograph showing the result of staining and observation.
- Th1 induction-related gene group is a graph showing the ratio of Th1 cells in the large intestine of WT mice, Myd88 ⁇ / ⁇ mice, Tlr4 ⁇ / ⁇ mice, and Myd88 ⁇ / ⁇ Trif ⁇ / ⁇ mice in which only Kp-2H7 is established.
- each point represents data for an individual mouse. Error bars indicate mean ⁇ standard deviation.
- FIG. 2 is a heat map showing differential gene expression in epithelial cells (EC) and dendritic cells (DC) of wild-type mouse colon colonized with only Kp-2H7 or BAA-2552 for 1 week.
- the heat map color represents a Z score obtained by normalizing each gene with an FPKM value.
- a graph showing the terms defined by gene ontology (GO) significantly increased in the gene set whose expression was enhanced in epithelial cells and dendritic cells of wild-type mouse colonized only with Kp-2H7 for 1 week. is there.
- the heat map color represents a Z score obtained by normalizing each gene with an FPKM value.
- FIG. 6 is a graph showing the results of analysis of each gene expression in the large intestine epithelial cells of mice in which only Kp-2H7 is established by qPCR over time. In the figure, the vertical axis indicates the value obtained by normalizing those of each gene by the expression of Gapdh.
- FIG. 6 is a graph showing the percentage of Th1 cells in colonic LPCD4 + cells of SPF WT mice or SPF Ifngr1 ⁇ / ⁇ mice treated with ampicillin (Amp) and forcibly administered Kp-2H7 or not. In the figure, each point represents data for an individual mouse.
- FIG. 10 is a graph showing the ratio of Th1 cells in colon LP of B6 mice in which a mixture of 13 strains (13-mix), Ef-11A1, Ka-11E12, or a mixture of 11 strains (11-mix) was established.
- each point represents data for an individual mouse. Error bars indicate mean ⁇ standard deviation. *** indicates P ⁇ 0.001 (based on one-way analysis of variance (ANOVA) followed by Tukey's post-hoc test).
- ANOVA analysis of variance
- Bacterial growth was determined based on measuring absorbance at a wavelength of 630 nm. Each data is also based on the results of at least 3 independent experiments. Expressed as mean ⁇ standard deviation. “Amp” is in the presence of ampicillin, “Tyrosin” is in the presence of tylosin (tyrosine), “MNZ” is in the presence of metronidazole, “VCM” is in the presence of vancomycin, “Spec” is in the presence of spectinomycin, and “MEPM” is in the mero In the presence of penem, “CAM” in the presence of clarithromycin, “TMP” in the presence of trimethoprim, “SM” in the presence of streptomycin, “GM” in the presence of gentamicin, “PL-B” in the presence of polymyxin B, “TC” indicates the culture result in the presence of tetracycline.
- ns indicates no significant difference (P> 0.05), *** indicates P ⁇ 0.001 (one-way analysis of variance (ANOVA) followed by Tukey's post-hoc test) based on).
- ANOVA one-way analysis of variance
- Tukey's post-hoc test based on.
- FIG. 6 is a graph showing the results of mapping the lead of each sample in the PRISM and UPen cohorts to Klebsiella species. It is a heat map figure which shows the result of having mapped the lead of each sample in the PRISM cohort to the gene arrangement
- the figure shows RPKM values for Th1-related genes. It is the schematic which shows the example of embodiment of this invention. It is a figure which shows having defined the difference with respect to the threshold value 25% in FIG. 21, 35, and 46 as a relative induction
- ⁇ Bacteria that induce Th1 cells in the intestinal tract As described above, when the present inventors establish 2H7 strain, 11E12 strain, 34E1 strain, BAA-1705 strain, 70000603 strain or 40B3 strain belonging to Klebsiella in the intestinal tract, significant induction of Th1 cells may occur. It was revealed. Thus, the present invention provides a bacterium that induces Th1 cell proliferation or activation in the intestine.
- the 2H7 strain, 11E12 strain, 34E1 strain, and 40B3 strain are bacteria (oral bacteria) that are usually present in the human oral cavity.
- the BAA-1705 strain and 7000060 strain are also bacteria usually present in the oral cavity of humans, but are bacteria (urinary bacteria) detected in human urine.
- the “Th1 cell” is a subgroup of CD4-positive helper T cells (Th cells) and means cells that enhance cellular immunity.
- Th1 cell activity refers to production of Th1 cytokines (IFN- ⁇ , etc.) by the cells, activation of cells such as macrophages, cytotoxic T cells (CTLs), etc. It means to include enhancement of sexual immunity.
- induction of proliferation or activation of Th1 cells is meant to include induction of differentiation from naive T cells to Th1 cells, which leads to proliferation or activation of Th1 cells. The effect of inducing proliferation or activation of Th1 cells in the intestinal tract can be evaluated by quantitatively detecting Th1 cell-specific markers (eg, CD4 and IFN- ⁇ ).
- Such quantitative detection can be performed by a known technique, for example, flow cytometry, imaging cytometry, ELISA method, radioimmunoassay, immunohistochemical staining method, immunoprecipitation method, immunoblotting, antibody array analysis method It can carry out by the method (immunological technique) which detects using antibodies, such as. Moreover, it can be determined using the screening method mentioned later, for example whether arbitrary microbes, a substance, etc. have the effect
- the Th1 cells in the intestinal tract have an action to induce proliferation or activation, and when it is 25% or more, the bacteria, substances, etc. proliferate the Th1 cells in the intestinal tract.
- the “bacteria that induce the proliferation or activation of Th1 cells in the intestinal tract” of the present invention is usually present in the oral cavity of humans, but by establishing in the intestinal tract, the proliferation or activation of Th1 cells is controlled. Inducing bacteria.
- the “bacteria that induce the proliferation or activation of Th1 cells in the intestinal tract” of the present invention preferably belongs to Klebsiella, more preferably belongs to Klebsiella pneumoniae or Klebsiella aeromobilis, and the growth or activity of Th1 cells in the intestinal tract. It is a bacterium that induces oxidization.
- the “bacteria that induce the proliferation or activation of Th1 cells in the intestinal tract” of the present invention is preferably easily established in an intestinal environment in which diversity is changed compared to a normal state by administration of an antibacterial agent. It is a bacterium.
- the “bacteria that induce the proliferation or activation of Th1 cells in the intestinal tract” of the present invention are bacteria that are easily established in the intestinal environment in which diversity has changed compared to a healthy state due to colitis.
- the “bacteria that induce the proliferation or activation of Th1 cells in the intestinal tract” of the present invention preferably have genes encoding at least 5 proteins selected from the following protein group, and more preferably: Possesses genes encoding at least 10 proteins selected from the protein group, more preferably possesses genes encoding at least 20 proteins selected from the following protein group, more preferably selected from the following protein group A gene encoding at least 30 proteins, and more preferably, a gene encoding at least 50 proteins selected from the following protein group.
- Mannose-1-phosphate guanyltransferase 1 Mannose-1-phosphate guanylyltransferase 1
- Multiphosphoryl transfer protein Multiphosphoryl transfer protein
- PTS system fructose-specific EIIABC component protein PTS system fructose-specific EIIABC component
- Phosphomannomutase / phosphoglucomutase Phosphomannmutase / phosphoglucomutase
- Mannosyl fructose-phosphate synthase 3-oxoacyl [acyl transport protein] reductase FabG (3-oxoacyl- [acyl-carrier-protein] reductase FabG)
- Rhamnosyl / mannosyltransferase rhamnosyl / mannosyltransferase
- Galactitol-1-phosphate 5-dehydrogenase galactitol-1-phosphate 5-dehydrogenase
- these proteins are specified by specific amino acid sequences (amino acid sequences defined by KEGG or UniProt IDs), but the proteins according to the present invention have these typical amino acid sequences. Not only the identified protein, but also functionally active derivatives thereof, functionally active fragments thereof, homologs thereof, hybridize to nucleic acids encoding this protein under conditions of high or low stringency. Also included are variants encoded by nucleic acids. Further, such a derivative, fragment, homologue or variant has at least 60% (preferably 70%, more preferably 80%, still more preferably 90%, more preferably) with respect to the specific amino acid sequence. 95%, particularly preferably 99%) proteins with homology.
- the proteins according to the present invention include proteins involved in the metabolism of mannose, fructose or galactose. Therefore, the “bacteria that induce the proliferation or activation of Th1 cells in the intestinal tract” of the present invention preferably express a gene involved in mannose, fructose, or galactose metabolism.
- the “bacteria that induce Th1 cell proliferation or activation in the intestinal tract” of the present invention preferably belongs to Klebsiella, does not form a capsule, and proliferates or activity of Th1 cells in the intestinal tract. More preferably, it belongs to Klebsiella pneumoniae, does not form a capsule, produces outer membrane vesicles (OMV) or OMV-like structures, and proliferates or activity of Th1 cells in the intestinal tract It is a bacterium that induces oxidization.
- OMV outer membrane vesicles
- the “bacteria that induce Th1 cell proliferation or activation in the intestinal tract” of the present invention is preferably a bacterium belonging to Klebsiella and having flagella, and preferably belonging to Klebsiella and stimulating to TLR5. It is a bacterium with
- the “bacteria that induce the proliferation or activation of Th1 cells in the intestinal tract” of the present invention are typically 2H7 strain, 11E12 strain, 34E1 strain, BAA-1705 strain, 7000060 strain, 40B3 strain belonging to Klebsiella. Is mentioned. For details of these bacteria, see Table 11.
- the nucleotide sequence specific to the 2H7 strain or 11E12 strain is not particularly limited, but preferably has 2H7 strain or 11E12 strain, but it is recognized in the BAA-2552 strain and 700721 strain belonging to Klebsiella as well as these strains. Nucleotide sequences that cannot be obtained (more preferably, nucleotide sequences that are not found in the BAA-2552 strain, the KCTC2242 strain, the KP-1, the 700721 strain, and the 13882 strain).
- the “bacteria that induce the proliferation or activation of Th1 cells in the intestinal tract” of the present invention include nucleotide sequences encoding 16S rRNA of 2H7 strain, 11E12 strain, 34E1 strain, BAA-1705 strain, 70000603 strain, or 40B3 strain.
- a bacterium containing a DNA having a nucleotide sequence having sex or identity, and more than 70% of nucleotide sequence specific to 2H7 strain, 11E12 strain, 34E1 strain, BAA-1705 strain, 70000603 strain or 40B3 strain Preferably 80% or more, more preferably 85% or more, still more preferably 90% or more, more preferably Ku is 95% (96% or more, 97% or more, 98% or more, 99% or more
- bacteria containing a DNA consisting of a nucleotide sequence having homology or identity preferably 80% or more, more preferably 85% or more, more preferably 90% or more, more preferably 95% or more (96% or more, 97% or more, 98% or more, 99% or more
- Th1 cell proliferation or activation can be induced by colonizing the above-described Th1 cell-inducing bacteria in the intestinal tract. Therefore, the present invention can provide a composition or method for inducing the proliferation or activation of Th1 cells.
- Th1 cells are CD4-positive helper T cells that produce IFN- ⁇ , play an important role in protecting against infection with pathogens such as Mycobacterium tuberculosis and Listeria, and monitor and eliminate cancerous cells. Also plays an important role.
- pathogens such as Mycobacterium tuberculosis and Listeria
- an immune checkpoint inhibitor anti-CTLA-4 antibody or anti-PD-L1
- the present invention can provide a composition or method for stimulating immunity.
- immunosal immunity activated or suppressed in the present invention includes not only mucosal immunity (eg, intestinal immunity) but also systemic immunity. Moreover, not only cellular immunity but also humoral immunity is included.
- the “bacteria that induce the proliferation or activation of Th1 cells in the intestinal tract” included as an active ingredient in the composition of the present invention is as described above, but it may be a live cell or a dead cell. May be. Moreover, according to the use of the active ingredient contained, the bacteria in which modification (genetic modification etc.) is performed may be sufficient. There is no restriction
- composition of the present invention only needs to contain one strain of bacteria that induces the proliferation or activation of Th1 cells in the intestinal tract, but may also include a plurality of strains.
- the composition can be used in combination, and as a result, when taken or absorbed together (in the case of a combined composition), the plurality of strains can also be present in two or more compositions (For example, each may be present in a separate composition).
- a physiologically active substance derived from a bacterium that induces proliferation or activation of Th1 cells in the intestinal tract refers to a substance contained in a bacterium that induces proliferation or activation of Th1 cells in the intestinal tract, the bacterium And more specifically, a polypeptide fraction, a polynucleotide fraction, a sugar chain fraction, a lipid fraction, a small molecule of the bacterium or a culture supernatant thereof. Metabolite fraction. Also included are outer membrane vesicles (OMV) or OMV-like structures produced by bacteria that induce Th1 cell proliferation or activation in the intestine.
- OMV outer membrane vesicles
- the physiologically active substance according to the present invention is preferably a substance that induces the proliferation or activation of Th1 cells in the intestinal tract, and more preferably a Toll-like receptor (TLR) involving MyD88 / Trif recognizes, It is a substance that functions as the ligand.
- TLR Toll-like receptor
- physiologically active substances include the bacteria, culture supernatants thereof, mouse intestinal samples (feces etc.), human oral samples (saliva etc.), human bladder samples, human From an intravaginal sample, a human ureteral sample (such as urine), a human intestinal sample, and the like, it is possible to identify by purifying the active ingredient by, for example, a screening method described later.
- composition of the present invention may be in the form of a pharmaceutical composition, a food or drink (including animal feed), or a reagent used for research purposes (eg, in vitro or in vivo experiments).
- a Th1 cell-inducing bacterium or a physiologically active substance derived from the bacterium which is an active ingredient of the composition of the present invention, induces the proliferation or activation of Th1 cells in the intestinal tract and stimulates immunity. Therefore, a pharmaceutical composition for treating, preventing or ameliorating infectious diseases such as tuberculosis, a food and drink, and a pharmaceutical composition for enhancing the anticancer effect by using in combination with an anticancer agent or an immune checkpoint inhibitor Furthermore, it can be suitably used as a pharmaceutical composition (vaccine adjuvant) for enhancing the immune response by using it together with a vaccine as a food or food / drink.
- “treatment” includes assistance for treatment.
- composition of the present invention can be formulated by a known pharmaceutical method.
- a known pharmaceutical method for example, capsule, tablet, pill, liquid, powder, granule, fine granule, film coating, pellet, troche, sublingual, chewing agent, buccal, paste, syrup, suspension, As elixirs, emulsions, coatings, ointments, plasters, cataplasms, transdermal preparations, lotions, inhalants, aerosols, injections, suppositories, etc.
- carriers that are acceptable as pharmacological or food and drink, specifically, sterile water and physiological saline, vegetable oils, solvents, bases, emulsifiers, suspending agents, surfactants, stabilizers, Flavoring agent, fragrance, excipient, vehicle, preservative, binder, diluent, tonicity agent, soothing agent, extender, disintegrant, buffering agent, coating agent, lubricant, colorant, sweetness Can be suitably combined with an agent, a thickening agent, a flavoring agent, a solubilizing agent or other additives.
- the composition of the present invention is incorporated in the intestinal tract. It may be combined with a composition that allows for efficient delivery.
- a composition that enables delivery into the intestinal tract is not particularly limited, and a known composition can be appropriately employed.
- a pH-sensitive composition a composition that suppresses release to the intestinal tract.
- Products cellulosic polymers, acrylic acid polymers and copolymers, vinyl acid polymers and copolymers, etc.
- bioadhesive compositions that specifically adhere to the intestinal mucosa (for example, US Pat. No. 6,368,586) Polymers described in the specification), protease inhibitor-containing compositions, and compositions that are specifically degraded by intestinal enzymes.
- compositions for inducing proliferation or activation of Th1 cells of the present invention when used as a pharmaceutical composition, known substances (antiviral agents, antibacterials) used for the treatment, prevention or improvement of infectious diseases and cancers are used. Agents, anticancer agents, immune checkpoint inhibitors, etc.) and may be used in combination with such substances.
- antiviral agents used for the treatment, prevention or improvement of infectious diseases and cancers.
- Agents, anticancer agents, immune checkpoint inhibitors, etc. and may be used in combination with such substances.
- vaccine adjuvant in addition to antigens that are active ingredients of vaccines (for example, antigens specific to bacteria or viruses, cancer-specific antigens), other known vaccine adjuvants and immunopotentiators
- the compositions of the invention may be included and used in combination with these substances.
- the food or drink is, for example, a health food, a functional food, a food for specified health use, a nutritional functional food, a functional indication food, a nutritional supplement, a food for the sick, or It can be an animal feed.
- Specific examples of food and drink include fermented beverages, oil-containing products, soups, milk beverages, soft drinks, tea beverages, alcoholic beverages, drinks, jelly-like beverages, etc .; carbohydrate-containing foods; livestock processed foods Processed fish food; processed vegetable food; semi-solid food; fermented food; confectionery; retort product;
- health foods and drinks prepared in the form of powder, granules, tablets, capsules, liquid, paste or jelly are also included.
- manufacture of the food-drinks in this invention can be implemented with a manufacturing technique well-known in the said technical field.
- an ingredient for example, nutrient etc.
- the product of the composition of the present invention (pharmaceutical product, food / beverage product, vaccine, reagent) or instructions thereof induces the proliferation or activation of Th1 cells, or treats, improves or prevents diseases such as infectious diseases and cancers. Therefore, it may be attached with an indication to be used.
- health function indications as health functional foods are provided in the present invention so that the form and the subject can be distinguished from general foods. It may be attached to a product of the composition.
- “labeled product or instructions” means that the product body, container, packaging, etc. are marked, or instructions, package inserts, promotional materials, or other printed materials that disclose product information. It means that the display is attached to.
- composition of the present invention may be in the form of a kit.
- kits include, for example, bacteria that induce Th1 cell proliferation or activation, or physiologically active substances derived from the bacteria, antiviral agents, antibacterial agents, anticancer agents, immune checkpoint inhibitors, antigens, and the like.
- Vaccine adjuvants and the like are usually present as two or more substances (compositions and the like), but may be mixed and prepared into a single composition before being ingested by a subject.
- the present invention is characterized in that a subject is ingested with a composition for inducing the proliferation or activation of Th1 cells, or a Th1 cell-inducing bacterium that is an active ingredient thereof, or a physiologically active substance derived from the bacterium.
- the present invention also provides a method for inducing proliferation or activation of Th1 cells in a subject, or a method for stimulating immunity in the subject.
- composition of the present invention or the active ingredient thereof can be used for animals including humans, but there are no particular restrictions on animals other than humans, and it can be used for various livestock, poultry, pets, laboratory animals, etc. It can be. Specific examples include, but are not limited to, pigs, cows, horses, sheep, goats, chickens, ducks, ostriches, ducks, dogs, cats, rabbits, hamsters, mice, rats, monkeys, and the like.
- the composition for inducing the proliferation or activation of Th1 cells of the present invention, or the target of ingestion thereof includes animals infected with viruses, bacteria, etc., regardless of their onset. It is done. From the viewpoint of prevention, the composition of the present invention may be ingested by an animal that is not infected or suspected of being infected with a virus, bacteria, or the like. Furthermore, from the viewpoint of recurrence prevention, the composition of the present invention can be suitably used for animals that carry viruses, bacteria, etc. that do not have symptoms. Similarly, the composition of the present invention can be suitably used not only for animals suffering from cancer but also for animals suspected of suffering from cancer and animals after anticancer therapy.
- the method for ingesting the composition of the present invention is not particularly limited, and may be oral administration or parenteral administration (for example, administration into the intestinal tract).
- the subject of intake of the composition of the present invention should reduce gastric acid production by ingesting a proton pump inhibitor (PPI) or the like.
- PPI proton pump inhibitor
- antibiotics eg, ampicillin, tylosin
- the amount of intake depends on the age, weight, disease symptoms, health condition of the subject, type of composition (pharmaceuticals, food and drink, etc.), ingestion method, etc. A person skilled in the art can select as appropriate.
- Th1 cell-inducing bacteria establish in the intestinal tract and induce Th1 cell proliferation or activation, thereby inducing diseases caused by Th1 cells such as Crohn's disease and ulcerative colitis. Therefore, if the bacteria are removed from the intestinal tract by inducing an immune response targeting the Th1 cell-inducing bacteria, the disease can be treated, ameliorated, or prevented. Therefore, the present invention can provide a vaccine composition or a method for inducing an immune response against the bacterium for treating, ameliorating or preventing a disease caused by Th1 cells.
- the “disease caused by Th1 cells” means a disease induced by the proliferation or activation of Th1 cells, and chronic diseases such as inflammatory bowel diseases (Crohn's disease, ulcerative colitis, inflammatory bowel disease) Inflammatory bowel disease and the like) and autoimmune diseases such as type 1 diabetes, rheumatoid arthritis, experimental immune encephalitis (EAE), multiple sclerosis, systemic lupus erythematosus, and chronic inflammatory diseases.
- inflammatory bowel diseases Crohn's disease, ulcerative colitis, inflammatory bowel disease
- autoimmune diseases such as type 1 diabetes, rheumatoid arthritis, experimental immune encephalitis (EAE), multiple sclerosis, systemic lupus erythematosus, and chronic inflammatory diseases.
- the “bacteria that induce the proliferation or activation of Th1 cells in the intestinal tract” included as an active ingredient in the composition of the present invention are as described above, and may be live or dead. Also good. Moreover, according to the use of the active ingredient contained, the bacteria in which modification (genetic modification etc.) is performed may be sufficient. There is no particular limitation on such modification, and when the composition of the present invention is a vaccine composition, modification that suppresses the action of inducing the proliferation or activation of Th1 cells in the intestinal tract and does not induce inflammation or the like. Is mentioned.
- the composition of the present invention only needs to contain one strain of bacteria that induces the proliferation or activation of Th1 cells in the intestinal tract, but may also include a plurality of strains. Alternatively, the composition can be used in combination, and as a result, when taken or absorbed together (in the case of a combined composition), the plurality of strains can also be present in two or more compositions .
- the “antigen specific to a bacterium that induces Th1 cell proliferation or activation in the intestinal tract” is a substance (polypeptide, polynucleotide, sugar chain, lipid, etc.) contained in the bacterium, It means a substance having antigenicity or immunogenicity.
- immunogenic means that a primary immune response or a memory immune response can be activated.
- Immunune response includes responses of CD4 positive T lymphocytes, CD8 positive T lymphocytes and B lymphocytes.
- T lymphocytes such responses can be proliferative and / or cytokines (eg, IL-2, IL-3, IL-4, IL-5, IL-6, IL-12, IL-13, IL -15, TNF- ⁇ , IFN- ⁇ ) production type.
- these responses may result in the production of cytotoxic T lymphocytes (CTL).
- CTL cytotoxic T lymphocytes
- a B lymphocyte reaction may result in antibody production by reacting B lymphocytes.
- “antigenic” means capable of being recognized by an antigen-specific T cell receptor (TCR) on an antibody molecule or an activated effector T cell (eg, cytokine producing T cell, CTL, etc.).
- a bacterium-specific antigen that induces Th1 cell proliferation or activation in the intestinal tract is recognized by an antibody that specifically recognizes the bacterium or the like, and is capable of binding to the substance.
- an appropriate antigen presenting cell (APC) and binding to an appropriate major histocompatibility complex (MHC) molecule it is recognized by the TCR on the effector T cell induced in response to the bacteria and the like. It is meant to include substances that can bind.
- bacteria-specific antigens by publicly known screening methods (for example, Paul WE, Fundamental ⁇ ⁇ ⁇ Immunology) using the reactivity with antigen-specific antisera and / or T lymphocytes as an index. 1993, 3rd edition, pages 243-247, Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring HarborLaboratory, 1998).
- the amino acid sequence of the bacteria-specific antigen (polypeptide) is analyzed using a computer program (for example, MHC-THREAD, EpiPredict, HLA-DR4 binding, ProPred, BIMAS, SVMHC, NetMHC, PREDICT, LpPep, SYFPEITHI, RankPep ).
- the vaccine composition of the present invention can be formulated by a known pharmaceutical method.
- Internal intramuscular, intravenous, intratracheal, intranasal, transdermal, intradermal, subcutaneous, intraocular, vaginal
- carriers that are acceptable as pharmacological or food and drink, specifically, sterile water and physiological saline, vegetable oils, solvents, bases, emulsifiers, suspending agents, surfactants, stabilizers, Flavoring agent, fragrance, excipient, vehicle, preservative, binder, diluent, tonicity agent, soothing agent, extender, disintegrant, buffering agent, coating agent, lubricant, colorant, sweetness Can be suitably combined with an agent, a thickening agent, a flavoring agent, a solubilizing agent or other additives.
- the vaccine composition of the present invention may contain a known vaccine adjuvant and immune enhancer.
- the vaccine adjuvant include aluminum hydroxide, KLH, MPL, QS21, complete Freund's adjuvant, incomplete Freund's adjuvant, aluminum phosphate, BCG, alum, TLR agonists such as CpG DNA, and the like.
- the aspect to which adjuvants, such as albumin, a wetting agent, and an emulsifier, are added as needed may be sufficient.
- the immunopotentiator include various cytokines (for example, IL-12, IL-18, GM-CSF, IFN ⁇ , IFN ⁇ , IFN ⁇ , Flt3 ligand).
- the product of the composition of the present invention (pharmaceutical, vaccine) or instructions thereof induces an immune response against bacteria that induces the proliferation or activation of Th1 cells in the intestine, and treats, ameliorates diseases caused by Th1 cells, or It may be an indication that it is used for prevention.
- labeled product or instructions means that the product body, container, packaging, etc. are marked, or instructions, package inserts, promotional materials, or other printed materials that disclose product information. It means that the display is attached to.
- composition of the present invention may be in the form of a kit.
- a kit for example, bacteria that induce the proliferation or activation of Th1 cells or antigens specific to the bacteria, vaccine adjuvants, immunopotentiators, etc. are usually present as two or more substances (compositions, etc.)
- the composition can be prepared by mixing or the like before ingesting the subject.
- the present invention also provides a method for inducing an immune response to a bacterium in a subject, characterized in that the subject ingests a vaccine composition, or a bacterium which is an active ingredient thereof or an antigen specific for the bacterium, or Also provided is a method for treating, ameliorating or preventing a disease caused by Th1 cells in a subject.
- composition of the present invention or the active ingredient thereof can be used for animals including humans, but there are no particular restrictions on animals other than humans, and it can be used for various livestock, poultry, pets, laboratory animals, etc. It can be.
- the subject of ingestion of the vaccine composition of the present invention, or an active ingredient thereof possesses a bacterium that induces the proliferation or activation of Th1 cells in the intestinal tract regardless of the onset of diseases caused by Th1 cells.
- a bacterium that induces the proliferation or activation of Th1 cells in the intestinal tract regardless of the onset of diseases caused by Th1 cells.
- the composition of the present invention may be ingested by an animal that does not have or is suspected to have the bacterium.
- the method of ingesting the composition of the present invention is not particularly limited, and may be oral administration or parenteral administration.
- the amount of intake is appropriately determined by those skilled in the art depending on the age, weight, symptom of the disease, health condition, dosage form of the composition, ingestion method, etc. You can choose.
- Th1 cell-inducing bacteria are established in the intestinal tract, induce Th1 cell proliferation or activation, and enhance the immune action, thereby causing Th1 cells such as Crohn's disease and ulcerative colitis. Induces disease. Therefore, if the bacteria are removed from the intestinal tract, the induction of Th1 cells is suppressed, and the immune action is suppressed, leading to the treatment of the disease and the like.
- a physiologically active substance derived from the bacteria that induces Th1 cell induction and immunostimulation is a substance that binds to the physiologically active substance (a substance corresponding to “test substance” in the figure). ), The induction of Th1 cells is suppressed and the immunity is suppressed, leading to the treatment of the disease and the like.
- the present invention provides a Th1 cell comprising, as an active ingredient, a substance having antibacterial activity against a bacterium that induces proliferation or activation of Th1 cells in the intestinal tract or a substance that binds to a physiologically active substance derived from the bacterium.
- the present invention provides a composition for suppressing the proliferation or activation of, a composition for suppressing immunity, or a composition for treating, ameliorating or preventing a disease caused by Th1 cells.
- the “substance having antibacterial activity against bacteria that induces the proliferation or activation of Th1 cells in the intestinal tract” contained as an active ingredient in the composition of the present invention is not particularly limited as long as it has the aforementioned activity.
- examples thereof include antibiotics, lytic substances (phages, lytic enzymes, etc.), antibodies that specifically recognize the bacteria, and the above-mentioned bacterial-specific antigens.
- the antibiotic include meropenem, tetracycline, polymyxin-B, trimethoprim, gentamicin and the like for Kp-2H7 and the like, and meropenem, tetracycline, trimethoprim, and the like for Ka-11E12 and the like. Ampicillin, gentamicin, streptomycin, spectinomycin and the like can be mentioned.
- other antibiotics shown in Tables 12 and 14 are sensitive to bacteria that induce proliferation or activation of Th1 cells in the intestinal tract.
- the physiologically active substance derived from bacteria that induces the proliferation or activation of Th1 cells in the intestinal tract is as described above.
- the substance that binds to the physiological activity may be any substance that binds to the physiologically active substance so that the induction of Th1 cells and the induction of immunostimulation by the physiologically active substance can be suppressed.
- low molecular weight compounds that bind to the physiologically active substance.
- a substance that binds to a binding site to TLR involving MyD88 / Trif in the physiologically active substance can be mentioned as a preferred embodiment.
- composition of the present invention may contain a plurality of substances having such antibacterial activity and / or antibodies that bind to the physiologically active substance.
- the composition can be used in combination, and as a result, when ingested or absorbed together (in the case of a combined composition), the plurality of substances can also be present in two or more compositions .
- composition of the present invention may be in the form of a pharmaceutical composition, a food or drink (including animal feed), or a reagent used for research purposes (eg, in vitro or in vivo experiments).
- the composition of the present invention suppresses Th1 induction and immunity in the intestinal tract of bacteria that induce Th1 cell proliferation or activation in the intestinal tract. Therefore, the treatment and prevention of the diseases caused by the above Th1 cells. Or it is used suitably as a pharmaceutical composition and food-drinks for improvement.
- composition of the present invention can be formulated by a known pharmaceutical method.
- a known pharmaceutical method for example, capsule, tablet, pill, liquid, powder, granule, fine granule, film coating, pellet, troche, sublingual, chewing agent, buccal, paste, syrup, suspension, As elixirs, emulsions, coatings, ointments, plasters, cataplasms, transdermal preparations, lotions, inhalants, aerosols, injections, suppositories, etc.
- carriers that are acceptable as pharmacological or food and drink, specifically, sterile water and physiological saline, vegetable oils, solvents, bases, emulsifiers, suspending agents, surfactants, stabilizers, Flavoring agent, fragrance, excipient, vehicle, preservative, binder, diluent, tonicity agent, soothing agent, extender, disintegrant, buffering agent, coating agent, lubricant, colorant, sweetness Can be suitably combined with an agent, a thickening agent, a flavoring agent, a solubilizing agent or other additives.
- the composition of the present invention is used in the preparation of the intestinal tract particularly in preparations for oral administration from the viewpoint of more efficiently suppressing the proliferation or activation of Th1 cells in the intestinal tract and immunity. May be combined with a composition that allows efficient delivery into the body.
- a composition that enables delivery into the intestinal tract is not particularly limited, and a known composition can be appropriately employed.
- a pH-sensitive composition a composition that suppresses release to the intestinal tract.
- Products (cellulosic polymers, acrylic acid polymers and copolymers, vinyl acid polymers and copolymers, etc.), bioadhesive compositions that specifically adhere to the intestinal mucosa (for example, US Pat. No. 6,368,586) Polymers described in the specification), protease inhibitor-containing compositions, and compositions that are specifically degraded by intestinal enzymes.
- composition for suppressing the growth or activation of Th1 cells or immunity of the present invention when used as a pharmaceutical composition, known substances (for example, used for treatment, prevention or improvement of diseases caused by Th1 cells) (Anti-inflammatory agent, immunosuppressive agent) may be further contained, and may be used in combination with such a substance.
- known substances for example, used for treatment, prevention or improvement of diseases caused by Th1 cells
- Anti-inflammatory agent, immunosuppressive agent may be further contained, and may be used in combination with such a substance.
- the food or drink is, for example, a health food, a functional food, a food for specified health use, a nutritional functional food, a functional indication food, a nutritional supplement, a food for the sick, or It can be an animal feed.
- foods and drinks include fermented beverages, oil-containing products, soups, milk beverages, soft drinks, tea beverages, alcoholic beverages, drinks, jelly-like beverages, etc .; carbohydrate-containing foods; livestock processed foods Processed fish food; processed vegetable food; semi-solid food; fermented food; confectionery; retort product;
- health foods and drinks prepared in the form of powder, granules, tablets, capsules, liquid, paste or jelly are also included.
- manufacture of the food-drinks in this invention can be implemented with a manufacturing technique well-known in the said technical field.
- the product of the composition of the present invention (medicine, food and drink, reagent) or instructions thereof suppress Th1 cell proliferation or activation, suppress immunity, or treat, ameliorate or prevent diseases caused by Th1 disease It may be attached with an indication that it is used for this purpose.
- health function indications as health functional foods are provided in the present invention so that the form and the subject can be distinguished from general foods. It may be attached to a product of the composition.
- labeled product or instructions means that the product body, container, packaging, etc. are marked, or instructions, package inserts, promotional materials, or other printed materials that disclose product information. It means that the display is attached to.
- the composition of the present invention may be in the form of a kit.
- the present invention provides a composition for inhibiting Th1 cell proliferation or activation, a composition for inhibiting immunity, or a substance having antibacterial activity against Th1 cell-inducing bacteria that are active ingredients thereof.
- a method for suppressing the proliferation or activation of Th1 cells in a subject a method for suppressing immunity in the subject, or the subject, characterized in that the subject ingests a substance that binds to a physiologically active substance derived from the bacterium
- the present invention also provides a method for treating, ameliorating or preventing a disease caused by Th1 cells.
- composition of the present invention or the active ingredient thereof can be used for animals including humans, but there are no particular restrictions on animals other than humans, and it can be used for various livestock, poultry, pets, laboratory animals, etc. It can be.
- composition for inducing the proliferation or activation of Th1 cells of the present invention may be Th1 in the intestinal tract regardless of the onset of diseases caused by Th1 cells.
- examples include animals carrying bacteria that induce cell proliferation or activation.
- the composition of the present invention may be ingested by an animal that does not have or is suspected to have the bacterium.
- the method for ingesting the composition of the present invention is not particularly limited, and may be oral administration or parenteral administration (for example, administration into the intestinal tract). From the viewpoint of further improving the effects of the composition of the present invention, the subject of intake of the composition of the present invention should reduce gastric acid production by ingesting a proton pump inhibitor (PPI) or the like. Is preferred.
- PPI proton pump inhibitor
- the amount of intake depends on the age, weight, disease symptoms, health condition of the subject, type of composition (pharmaceuticals, food and drink, etc.), ingestion method, etc. A person skilled in the art can select as appropriate.
- ⁇ Method 1 for screening bacteria that induce or suppress Th1 cells in intestinal tract it was possible to identify a bacterium that induces the proliferation or activation of Th1 cells in the intestine by ingesting human saliva into a sterile mouse. Therefore, the present invention provides the following screening method.
- a method for screening a bacterium that induces Th1 cell proliferation or activation in the intestinal tract comprising: ingesting a test sample into a non-human sterile animal; and the number or activity of Th1 cells in the intestinal tract of the non-human sterile animal. And a step of isolating bacteria from an intestinal sample of a non-human sterile animal in which Th1 cell proliferation or activation has been detected in the step.
- the “test sample” may be a human-derived sample.
- a human oral sample saliva etc.
- a human intravesical sample a human vaginal sample
- a human ureteral sample urine
- human intestinal samples or cultures thereof.
- an oral sample derived from a human suffering from a disease caused by Th1 cells is preferably used.
- non-human sterile animal means a non-human animal that is born and grows under aseptic conditions.
- animals other than humans include, but are not limited to, mice, rats, monkeys, pigs, cows, horses, sheep, goats, chickens, ducks, ostriches, ducks, dogs, cats, rabbits, hamsters and the like. . In these animals, mice are preferably used.
- “detection” of proliferation or activation of Th1 cells in the intestinal tract can be performed by detecting Th1 cell-specific markers (eg, CD4 and IFN- ⁇ ).
- Th1 cell-specific markers eg, CD4 and IFN- ⁇
- detection can be performed by a known technique, for example, antibodies such as flow cytometry, imaging cytometry, ELISA method, radioimmunoassay, immunohistochemical staining method, immunoprecipitation method, immunoblotting, antibody array analysis method, etc.
- the method of detecting using (immunological technique) is mentioned.
- limiting in particular as a timing of detection If it is an expert, it can adjust suitably according to the kind etc. of animal to be used.
- Th1 cell proliferation if a significant increase in Th1 cell-specific marker is observed compared to a control (eg, a non-human sterile animal that has not been ingested the test sample), Th1 cell proliferation or Although it can be determined that activation has been detected, the control and determination method is not limited thereto.
- a control eg, a non-human sterile animal that has not been ingested the test sample
- the “intestinal sample” may be a sample containing Th1 cell-inducing bacteria established in a non-human sterile animal, and examples thereof include a stool sample of the animal or a culture thereof.
- the method for “isolating” bacteria from an intestinal sample is not particularly limited, and includes known methods (dilution culture, single colony culture by plate culture).
- the intestinal tract containing the obtained bacterium Bacteria that induce Th1 cell proliferation or activation in the intestinal tract can be isolated by ingesting a sample into a new non-human sterile animal instead of the test sample and performing the above-described screening multiple times. .
- the present invention can also provide the following screening method.
- a method of screening for bacteria that suppress the proliferation or activation of Th1 cells in the intestinal tract comprising a step of ingesting a test sample into a non-human sterile animal, and the number or activity of Th1 cells in the intestinal tract of the non-human sterile animal. And a step of isolating bacteria from an intestinal sample of a non-human sterile animal in which suppression of Th1 cell proliferation or activation was detected in the step.
- a physiologically active substance responsible for induction can be screened from bacteria that induce Th1 cell proliferation or activation in the intestinal tract. That is, the present invention also provides the following screening method.
- a method for screening a physiologically active substance that induces proliferation or activation of Th1 cells in the intestinal tract wherein a physiologically active substance derived from a bacterium that induces proliferation or activation of Th1 cells in the intestinal tract is treated with a non-human sterile animal Ingesting, the step of detecting the number or activity of Th1 cells in the intestinal tract of the non-human sterile animal, and when the proliferation or activation of Th1 cells is detected in the step, the physiologically active substance is Determining that the substance is a physiologically active substance that induces the proliferation or activation of Th1 cells in the intestinal tract.
- the “physiologically active substance” used in the method means a substance contained in a bacterium that induces the proliferation or activation of Th1 cells in the intestinal tract, a secreted product of the bacterium, and a metabolite by the bacterium. May be a single isolated substance, or a fraction containing a plurality of substances (for example, a polypeptide fraction, a polynucleotide fraction, a sugar chain of the bacterium or a culture supernatant thereof) Fraction, lipid fraction, and low-molecular-weight metabolite fraction).
- the other elements in the screening method are the same as in ⁇ Method 1 for screening bacteria that induce or suppress Th1 cells in the intestinal tract>.
- the present invention can also provide the following screening method.
- a method for screening a physiologically active substance that suppresses the proliferation or activation of Th1 cells in the intestinal tract wherein a physiologically active substance derived from a bacterium that inhibits the proliferation or activation of Th1 cells in the intestinal tract is treated with a non-human sterile animal
- the physiologically active substance when the suppression of the proliferation or activation of Th1 cells is detected in the step the step of detecting the number or activity of Th1 cells in the intestinal tract of the non-human sterile animal, And a step of determining that it is a physiologically active substance that suppresses the proliferation or activation of Th1 cells in the intestinal tract.
- a bacterium that induces the proliferation or activation of Th1 cells in the intestinal tract is orally ingested by a mouse, so that the bacteria are established in the intestinal tract, and Th1 cells are induced, It has become clear that inflammation is caused.
- the present invention provides a non-human animal or a non-human animal model of a disease caused by Th1 cells, in which bacteria that induce the proliferation or activation of Th1 cells in the intestinal tract are established in the intestinal tract.
- such an animal can be produced by ingesting a bacterium that induces the proliferation or activation of Th1 cells in the intestinal tract into a non-human animal and fixing the bacterium in the intestinal tract of the animal. Therefore, the present invention also provides a manufacturing method thereof.
- the “bacteria that induce the proliferation or activation of Th1 cells in the intestinal tract” to be ingested by non-human animals is as described above, and may be live or dead. Moreover, according to the use of the active ingredient contained, the bacteria in which modification (genetic modification etc.) is performed may be sufficient. There is no restriction
- mice are preferably used.
- the “non-human animal” is preferably a non-human aseptic animal that is born and grows under aseptic conditions from the viewpoint that the bacteria are more likely to settle.
- non-human animal a non-human animal in which the activity of IL-10 is suppressed is preferable from the viewpoint of facilitating inflammation.
- suppression of IL-10 activity includes not only suppression of its function but also suppression of expression (expression at the translation level or transcription level).
- the suppression of the function can be performed by administering an IL-10 specific antibody, aptamer or the like to a non-human animal.
- suppression of expression can be performed by gene recombination (so-called knockout), genome editing, or administration of siRNA, shRNA, or antisense RNA.
- the method for “ingesting” the bacterium to a non-human animal is not particularly limited, and is usually performed by oral administration, but may be parenteral administration (for example, administration into the intestinal tract).
- oral administration from the standpoint that the bacteria are more likely to settle, non-human animals can reduce gastric acid production by ingesting a proton pump inhibitor (PPI) or the like, or antibiotics It is preferable to take in.
- PPI proton pump inhibitor
- a non-human animal may ingest one strain of the bacterium, but may ingest a plurality of strains.
- Th1 cell proliferation or activation is induced in the intestinal tract. It can be suitably used as a non-human model animal for diseases caused by Th1 cells. Therefore, the present invention also provides the following screening method using this model animal.
- a screening method for a substance that induces the proliferation or activation of Th1 cells in the intestinal tract wherein a test substance is applied to a non-human animal in which bacteria that induce the proliferation or activation of Th1 cells in the intestinal tract are established.
- a step of determining that the test substance is a substance that induces proliferation or activation of Th1 cells in the intestinal tract when it is increased.
- test substance a synthetic low molecular weight compound, an antibody, polypeptide, polynucleotide, lipid, saccharides (monosaccharide, disaccharide, oligosaccharide, sugar chain etc.) and Libraries composed of these substances, cell (bacteria, plant cells, animal cells) extracts and cultures (culture supernatants, etc.), bacterial secretion products, bacterial metabolites, marine organisms, plants or animals Extracts, soils, random phage peptide display libraries.
- the “bacteria that induce the proliferation or activation of Th1 cells in the intestinal tract” are as described above.
- the method of “ingesting” a test substance to a non-human animal is not particularly limited, and is usually performed by oral administration, but may be parenteral administration (for example, administration into the intestinal tract).
- parenteral administration for example, administration into the intestinal tract.
- PPI proton pump inhibitor
- the other elements in the screening method are the same as in ⁇ Method 1 for screening bacteria that induce or suppress Th1 cells in the intestinal tract>.
- the present invention can also provide the following screening method.
- a method for screening a substance having an activity of inducing or exacerbating a disease caused by Th1 cells, wherein a bacterium that induces the proliferation or activation of Th1 cells in the intestinal tract is established in the intestinal tract.
- a step of causing a non-human model animal having a disease caused by a cell to ingest a test substance, a step of detecting a degree of a lesion caused by a Th1 cell in the non-human animal, and a degree of the lesion detected in the step Determining that the test substance is a substance having an activity of inducing or exacerbating a disease caused by Th1 cells when the test substance is increased in comparison with the case where the test substance is not ingested. , Including methods.
- a screening method for a substance that suppresses the proliferation or activation of Th1 cells in the intestinal tract wherein a test substance is applied to a non-human animal in which bacteria that induce the proliferation or activation of Th1 cells in the intestinal tract are established.
- a step of determining that the test substance is a substance that suppresses the proliferation or activation of Th1 cells in the intestinal tract when it is reduced further.
- a step of ingesting a test substance into a non-human animal model of a disease caused by Th1 cells; a step of detecting the degree of a disease lesion caused by Th1 cells in the non-human animal; and the lesion detected in the step When the degree is lower than that in the case where the test substance is not ingested, the test substance is determined to be a substance having an activity of treating, ameliorating or preventing a disease caused by Th1 cells.
- the “lesion” of the disease caused by Th1 cells is not particularly limited, and those skilled in the art can appropriately select the disease according to the target disease.
- an inflammatory bowel disease such as ulcerative colitis
- it can be evaluated by observing the degree of inflammation in the intestinal tract, as shown in FIGS.
- a disease score determined for each disease for example, “Disease Activity Index (DAI)” in ulcerative colitis etc., S. Wirtz, C. Neufert, B. Weigmann, MF Neuroth, Nat Protoc. 2, 541 (2007)
- DAI Disease Activity Index
- Th1 cell-inducing bacteria or physiologically active substances derived from the bacteria are taken up by intestinal epithelial cells and transferred to dendritic cells, or the physiological activity It is envisaged that the substance will be captured directly by the dendritic cells. Furthermore, it is assumed that differentiation from naive T cells to Th1 cells is induced by cytokines produced by dendritic cells presenting the physiologically active substance as an antigen.
- the present invention also provides the following screening method.
- a method for screening a bacterium that induces proliferation or activation of Th1 cells in the intestinal tract comprising adding a test bacterium to the intestinal epithelial cell in a system comprising the intestinal epithelial cell and peripheral blood mononuclear cells; A step of detecting the number or activity of Th1 cells in the system, and when the induction of Th1 cell proliferation or activation is detected in the step, the test bacteria are treated with Th1 cell proliferation or activity in the intestine. Determining that the bacterium induces oxidization.
- test bacteria is not particularly limited, and examples thereof include human oral bacteria, human bladder bacteria, human vaginal bacteria, human ureteral bacteria, human urinary bacteria, and human intestinal bacteria.
- intestinal epithelial cell means a cell that exists on the luminal surface in the intestinal tract and is involved in nutrient absorption and immune response in the intestinal tract.
- peripheral blood mononuclear cell means a cell group (PBMC) containing lymphocytes and monocytes derived from peripheral blood, and in the present invention, peripheral blood itself may be used.
- PBMC cell group
- the origin of intestinal epithelial cells and peripheral blood mononuclear cells is not particularly limited, and examples include animals including humans (human, mouse, rat, monkey), but human-derived cells are preferably used.
- any culture system containing these cells may be used, but the intestinal epithelial cells and peripheral blood mononuclear cells are preferably in contact with each other. More preferably, the culture system is such that the layer containing peripheral blood mononuclear cells is the lower layer and the layer containing intestinal epithelial cells is the upper layer.
- Such a culture system can be constructed by, for example, laminating intestinal epithelial cells on peripheral blood mononuclear cells, or using a commercially available complex culture system (Transwell (registered trademark) culture system or the like) It can also be constructed by, for example, monolayer culture of intestinal epithelial cells in the compartment and peripheral blood mononuclear cells in the lower compartment.
- Transportwell registered trademark
- the other elements (method, conditions, etc.) in the screening method are the same as in ⁇ Method 1 for screening bacteria that induce or suppress Th1 cells in the intestinal tract>.
- the present invention can also provide the following screening method.
- a method for screening a bacterium that suppresses the proliferation or activation of Th1 cells in the intestinal tract comprising adding a test bacterium to the intestinal epithelial cell in a system comprising intestinal epithelial cells and peripheral blood mononuclear cells; A step of detecting the number or activity of Th1 cells in the system, and, when suppression of Th1 cell proliferation or activation is detected in the step, the test bacteria are Determining that the bacterium suppresses activation.
- a physiologically active substance responsible for induction can be screened from bacteria that induce Th1 cell proliferation or activation in the intestinal tract. That is, the present invention also provides the following screening method.
- a method for screening for a physiologically active substance that induces the proliferation or activation of Th1 cells in the intestinal tract wherein the system comprises intestinal epithelial cells and peripheral blood mononuclear cells.
- a step of adding a physiologically active substance derived from a bacterium that induces the growth or activation of Th1 cells, a step of detecting the number or activity of Th1 cells in the system, and the growth or activation of Th1 cells is detected in said step If it is, the method includes the step of determining that the physiologically active substance is a physiologically active substance that induces proliferation or activation of Th1 cells in the intestinal tract.
- the “bioactive substance” used in the method is as described in ⁇ Method 1 for screening bioactive substances that induce or suppress Th1 cells in the intestinal tract>.
- the other elements (method, conditions, etc.) in the screening method are the same as in ⁇ Method 1 for screening bacteria that induce or suppress Th1 cells in the intestinal tract>.
- the present invention can also provide the following screening method.
- a method for screening for a physiologically active substance that suppresses the proliferation or activation of Th1 cells in the intestinal tract wherein the system comprises intestinal epithelial cells and peripheral blood mononuclear cells.
- a step of adding a physiologically active substance derived from a bacterium that suppresses the growth or activation of a step of detecting the number or activity of Th1 cells in the system, and the suppression of the growth or activation of Th1 cells in the step
- a step of determining that the physiologically active substance is a physiologically active substance that suppresses the proliferation or activation of Th1 cells in the intestinal tract when detected.
- Th1 cell induction in the intestinal tract can be reproduced in vitro.
- the present invention provides a kit for evaluating the proliferation or activation of Th1 cells, comprising intestinal epithelial cells and peripheral blood mononuclear cells, or a bacterium that induces the proliferation or activation of Th1 cells in the intestinal tract. And a kit for evaluating the proliferation or activation of Th1 cells, comprising intestinal epithelial cells and peripheral blood mononuclear cells.
- the bacteria, intestinal epithelial cells, and peripheral blood mononuclear cells are as described in ⁇ Method 2 for screening bacteria that induce or suppress Th1 cells in the intestinal tract>.
- the evaluation kit includes a medium for maintaining or culturing these cells, a culture system (plate, etc.), a reagent for detecting Th1 cells (CD4 antibody, IFN- ⁇ antibody, two Secondary antibodies, labeling substances, etc.) may be included. Further, such an evaluation kit can include instructions for use of the kit.
- ⁇ Method 2 for screening a substance that induces or suppresses Th1 cells in the intestinal tract a system including intestinal epithelial cells and peripheral blood mononuclear cells, and a system including a bacterium that induces the proliferation or activation of Th1 cells in the intestinal tract is used to evaluate the proliferation or activation of Th1 cells. It can be preferably used. Therefore, the present invention also provides the following screening method using such a system.
- a method for screening a substance that induces proliferation or activation of Th1 cells in the intestinal tract comprising: intestinal epithelial cells and peripheral blood mononuclear cells; A step of adding a bacterium that induces activation and a test substance, a step of detecting the number or activity of Th1 cells in the system, and the number or activity of the Th1 cells detected in the step are determined by the test substance Determining that the test compound is a substance that induces the proliferation or activation of Th1 cells in the intestinal tract when the amount of the test compound is higher than that in the case where no is added.
- a method for screening a substance that suppresses the proliferation or activation of Th1 cells in the intestinal tract comprising: intestinal epithelial cells and peripheral blood mononuclear cells; A step of adding a bacterium that induces activation and a test substance, a step of detecting the number or activity of Th1 cells in the system, and the number or activity of Th1 cells detected in the step are the test compound And the step of determining that the test substance is a substance that suppresses the proliferation or activation of Th1 cells in the intestinal tract when the amount of the test substance is lower than that in the case where no is added.
- ⁇ Method for screening bacteria that induce or suppress Th1 cells in the intestinal tract 2> ⁇ Th1 cells in the intestinal tract As described in Method 1> for screening for substances to be induced or suppressed.
- composition 1 for testing diseases caused by Th1 cells As described above, in the present invention, a bacterium that induces proliferation or activation of Th1 cells in the intestinal tract is established in the intestinal tract, whereby Th1 cells are induced and a disease caused by Th1 cells is induced. It was revealed. Therefore, by detecting the presence of the bacterium, it is possible to examine a disease caused by Th1 cells.
- the present invention provides a composition for examining a disease caused by the following Th1 cells.
- a composition for examining a disease caused by Th1 cells comprising an antibody specifically recognizing a bacterium that induces proliferation or activation of Th1 cells in the intestinal tract.
- a composition for examining a disease caused by Th1 cells comprising a polynucleotide for detecting a nucleotide sequence specific to bacteria that induces proliferation or activation of Th1 cells in the intestinal tract.
- an antibody specifically recognizing a bacterium that induces the proliferation or activation of Th1 cells in the intestinal tract may be a polyclonal antibody or a monoclonal antibody as long as the bacterium can be specifically recognized. It may also be a functional fragment of an antibody (eg, Fab, Fab ′, F (ab ′) 2, variable region fragment (Fv), disulfide bond Fv, single chain Fv (scFv), sc (Fv) 2 A diabody, a multispecific antibody, or a polymer thereof).
- an immunized animal can be treated with an antigen (a bacterium that induces proliferation or activation of Th1 cells in the intestinal tract, a polypeptide derived from the bacterium, a polynucleotide, a sugar chain, a lipid, etc.). Immunized and purified from the antiserum by conventional means (eg salting out, centrifugation, dialysis, column chromatography, etc.). Monoclonal antibodies can be prepared by a hybridoma method or a recombinant DNA method.
- an antibody bound with a labeling substance can be used as an antibody used in the test of the present invention.
- detecting the labeling substance it is possible to directly measure the amount of antibody bound to the bacterium or the substance derived from the bacterium.
- the labeling substance is not particularly limited as long as it can bind to an antibody and can be detected by a chemical or optical method.
- a fluorescent dye GFP, etc.
- HRP enzyme
- radioactive Substances radioactive Substances.
- the test composition of the present invention can contain other components acceptable as a composition in addition to the antibody component.
- other components include carriers, excipients, disintegrants, buffers, emulsifiers, suspension agents, stabilizers, preservatives, preservatives, physiological saline, labeling substances, and secondary antibodies.
- a substrate necessary for detection of a labeling substance, a positive control or negative control, a buffer used for dilution or washing of a sample, a tube or a plate used for the reaction of the sample with the antibody of the present invention Can be combined, and a kit for testing diseases caused by Th1 cells can also be obtained.
- test kit for a disease caused by Th1 cells can include an instruction manual for the kit.
- an apparatus for detecting the antibody of the present invention can be combined with the test composition of the present invention.
- examples of such an apparatus include a flow cytometry apparatus and a microplate reader.
- the “polynucleotide for detecting a nucleotide sequence specific to a bacterium that induces proliferation or activation of Th1 cells in the intestinal tract” is not particularly limited as long as a sequence specific to the bacterium is detected.
- a polynucleotide having any one of the following (a) to (b) having a chain length of at least 15 nucleotides can be mentioned.
- b) a polynucleotide which is a primer or probe that hybridizes to a nucleotide sequence containing the specific nucleotide sequence is not particularly limited as long as a sequence specific to the bacterium is detected.
- Such a polynucleotide of the present invention has a base sequence complementary to the nucleotide sequence of a bacterium that induces the proliferation or activation of Th1 cells in the intestinal tract.
- “complementary” does not need to be completely complementary as long as it hybridizes.
- These polynucleotides usually have a homology of 80% or more, preferably 90% or more, more preferably 95% or more, and particularly preferably 100% with respect to the nucleotide sequence.
- the “chain length” in the polynucleotide of the present invention is usually 15 to 100 nucleotides, preferably 17 to 30 nucleotides, more preferably 20 to 25 nucleotides when used as a primer. When used as a probe, it is usually 15 to 1000 nucleotides, preferably 20 to 100 nucleotides.
- a polynucleotide (a pair of primers) for detecting a nucleotide sequence specific to the 2H7 strain belonging to Klebsiella pneumoniae shown below can be mentioned.
- the polynucleotide of the present invention may be DNA or RNA, and in part or in whole, LNA (registered trademark, cross-linked nucleic acid), ENA (registered trademark, 2′-O, 4′- Nucleotides may be substituted by artificial nucleic acids such as C-Ethylene-bridged nucleic acid), GNA (glycerol nucleic acid), TNA (threonucleic acid), PNA (peptide nucleic acid) and the like.
- LNA registered trademark, cross-linked nucleic acid
- ENA registered trademark, 2′-O, 4′- Nucleotides
- GNA glycerol nucleic acid
- TNA threonucleic acid
- PNA peptide nucleic acid
- the polynucleotide of the present invention can be chemically synthesized using a commercially available nucleotide automatic synthesizer or the like.
- a polynucleotide used in the test of the present invention a polynucleotide to which a labeling substance is bound can be used.
- the labeling substance is not particularly limited as long as it can bind to a polynucleotide and can be detected by a chemical or optical method.
- a fluorescent dye (DEAC, FITC, R6G, TexRed, Cy5, etc.)
- Dyes such as DAB
- enzymes enzymes
- radioactive substances in addition to fluorescent dyes.
- test composition of the present invention may contain other pharmacologically acceptable components in addition to the aforementioned polynucleotide.
- examples of such other components include a buffer, an emulsifier, a suspending agent, a stabilizer, a preservative, and physiological saline.
- a tube or a plate used for the reaction with the polynucleotide can be combined, and a test kit for a disease caused by Th1 cells can also be obtained.
- the kit for testing diseases caused by Th1 cells can include instructions for using the kit.
- test composition of the present invention can be combined with a device for detecting a nucleotide sequence specific to bacteria that induces the proliferation or activation of Th1 cells in the intestinal tract.
- a device for detecting a nucleotide sequence specific to bacteria that induces the proliferation or activation of Th1 cells in the intestinal tract examples include a PCR apparatus, a sequencer, and a microarray.
- the present invention also provides a method for testing diseases caused by Th1 cells using the above-described antibody, polynucleotide, or test composition. That is, a step of bringing the antibody, polynucleotide, or test composition into contact with a sample isolated from a subject, and bacteria that induce Th1 cell proliferation or activation in the intestinal tract by the contact.
- the present invention provides a method for examining a disease caused by Th1 cells, comprising the step of detecting the presence or absence.
- the subject is not particularly limited, and examples thereof include animals such as humans suspected of suffering from diseases caused by Th1 cells.
- the sample isolated from such a subject is not particularly limited, but a stool sample of the subject, a culture thereof, or a polypeptide, polynucleotide, sugar chain, lipid and the like extracted from the present invention are included in the present invention. It is suitably used in the method.
- Examples of the method for detecting the presence or absence of bacteria that induce the proliferation or activation of Th1 cells in the intestinal tract by bringing the antibody of the present invention or a test composition containing the antibody into contact with the sample include, for example, , ELISA method, immunoblotting, antibody array analysis method, immunohistochemical staining method, flow cytometry, imaging cytometry, radioimmunoassay, immunoprecipitation, etc. It is done.
- PCR real-time PCR, quantitative PCR
- DNA microarray analysis method DNA microarray analysis method
- Northern blotting next-generation sequencing method (sequencing-by-synthesis, eg, Solexa Genome Analyzer manufactured by Illumina) Or sequencing by Hiseq (registered trademark) 2000), pyrosequencing method (for example, sequencing by sequencer GSRX or FLX manufactured by Roche Diagnostics (454) (so-called 454 sequencing) )
- Ligase reaction sequencing method for example, sequencing by Life Technology's SoliD (registered trademark) or 5500xl
- bead array method in situ hybridization
- dot blot RNase protection assay method
- mass spectrometry method Genomic PCR and Southern blotting
- “examination” of a disease caused by Th1 cells includes not only the presence / absence of the onset of the disease but also examination of the risk of the onset of the disease. If the presence of a bacterium that induces proliferation or activation is detected, it can be determined that a disease caused by Th1 cells has developed or has a high risk of onset.
- Diagnosis of a disease caused by Th1 cells in a subject is usually performed by a doctor (including those who have received instructions from the doctor), but the data obtained by the method of the present invention is useful for diagnosis by the doctor. . Therefore, the method of the present invention can also be expressed as a method of collecting and presenting data useful for diagnosis by a doctor.
- a companion diagnostic method using the above-described inspection method and its drug can also be provided. That is, the present invention also provides the following.
- a method for determining the effectiveness of a substance having antibacterial activity against a bacterium that induces proliferation or activation of Th1 cells in the intestinal tract in the treatment, amelioration or prevention of a disease caused by Th1 cells, the antibody A step of contacting a polynucleotide or a test composition with a sample isolated from a subject, and detecting the presence or absence of a bacterium that induces proliferation or activation of Th1 cells in the intestinal tract by the contact. In the step, in the step, if the presence of the bacterium is detected, it is determined that the substance in the subject is highly effective in treating, improving or preventing the disease.
- a method for treating, ameliorating or preventing a disease caused by Th1 cells, wherein a substance having antibacterial activity against bacteria that induces proliferation or activation of Th1 cells in the intestinal tract by the determination method is highly effective
- a method comprising the step of causing the determined patient to take the substance.
- a composition for treating, ameliorating or preventing a disease caused by Th1 cells comprising as an active ingredient a substance having antibacterial activity against bacteria that induces proliferation or activation of Th1 cells in the intestinal tract,
- a composition ingested by a subject determined to be highly effective by the determination method.
- mice C57BL / 6 mice, BALB / c mice and IQI mice were purchased from Sankyo Lab Service (Japan), SLC (Japan) or Claire (Japan), which were maintained under SPF or GF conditions.
- SPF pathogen-free condition
- GF germ-free condition
- mice and pure isolation group (notobiotic) mice were bred and maintained in the gnotobiotic facility of Keio University School of Medicine, graduate School of Medicine or RIKEN Institute for Integrated Biomedical Sciences (IMS).
- IMS Integrated Biomedical Sciences
- Il10 ⁇ / ⁇ mice and Ifngr1 ⁇ / ⁇ mice were purchased from Jackson Laboratories.
- Myd88 ⁇ / ⁇ mice, Tlr4 ⁇ / ⁇ mice and Myd88 ⁇ / ⁇ Trif ⁇ / ⁇ mice were purchased from Oriental Bioservice (Japan).
- the frozen sample was thawed, and 100 ⁇ L of suspension, TE10 (10 mM) containing RNase A (final concentration 100 ⁇ g / mL, manufactured by Invitrogen) and lysosome (final concentration 3.0 mg / mL, manufactured by Sigma) were used.
- the resulting suspension was incubated while gently mixing at 37 ° C. for 1 hour.
- Purified achromopeptidase (Wako) was added to a final concentration of 2000 units / mL, and the mixture was further incubated at 37 ° C. for 30 minutes.
- the high molecular weight DNA was extracted with phenol-chloroform-isoamyl alcohol (25: 24: 1), precipitated with isopropanol, washed with 70% ethanol, and resuspended in 200 ⁇ L of TE.
- PCR for amplifying the V1-V2 region of 16S rRNA was performed using ExTaq (manufactured by Takara) and the following primer set.
- the 454primer A [5′-CCATCTCATCCCTGCGTGTCTCCGAACTCAG [SEQ ID NO: 7] (454 adapter sequence) + Barcode (10 bases) + AGRGTTTGATYMTGGCTCAG [SEQ ID NO: 8] -3 ′ (27Fmod)]
- the 454 primer B [5′-CCTATCCCCTTGTGGCCTTGGCAGTCTCAG [SEQ ID NO: 35] (454 adapter sequence) + TGCTGCCTCCCGTAGGAGT [SEQ ID NO: 9] -3 ′ (338R)].
- the amplification product ( ⁇ 330 bp) obtained from each sample was purified using AMPure XP (manufactured by Beckman Coulter), and the DNA was Quant-iT Picogreen dsDNA assay kit (manufactured by Invitrogen) and TBS-380 mini- Quantification was performed using a fluorometer (Turner Biosystems).
- Reads that passed the quality filter were obtained by excluding reads that did not have both primer sequences, had an average quality value of ⁇ 25, and could be chimeric products. From the reads that passed the filter, 3000 reads from which both primer sequences in each sample were deleted were subjected to OTU analysis using a cutoff similarity of 96%.
- Saliva samples were selected from the following human samples as representative examples of each group (healthy, CD and UC) based on the results of principal coordinate analysis of 16S rRNA sequences in the salivary microbiota.
- CD # 1 IBD029, 50 years old, Japanese male, IOIBD score 3 (active period)
- CD # 2 patient IBD 121, 52 years old, Japanese male, IOIBD score 1 (remission period)
- Saliva samples were suspended in an equal volume (w / v) of PBS containing 20% glycerol / PBS, rapidly frozen in liquid nitrogen, and stored at ⁇ 80 ° C. until use.
- the frozen stock was thawed, centrifuged at 3300 g for 10 minutes at 4 ° C., suspended in PBS, and orally administered to GF mice (100 ⁇ L per mouse).
- GF mice 100 ⁇ L per mouse.
- the cecum contents of GF + CD # 2 and GF + UC # 2 were serially diluted in PBS and plated on non-selective and selective agar plates. Subsequently, after culturing at 37 ° C. for 2 days or 4 days in an anaerobic chamber (Coy Laboratory Products) under anaerobic conditions (80% N 2 , 10% H 2 , 10% CO 2 ), independent colonies were isolated. Collected.
- the 16S rRNA gene region was amplified using the following universal primer set, and the sequence was determined. (27F: 5′-AGRGTTTGATYMTGGCTCAG-3 ′ [SEQ ID NO: 8], 1492R: 5′-GGYTACCTTTGTACCACTACT-3 ′ [SEQ ID NO: 10]) Samples for which sequences were obtained in the culture collection were classified as “strains” when their 16S rRNA gene sequences were 100% identical.
- the sequence of the obtained strain was compared with OTUs detected from the RDP database and GF + CD # 2 and GF + UC # 2 stool samples to determine related species or strains and corresponding OTUs.
- the bacterial strains were individually cultured until confluence, and mixed to obtain an equal volume of medium.
- the individual cultures were performed in Schadler broth for Kp-2H7 and Ec-2B1, in PYG broth for 2D5, Ve-2E1, 2G7, and 2E4, and in EGF broth for Fu-21f and 2B11.
- the mixture of isolates was orally administered to GF mice (200 ⁇ L medium containing about 1-2 ⁇ 10 8 CFU of total bacteria per mouse). All mice fed the mixture were maintained on a single gnotobiotic isolator.
- Bacterial strain K. pneumoniae, BAA-2552, BAA-1705, 700721, 7000060 and 13882 were purchased from the American Type Culture Collection (Manassas, VA, USA).
- K. pneumoniae KP-1 is the Scott A. It was isolated at Rice Laboratories (see KW Lee et al., The ISME journal 8, 894 (Apr, 2014)).
- KCTC2242 was obtained from the Korean Microbial Culture Collection (KCTC, Dajeon, Korea).
- K. pneumoniae 34E1 was isolated from the cecum contents of SPF mice treated with ampicillin in the laboratory of the present inventor Kenya Honda.
- the pneumonia strain was cultured at 37 ° C. in Schaedler broth, Luria Bertani (LB) broth or LB agar plate.
- Kp-2H7 was cultured overnight, washed with autoclaved water, and heat-sterilized at 105 ° C. for 30 minutes. It was ingested by GF mice through drinking water for 3 weeks (5 ⁇ 10 7 equivalent CFU / mL).
- HBSS Hank's balanced salt solution
- the sample was cut into small pieces, 4% fetal bovine serum and 0.5 mg / mL collagenase D, 0.5 mg / mL dispase II and 40 ⁇ g. / ML DNase I (all manufactured by Roche Diagnostics) -containing RPMI 1640 (10 mL) was incubated for 45 minutes in a shaking water bath set at 37 ° C.
- the digested tissue was washed with 5 mM EDTA-containing HBSS, resuspended in 5 mL of 40% percoll (manufactured by GE Healthcare), and overlaid on 2.5 mL of 80% percoll in a 15 mL falcon tube. Then, Percoll density gradient separation was performed by subjecting to centrifugation at 25 ° C. and 850 g for 25 minutes. Lymphocytes were collected from the boundary surface of the Percoll density gradient, washed with 10% FBS-containing RPMI 1640, and in the presence of Golgistop (registered trademark, manufactured by BD Biosciences) at 37 ° C. for 4 hours, 50 ng / mL PMA and 750 ng. / ML Ionomycin (both manufactured by Sigma) was stimulated.
- Golgistop registered trademark, manufactured by BD Biosciences
- the dead cells were labeled with ghost Dye 780 (manufactured by Tonbo Biosciences), and the cells were subjected to permeabilization, and anti-CD3e antibody (BV605; manufactured by Biolegend), anti-CD4 antibody (BV510; manufactured by Biolegend), anti-CD8a Antibody (PE / Cy7; manufactured by Biolegend), anti-TCR ⁇ antibody (BV421; manufactured by Biolegend), anti-TCRgd antibody (PE; manufactured by Biolegend), anti-CD44 antibody (BV785; manufactured by Biolegend), anti-IFN- ⁇ antibody ( FITC or PE / Cy7; manufactured by Biolegend), anti-IL-17A antibody (eFluor660; manufactured by eBioscience), anti-T-Bet antibody (PE / Cy7; manufactured by Biolegend), anti-ROR ⁇ t antibody (P or APC; eBioscience, Inc.) and anti-Foxp3 antibodies (PerCpCy5.5; by eBioscience, Inc.) was
- CD4 + TCR ⁇ + CD3e + subset in the raw lymphocyte gate was defined as CD4 + T cells.
- ⁇ Scanning electron microscope> The intestine was washed with PBS, fixed with 50 mM phosphate buffer (pH 7.2) containing 2.5% glutaraldehyde, and post-fixed with 50 mM phosphate buffer (pH 7.2) containing 1% osmium tetroxide.
- the sample was dehydrated in an ethanol system and replaced with isoamyl acetate. After dehydration, the sample was subjected to a critical point dryer (CPD 030; Leica Microsystems), coated with platinum, and observed at 5 kV or 10 kV using a scanning electron microscope (SU-1510; Hitachi High-Technologies). All scanning microscope analyzes were performed on at least 10 areas per animal. Each group is composed of 3 to 5 animals.
- ⁇ Transmission electron microscope> In order to visualize the structure of bacteria, a metal contact freezing method and a freeze replacement method were used.
- As the freezing apparatus type VFZ-101 (manufactured by Vacuum Device Inc., Ibaraki, Japan) equipped with liquid nitrogen as a freezing agent was used. Freeze substitution was performed with acetone containing 2% OsO 4 at ⁇ 80 ° C. for 120 hours and gradually returned to room temperature (the temperature was raised from ⁇ 80 ° C. to ⁇ 55 ° C. over 12.5 hours, and then ⁇ 55 ° C. The temperature was maintained for 8 hours, the temperature was increased from ⁇ 55 ° C. to ⁇ 25 ° C. over 10 hours, and then ⁇ 25 ° C. was maintained for 8 hours, and the temperature was increased from ⁇ 25 ° C. to 0 ° C. over 5 hours. ).
- Bacterial cells were embedded in low-viscosity epoxy resin (Nissin EM) and ultrathin sections were prepared using an ultramicrotome (EM-UC7; Leica Microsystems) for 12 minutes in 4% uranyl acetate aqueous solution, Reynolds' Sections were stained for 1 minute with a lead chelate solution (see ES Reynolds, The Journal of cell biology 17, 208 (Apr, 1963)). And it observed at 80 kV using the transmission electron microscope (JEM-1400; Jeol, Tokyo, Japan).
- the bacteria were cultured on an agar plate and fixed with 1% ammonium acetate containing 2.5% glutaraldehyde.
- the sample was placed on a Formvar-coated hydrophilic pretreated copper grid (200 mesh), and the grid was immediately brought into contact with the filter paper from the edge to remove excess sample.
- Samples were stained with phosphotungstic acid (pH 7.0) for 10-20 seconds, blotted dry, and observed with JEM-1400.
- mice SPF mice (WT B6, Il10 ⁇ / ⁇ or Ifngr1 ⁇ / ⁇ ) were administered with the following antibiotics via drinking water for 4 days prior to the forced intake of bacteria. In addition, mice that did not receive these antibiotics were also prepared.
- Antibiotics ampicillin (200 mg / L), tylosin (500 mg / L), metronidazole (500 mg / L), spectinomycin (200 mg / L), vancomycin (200 mg / L).
- Kp-2H7 or Ka-11E12 was cultured to the log phase in LB broth and 1-2 ⁇ 10 8 CFUs were used for inoculation of mice.
- ⁇ Preparation of large intestine EC and DC> Collect large intestine tissue, cut longitudinally, wash well with ice-cold PBS, peel epithelial cells (ECs) using a glass slide, immediately freeze in liquid nitrogen, and keep at -80 ° C until analysis saved.
- the remaining tissue was incubated with 5 mM EDTA-containing HBSS at 37 ° C. for 20 minutes with shaking to completely remove ECs.
- the tissue was then cut into small pieces, 4% fetal bovine serum, 0.5 mg / mL collagenase D, 0.5 mg / mL dispase II and 40 ⁇ g / mL DNase I in RPMI 1640 for 45 minutes in a 37 ° C. shaking water bath. Incubated.
- CD11 positive cells were stained with an anti-CD11c antibody (APC; manufactured by Biolegend), and concentrated with MACS using anti-APC beads (manufactured by Miltenyi Biotec). Cells selected as positive were further subjected to sorting with FACSAria II to a purity of about 97%.
- APC anti-CD11c antibody
- RNA-seq and qPCR analysis Total RNA was isolated from EC and DC of the large intestine using TRIzol reagent (manufactured by Invitrogen) according to the instructions.
- cDNA was synthesized using RiverTra Ace (registered trademark) qPCR RT master mix (manufactured by TOYOBO). Using a Thunderbird (registered trademark) SYBR qPCR mix (manufactured by TOYOBO), LightCycler 480 (Roche). QPCR was performed. The following primers were used.
- RNA library was prepared using a NENB Ultra Ultra RNA library preparation kit (manufactured by New England Biolabs) for Illumina according to the manufacturer's instructions.
- sequencing was performed with a single-end 50 bp read by HiSeq 1500 system (manufactured by Illumina).
- sequenced reads were mapped to the mouse reference genome (mm9, NCBI build 37) and normalized to the number of fragments (FPKM) per million reads / kbp using the Tophat & Cufflinks software pipeline.
- the heat map in FIG. 25 shows a gene (> 2-fold, FPKM value 0. 0) in which the expression was improved in mice in which only Kp-2H7 was established, compared to mice in which only GF mice and BAA2552 were established. 1 or more) relative intensity (Z-score).
- the heat map in FIG. 27 shows that the gene in which only Kp-2H7 was established was improved in expression compared to Myd88 ⁇ / ⁇ mouse in which only Kp-2H7 was established (> 2-fold, The relative intensity (Z-score) of FPKM value 0.1 or higher is shown.
- the gene whose expression was improved (the gene shown on the left of FIG. 26, the gene shown on the right of FIG. 26 or the gene shown in FIG. 28) is DAVID bioinformatics resource 6.8 (W. Huang da et al., Nature protocols). 4, 44 (2009)) and subjected to gene ontology enrichment analysis.
- mice were sacrificed 5 weeks after oral inoculation or 7 days after intratracheal injection.
- the large intestine and lung were fixed with 4% paraformaldehyde, embedded in paraffin, and stained with hematoxylin and eosin.
- the degree of colitis was classified according to the following criteria. Inflammatory cell infiltration (score: 0-4) Mucosal thickening (score: 0-4) Goblet cell loss (score: 0-4) Crypt abscess (score: 0-4) Structural destruction (score: 0-4). The final histological score was determined by the score sum of these parameters.
- the pneumonia score was determined by summing the scores of the following two sections. Alveoli (no change: 0, edema: 1, detection of inflammatory cells in alveolar space: 2, inflammatory destruction of alveoli with lung abscess) Bronchioles (no change: 0, mild inflammation in the wall: 1, severe inflammation in the wall with luminal scab: 2, severe inflammation with luminal scab and peribronchiolitis: 3) .
- ⁇ Bacterial genome sequencing> The genome of the Klebsiella strain was extracted by the same method as described in 16S rRNA gene pyrosequencing. Then, the genome sequence was determined by the whole genome shotgun sequencing method using PacBioRSII and Illumina MiSeq sequencer.
- Genomic DNA was cleaved to obtain a DNA fragment.
- Template DNA was prepared according to the protocol by each supplier.
- the obtained RSII lead was subjected to a de novo assembly using HGAP3.
- MiSeq reads (2 ⁇ 300 nt) were mapped onto contigs obtained by assembling RSII and corrected for low quality region sequences.
- sequence-based capsule (K) typing was performed based on sequencing of wzi or wzc genes.
- Bacterial kinetic analysis was performed using semi-solid LB medium containing 0.25% in a 14 mL tube. Bacteria cultivated in LB broth medium were inoculated using a straight wire in semi-solid LB medium and pierced in the middle of the tube to half the depth of the medium. Bacteria that were diffused and vaguely diffused throughout the medium after incubation at 37 ° C. for 24 hours were determined as motile bacteria. Bacterial flagella were detected using HEK-Blue mTLR5 cells (InvivoGen) according to the manufacturer's protocol.
- Example 1 The present inventors forcibly orally administer saliva samples from Crohn's disease (CD) 2 patients to C57BL / 6 (B6) sterile (GF) mice to produce gnotobiotic mice. Each group of mice was then kept in a pure isolation isolator for 6 weeks and examined for immune cells in the small intestine and colon lamina intestinal (LP).
- CD Crohn's disease
- B6 C57BL / 6
- GF sterile mice
- the population composition was compared by 16S rRNA sequencing in the salivary microbiota before administration of GF mice and the fecal microbiota of the animal (exGF mice) in which the microbiota was established.
- the saliva samples of both patients showed a remarkable difference between the stool microbiota of GF + CD # 2 mice and GF + CD # 1 mice, although they showed similar microbial composition. It was. Importantly, the majority of bacterial species observed in the fecal microbiota of exGF mice were bacterial species that were slightly present in the salivary microflora.
- these colonies include the following 8 strains composed of various genera, and occupy the main bacterial species of the gastrointestinal microbiota established in GF + CD # 2 mice. It was revealed. Gemella, Bifidobacterium, Streptococcus, Escherichia, Fusobacterium, Velilella, Anaerococcus, Klebsiella.
- mice in which these two strains were established were prepared. However, as shown in FIG. 4, only a few Th1 cells were observed in mice in which these bacterial strains were established.
- Klebsiella pneumoniae strain ID 2H7 (Kp-2H7), which showed the highest composition ratio in the microbial flora of GF + CD # 2 mice, was tested.
- the Kp-2H7 strain is a bacterial strain that mainly contributes to the accumulation of Th1 cells observed in GF + CD # 2 mice.
- Kp-2H7 The induction effect by Kp-2H7 was relatively Th1 cell-specific in the tested immune cells (see FIG. 5).
- the induced Th1 cells showed a negative response to interleukin 17, ROR ⁇ t, and Foxp3, while they showed a positive response to T-bet and CD44 (see FIG. 6).
- These results suggest that chronically activated / memory Th1 cells have been produced (see S. Okada et al., Science 349, 606-613 (2015)).
- Kp-2H7 was mainly settled in the large intestine, reflecting that Th1 cells were induced more predominantly in the large intestine than in the small intestine (see FIGS. 7 and 8).
- Kp-2H7 isolated by the present inventors is effective against various antibiotics such as ampicillin (Amp), tylosin (Tyl), spectinomycin (Spc) and metronidazole (MNZ) as shown in FIG. 11 and Table 12. Showed resistance.
- Amicillin Amicillin
- Tyl tylosin
- Spc spectinomycin
- MNZ metronidazole
- FIG. 12 the firm establishment of Kp-2H7 was accompanied by a marked increase in Th1 cells in the intestine.
- K-2H7's ability to enter enteritis As a result, as shown in FIGS. 13 to 16, although Th1 cells were induced, Kp-2H7 was treated with a wild type (WT) host, a B6 GF mouse in which only the bacteria were established, or after Amp treatment. It did not cause inflammatory changes in the gut of established mice.
- WT wild type
- Kp-2H7 or Escherichia coli strain ID 2B1 (Ec-2B1) was orally administered to GF Il10 ⁇ / ⁇ mice or Amp-treated SPF Il10 ⁇ / ⁇ mice.
- Ec-2B1 was isolated from GF + CD # 2 mice, but is a bacterium with weak ability to induce Th1 cells.
- E. coli belongs to Enterobacteriaceae, and this family includes bacteria that are involved in the development of IBD (see Non-Patent Document 2, XC Morgan et al., Genome biology 13, R79 (2012)).
- Kp-2H7 acts as a gastrointestinal pathogenic symbiosis by inducing a Th1 cell response and possibly an accompanying pro-inflammatory effect in a species-specific and host genotype-specific manner. It is suggested that
- Kp-2H7 and 8 K.P. are phylogenetic by obtaining new sequences or obtaining sequences from public databases and performing sequence and core genome phylogenetic analysis based on multilocus sequencing (MLST), capsule (K) typing It was clarified that they are different from each other (see Table 13 and FIG. 20).
- MLST multilocus sequencing
- K capsule
- Th1 cell induction equivalent to Kp-2H7 was observed in some bacterial strains such as ATCC700703, BAA-1705 and 34E1, but the induction ability was low in other strains.
- KCTC2242 and BAA2552 had no or little effect on Th1 cell frequency. This suggests that the induction of Th1 cells is bacterial strain specific.
- Th1 cells did not depend on the amount of bacteria and was not accompanied by inflammation (see FIG. 22). Furthermore, there was no relationship between the induction of Th1 cells and MLST, K-typing and lineage (see FIGS. 20, 21 and Table 13).
- the gene group included genes predicted to encode enzymes involved in absorption and metabolism processes associated with fructose, galactitol and mannose (see FIG. 23 and Tables 1-10).
- Non-Patent Document 1 XC Morgan et al., Genome biology 13, R79 (2012), AR Records, Molecular plant-microbe interactions: MPMI 24, 751-757 (2011), S. Fukuda et al., Nature 469, 543-547 (2011), A. N. Thorburn, L. Macia, C. R. Mackay, Immunity 40, 833-842 (2014)).
- the inventors tried to elucidate signal transduction in host cells that regulate Th1 induction.
- the involvement of the innate immune signal pathway was examined using GF Myd88 ⁇ / ⁇ mice, Myd88 ⁇ / ⁇ Trif ⁇ / ⁇ mice, and Tlr4 ⁇ / ⁇ mice in which only Kp-2H7 was established.
- Tlr4 - / - was partially attenuated in mice (see Figure 24). This suggests that innate immune signaling pathways are involved, including Toll-like receptors (TLRs), and possibly other MyD88-dependent receptors, including IL-1b and IL-18. It is suggested.
- TLRs Toll-like receptors
- IFN-inducible (IFI) genes such as major histocompatibility complex-related molecules involved in antigen processing / presentation (eg, H2-DMb1, H2-Ab1 and Tap1), and guanylate binding proteins (GBPs) are GF significantly increased in large intestine EC and DC derived from WT + Kp-2H7 mice. Moreover, as shown in FIG. 29, the difference in expression was confirmed by qPCR analysis. As shown in FIGS. 25 and 29-31, the up-regulation of the IFI gene in EC and DC, and the Ifng gene in DC, in particular, began in the early stage (one week) of establishment where Th1 induction was restricted. This suggests that the enhanced expression of IFN-g and IFI is not only a result but also involved in the induction of Th1 cells.
- IFI IFN-inducible
- Th1 cell induction by Kp-2H7 colonization was incomplete in IFN-g receptor 1-deficient (IFNgR1 ⁇ / ⁇ ) mice.
- K.K. The pneumoniae strain produces certain innate immune system ligands. It can penetrate the mucin layer and access the intestinal EC and DC. As a result, it is considered that MyD88 and IFI-dependent signal transduction pathways are activated and Th1 cells are induced.
- Example 3 To further confirm the association between oral-derived bacteria and Th1 cell induction, two healthy donors (He # 1 and He # 2) and two active ulcerative colitis (UC) patients (UC) Additional saliva samples were obtained from # 1 and UC # 2) and were orally administered to GF WT B6 mice.
- K.K A reclassification from Aerobilis Enterobacter aerogenes has been proposed recently.
- the bacterium is K.
- the identity of the 16S rRNA gene sequence with pneumoniae is 99%, which is very close to the phylogeny (see SM Diene et al., Molecular biology and evolution 30, 369-383 (2013)).
- Ef-11A1, Ka-11E12, or a mixture of 11 other bacterial strains (11-mix) was forcibly administered to GF mice.
- Ka-11E12 significantly induced Th1 cell accumulation in the large intestine as observed in GF + 13-mix mice and GF + UC # 2 mice.
- no significant induction was observed in Ef-11A1 and 11-mix.
- Ka-11E12 is likely to be a major factor in Th1 cell induction observed in GF + UC # 2 mice, even though it is only a minority of bacteria in the gut microbiota of GF + UC # 2 mice (Fig. 34).
- Ka-11E12 showed resistance to various antibiotics.
- F-11 revealed that Ka-11E12, like Kp-2H7, settled away from the large intestine EC.
- Ka-11E12 has a flagellar assembly system, exhibits a high degree of motility, and has TLR5 stimulation.
- Kp-2H7 does not form a capsule and produces outer membrane vesicles (OMV) or OMV-like structures.
- K. cerevisiae has settled in the healthy human oral cavity. It was revealed that pneumoniae strains can also induce Th1 induction when established in the intestine.
- Example 4 We next examined whether Klebsiella was abundant in patients with IBD and other diseases.
- the number of reads per 1 million bp and the number of reads per 1 kbp (RPKM) in Th1-related genes were generally higher in patient samples than in non-IBD samples.
- 9CD, 5UC and 3non-IBD carried Klebsiella.
- the 7CD, 4UC and 0 non-IBD controls were positive for Th1-related genes in the intestinal microbiota near the threshold where the average RPKM was> 1 (see FIG. 49).
- Th1 cell proliferation or activation can be induced in the intestinal tract by bacteria such as 2H7 strain, 11E12 strain, 34E1 strain, BAA-1705 strain, 70000603 strain, and 40B3 strain belonging to Klebsiella.
- Th1 cell-inducing bacterium a bacterium that induces the proliferation or activation of Th1 cells in such an intestinal tract
- the growth of the bacterium is suppressed or killed, thereby causing Th1
- Th1 It becomes possible to suppress cell proliferation or activation.
- Th1 cell-inducing bacteria or physiologically active substances thereof by using such Th1 cell-inducing bacteria or physiologically active substances thereof, the proliferation or activation of Th1 cells is induced, and immunity is activated, thereby treating infectious diseases, It is also possible to enhance the anticancer effect.
- the present invention is extremely useful in drug development, treatment, improvement, prevention or diagnosis of various diseases.
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Abstract
Description
<1> 腸管内でTh1細胞の増殖又は活性化を誘導する細菌。
<2> 更に、Klebsiella pneumoniae又はKlebsiella aeromobilisに属することを特徴とする、<1>に記載の細菌。
<3> 更に、下記タンパク質群から選択される少なくとも5つのタンパク質をコードする遺伝子を保有することを特徴とする、<1>又は<2>に記載の細菌
タンパク質群:
マンノース-1-リン酸グアニルトランスフェラーゼ1、
マルチホスホリル転移タンパク質、
PTS系フルクトース特異的EIIABC構成タンパク質、
ホスホマンノムターゼ/ホスホグルコムターゼ、
マンノシルフルクトース-リン酸合成酵素、
3-オキソアシル[アシル輸送タンパク質]レダクターゼ FabG、
ラムノシル/マンノシルトランスフェラーゼ、
ガラクチトール-1-リン酸5-デヒドロゲナーゼ、
ガラクチトールパーミアーゼIIC構成タンパク質、
ガラクチトール特異的ホスホトランスフェラーゼ酵素IIB構成タンパク質、
D-タガトース-1,6-ビスリン酸アルドラーゼサブユニット GatZ、
タガトース-6-リン酸キナーゼ、
D-タガトース-1,6-ビスリン酸アルドラーゼサブユニット GatY、
ガラクチトールパーミアーゼIIC構成タンパク質、
GDP-マンノース-依存性 α-(1-2)-ホスファチジルイノシトール マンノシルトランスフェラーゼ、
L-キシルロース/3-ケト-L-グロン酸キナーゼ、
2-デヒドロ-3-デオキシグルコノキナーゼ、
莢膜グルカン合成酵素、
3-オクタプレニル-4-ヒドロキシベンゾエートカルボキシ-リアーゼ パートナータンパク質、
2-オクタプレニルフェノールヒドロキシラーゼ、
フェノール酸デカルボキシラーゼ サブユニットC、
オキサロ酢酸デカルボキシラーゼβ鎖、
アコニット酸ヒドラターゼ2、
推定アルドラーゼ LsrF、
推定アセチルトランスフェラーゼ、
プロパンジオール利用タンパク質 PduA、
推定グリコシルトランスフェラーゼ EpsF、
へミン結合ペリプラズムタンパク質 HmuT前駆体、
タイコ酸排出ATP結合タンパク質 TagH、
タイコ酸移行透過タンパク質 TagG
外膜タンパク質 TolC前駆体
多剤輸送体 EmrE
マグネシウム及びコバルト排出タンパク質 CorC
内膜タンパク質 YibH
アスパラギン酸/アラニン交換輸送体
鉄エンテロバクチン受容体前駆体
シグナル伝達ヒスチジン-プロテインキナーゼ BarA
ヘモリシン輸送タンパク質 ShlB前駆体
オリゴペプチド輸送ATP結合タンパク質 OppD
ヒ素ポンプ-駆動ATPアーゼ
推定抗シグマ因子アンタゴニスト
推定膜タンパク質 YdfK
推定ヘモグロビン及びヘモグロビン-へパトグロビン結合タンパク質2前駆体
(2R)-3-スルホ乳酸デヒドロゲナーゼ(NADP(+))
ぺプチダーゼE
オリゴペプチダーゼA
ホスフィノトリシン N-アセチルトランスフェラーゼ
推定2-ヒドロキシ酸デヒドロゲナーゼ YoaD
mRNAインターフェラーゼ RelE
一本鎖DNA特異的エクソヌクレアーゼ RecJ
チロシンリコンビナーゼ XerD_6
チロシンリコンビナーゼ XerD
グルシトールオペロンリプレッサー
ギ酸塩ヒドロゲンリアーゼ転写活性因子
HTH-タイプ転写制御因子 TdfR
HTH-タイプ転写制御因子 CatM
転写制御タンパク質 tctD
HTH-タイプ転写抑制因子 AseR
サイクリックdi-GMPホスホジエステラーゼ YahA
セリン-プロテインキナーゼ RsbW
繊維状赤血球凝集素
ジヒドロプテロイン酸合成酵素
δ-アミノレブリン酸デヒドラターゼ
好気呼吸制御タンパク質 ArcA。
<4> 更に、マンノース、フルクトース及びガラクトースのうちの少なくとも1の糖の代謝に関与する遺伝子を保有することを特徴とする、<1>~<3>のうちのいずれか一項に記載の細菌。
<5> <1>~<4>のうちのいずれか一項に記載の細菌又は該細菌に由来する生理活性物質を有効成分として含む、免疫を賦活させるための組成物。
<6> 更に、Th1細胞の増殖又は活性化を誘導するための組成物であることを特徴とする、<5>に記載の組成物。
<7> <1>~<4>のうちのいずれか一項に記載の細菌又は該細菌に由来する生理活性物質を、対象に摂取させ、該対象におけるTh1細胞の増殖又は活性化を誘導する方法。
<8> <1>~<4>のうちのいずれか一項に記載の細菌又は該細菌に由来する生理活性物質を、対象に摂取させ、該対象における免疫を賦活する方法。
<9> <1>~<4>のうちのいずれか一項に記載の細菌又は該細菌に特異的な抗原を有効成分として含む、ワクチン組成物。
<10> <1>~<4>のうちのいずれか一項に記載の細菌又は該細菌に特異的な抗原を、対象に摂取させ、該対象における前記細菌に対する免疫応答を誘導する方法。
<11> <1>~<4>のうちのいずれか一項に記載の細菌に対して抗菌活性を有する物質又は<1>~<4>のうちのいずれか一項に記載の細菌に由来する生理活性物質に結合する物質を、有効成分として含む、免疫を抑制するための組成物。
<12> 更に、Th1細胞の増殖又は活性化を抑制するための組成物であることを特徴とする、<11>に記載の組成物。
<13> 更に、Th1細胞に起因する疾患を治療、改善又は予防するための組成物であることを特徴とする、<11>又は<12>に記載の組成物。
<14> <1>~<4>のうちのいずれか一項に記載の細菌に対して抗菌活性を有する物質又は<1>~<4>のうちのいずれか一項に記載の細菌に由来する生理活性物質に結合する物質を、対象に摂取させ、該対象におけるTh1細胞の増殖又は活性化を抑制する方法。
<15> <1>~<4>のうちのいずれか一項に記載の細菌に対して抗菌活性を有する物質又は<1>~<4>のうちのいずれか一項に記載の細菌に由来する生理活性物質に結合する物質を、対象に摂取させ、該対象における免疫を抑制する方法。
<16> <1>~<4>のうちのいずれか一項に記載の細菌に対して抗菌活性を有する物質又は<1>~<4>のうちのいずれか一項に記載の細菌に由来する生理活性物質に結合する物質を、対象に摂取させ、該対象におけるTh1細胞に起因する疾患を治療、改善又は予防する方法。
<17> <1>~<4>のうちのいずれか一項に記載の細菌が、腸管内に定着している、非ヒト動物。
<18> 更に、Th1細胞に起因する疾患の非ヒトモデル動物であることを特徴とする、<17>に記載の非ヒト動物。
<19> <17>又は<18>に記載の非ヒト動物の製造方法であって、
腸管内でTh1細胞の増殖又は活性化を誘導する細菌を、非ヒト動物に摂取させ、該細菌を該動物の腸管内に定着させる工程を含む、方法。
<20> <1>~<4>のうちのいずれか一項に記載の細菌と、腸管上皮細胞と、末梢血単核細胞とを含む、Th1細胞の増殖又は活性化を評価するためのキット。
<21> 腸管上皮細胞と、末梢血単核細胞とを含む、Th1細胞の増殖又は活性化を評価するためのキット。
<22> 腸管内でTh1細胞の増殖又は活性化を誘導する細菌をスクリーニングする方法であって、
被験試料を非ヒト無菌動物に摂取させる工程と、
該非ヒト無菌動物の腸管内におけるTh1細胞の数又は活性を検出する工程と、
前記工程にてTh1細胞の増殖又は活性化が検出された非ヒト無菌動物の腸管内試料から細菌を単離する工程とを、含む方法。
<23> 腸管内でTh1細胞の増殖又は活性化を誘導する生理活性物質をスクリーニングする方法であって、
腸管内でTh1細胞の増殖又は活性化を誘導する細菌に由来する生理活性物質を、非ヒト無菌動物に摂取させる工程と、
該非ヒト無菌動物の腸管内におけるTh1細胞の数又は活性を検出する工程と、
前記工程にてTh1細胞の増殖又は活性化が検出された場合に、前記生理活性物質を、腸管内でTh1細胞の増殖又は活性化を誘導する生理活性物質であると判定する工程とを、含む方法。
<24> 腸管内でTh1細胞の増殖又は活性化を誘導する細菌をスクリーニングする方法であって、
腸管上皮細胞と末梢血単核細胞とを含む系において、該腸管上皮細胞に被験細菌を添加する工程と、
該系におけるTh1細胞の数又は活性を検出する工程と、
前記工程にてTh1細胞の増殖又は活性化が検出された場合には、前記被験細菌を、腸管内でTh1細胞の増殖又は活性化を誘導する細菌であると判定する工程とを、含む方法。
<25> 腸管内でTh1細胞の増殖又は活性化を誘導する生理活性物質をスクリーニングする方法であって、
腸管上皮細胞と末梢血単核細胞とを含む系において、該腸管上皮細胞に、腸管内でTh1細胞の増殖又は活性化を誘導する細菌に由来する生理活性物質を添加させる工程と、
該系におけるTh1細胞の数又は活性を検出する工程と、
前記工程にてTh1細胞の増殖又は活性化が検出された場合には、前記生理活性物質を、腸管内でTh1細胞の増殖又は活性化を誘導する生理活性物質であると判定する工程とを、含む方法。
<26> 腸管内でTh1細胞の増殖又は活性化を誘導する物質のスクリーニング方法であって、
腸管上皮細胞と末梢血単核細胞とを含む系において、該腸管上皮細胞に、腸管内でTh1細胞の増殖又は活性化を誘導する細菌と、被験物質とを添加する工程と、
該系におけるTh1細胞の数又は活性を検出する工程と、
前記工程にて検出されたTh1細胞の数又は活性が、前記被験物質を添加しなかった場合におけるそれよりも増加している場合に、前記被験化合物は、腸管内でTh1細胞の増殖又は活性化を誘導する物質であると判定する工程とを、含む方法。
<27> 腸管内でTh1細胞の増殖又は活性化を誘導する物質のスクリーニング方法であって、
<17>に記載の非ヒト動物に、被験物質を摂取させる工程と、
該非ヒト動物の腸管内におけるTh1細胞の数又は活性を検出する工程と、
前記工程にて検出されたTh1細胞の数又は活性が、前記被験物質を摂取させなかった場合におけるそれよりも増加している場合に、前記被験物質は、腸管内でTh1細胞の増殖又は活性化を誘導する物質であると判定する工程とを、含む方法。
<28> <22>~<27>のうちのいずれか一項に記載のスクリーニング方法によって得られる、細菌、生理活性物質又は物質を、有効成分として含む、免疫を賦活するための組成物。
<29> 更に、Th1細胞の増殖又は活性化を誘導するための組成物であることを特徴とする、<28>に記載の組成物。
<30> <22>若しくは<24>に記載のスクリーニング方法によって得られる細菌又は該細菌に特異的な抗原を有効成分として含む、ワクチン組成物。
<31> Th1細胞に起因する疾患を誘発又は憎悪させる活性を有する物質を、スクリーニングするための方法であって、
<18>に記載の非ヒト動物に、被験物質を摂取させる工程と、
該非ヒト動物におけるTh1細胞に起因する疾患の病変の程度を検出する工程と、
前記工程にて検出された病変の程度が、前記被験物質を摂取させなかった場合におけるそれよりも増加している場合に、前記被験物質は、Th1細胞に起因する疾患を誘発又は憎悪させる活性を有する物質であると判定する工程とを、含む方法。
<32> <31>に記載のスクリーニング方法によって得られる物質を、有効成分として含む、Th1細胞に起因する疾患を誘発又は憎悪させるための組成物。
<33> 腸管内でTh1細胞の増殖又は活性化を抑制する細菌をスクリーニングする方法であって、
被験試料を非ヒト無菌動物に摂取させる工程と、
該非ヒト無菌動物の腸管内におけるTh1細胞の数又は活性を検出する工程と、
前記工程にてTh1細胞の増殖又は活性化の抑制が検出された非ヒト無菌動物の腸管内試料から細菌を単離する工程とを、含む方法。
<34> 腸管内でTh1細胞の増殖又は活性化を抑制する生理活性物質をスクリーニングする方法であって、
腸管内でTh1細胞の増殖又は活性化を抑制する細菌に由来する生理活性物質を、非ヒト無菌動物に摂取させる工程と、
該非ヒト無菌動物の腸管内におけるTh1細胞の数又は活性を検出する工程と、
前記工程にてTh1細胞の増殖又は活性化の抑制が検出された場合に、前記生理活性物質を、腸管内でTh1細胞の増殖又は活性化を抑制する生理活性物質であると判定する工程とを、含む方法。
<35> 腸管内でTh1細胞の増殖又は活性化を抑制する細菌をスクリーニングする方法であって、
腸管上皮細胞と末梢血単核細胞とを含む系において、該腸管上皮細胞に被験細菌を添加する工程と、
該系におけるTh1細胞の数又は活性を検出する工程と、
前記工程にてTh1細胞の増殖又は活性化の抑制が検出された場合には、前記被験細菌を、腸管内でTh1細胞の増殖又は活性化を抑制する細菌であると判定する工程とを、含む方法。
<36> 腸管内でTh1細胞の増殖又は活性化を抑制する生理活性物質をスクリーニングする方法であって、
腸管上皮細胞と末梢血単核細胞とを含む系において、該腸管上皮細胞に、腸管内でTh1細胞の増殖又は活性化を抑制する細菌に由来する生理活性物質を添加する工程と、
該系におけるTh1細胞の数又は活性を検出する工程と、
前記工程にてTh1細胞の増殖又は活性化の抑制が検出された場合には、前記生理活性物質を、腸管内でTh1細胞の増殖又は活性化を抑制する生理活性物質であると判定する工程とを、含む方法。
<37> 腸管内でTh1細胞の増殖又は活性化を抑制する物質のスクリーニング方法であって、
腸管上皮細胞と末梢血単核細胞とを含む系において、該腸管上皮細胞に、腸管内でTh1細胞の増殖又は活性化を誘導する細菌と、被験物質とを添加する工程と、
該系におけるTh1細胞の数又は活性を検出する工程と、
前記工程にて検出されたTh1細胞の数又は活性が、前記被験化合物を添加しなかった場合におけるそれよりも低減している場合に、前記被験物質は、腸管内でTh1細胞の増殖又は活性化を抑制する物質であると判定する工程とを、含む方法。
<38> 腸管内でTh1細胞の増殖又は活性化を抑制する物質のスクリーニング方法であって、
<17>に記載の非ヒト動物に、被験物質を摂取させる工程と、
該非ヒト動物の腸管内におけるTh1細胞の数又は活性を検出する工程と、
前記工程にて検出されたTh1細胞の数又は活性が、前記被験物質を摂取させなかった場合におけるそれよりも低減している場合に、前記被験物質は、腸管内でTh1細胞の増殖又は活性化を抑制する物質であると判定する工程とを、含む方法。
<39> <33>~<38>のうちのいずれか一項に記載のスクリーニング方法によって得られる、細菌、生理活性物質又は物質を、有効成分として含む、免疫を抑制するための組成物。
<40> 更に、Th1細胞の増殖又は活性化を抑制するための組成物であることを特徴とする、<39>に記載の組成物。
<41> 更に、Th1細胞に起因する疾患を治療、改善又は予防するための組成物であることを特徴とする、<39>又は<40>に記載の組成物。
<42> Th1細胞に起因する疾患を治療、改善又は予防する活性を有する物質をスクリーニングするための方法であって、
<18>に記載の非ヒト動物に、被験物質を摂取させる工程と、
該非ヒト動物におけるTh1細胞に起因する疾患の病変の程度を検出する工程と、
前記工程にて検出された病変の程度が、前記被験物質を摂取させなかった場合におけるそれよりも低減している場合に、前記被験物質は、Th1細胞に起因する疾患を治療、改善又は予防する活性を有する物質であると判定する工程とを、含む方法。
<43> <42>に記載のスクリーニング方法によって得られる物質を、有効成分として含む、Th1細胞に起因する疾患を治療、改善又は予防するための組成物。
<44> <1>~<4>のうちのいずれか一項に記載の細菌を特異的に認識する抗体を含む、Th1細胞に起因する疾患を検査するための組成物。
<45> <1>~<4>のうちのいずれか一項に記載の細菌に特異的なヌクレオチド配列を検出するためのポリヌクレオチドを含む、Th1細胞に起因する疾患を検査するための組成物。
<46> <1>~<4>のうちのいずれか一項に記載の細菌に由来する生理活性物質。
<47> <1>~<4>のうちのいずれか一項に記載の細菌に特異的な抗原。
<48> <1>~<4>のうちのいずれか一項に記載の細菌を特異的に認識する抗体。
<49> <1>~<4>のうちのいずれか一項に記載の細菌に特異的なヌクレオチド配列を検出するためのポリヌクレオチド。
上述の通り、本発明者らによって、Klebsiellaに属する2H7株、11E12株、34E1株、BAA-1705株、700603株又は40B3株が、腸管内に定着すると、Th1細胞の顕著な誘導が生じることが明らかになった。したがって、本発明は、腸管内でTh1細胞の増殖又は活性化を誘導する細菌を、提供する。
マンノース-1-リン酸グアニルトランスフェラーゼ1(Mannose-1-phosphate guanylyltransferase 1)、
マルチホスホリル転移タンパク質(Multiphosphoryl transfer protein)、
PTS系フルクトース特異的EIIABC構成タンパク質(PTS system fructose-specific EIIABC component)、
ホスホマンノムターゼ/ホスホグルコムターゼ(Phosphomannomutase/phosphoglucomutase)、
マンノシルフルクトース-リン酸合成酵素(Mannosylfructose-phosphate synthase)、
3-オキソアシル[アシル輸送タンパク質]レダクターゼ FabG(3-oxoacyl-[acyl-carrier-protein] reductase FabG)、
ラムノシル/マンノシルトランスフェラーゼ(rhamnosyl/mannosyltransferase)、
ガラクチトール-1-リン酸5-デヒドロゲナーゼ(Galactitol-1-phosphate 5-dehydrogenase)、
ガラクチトールパーミアーゼIIC構成タンパク質(Galactitol permease IIC component)、
ガラクチトール特異的ホスホトランスフェラーゼ酵素IIB構成タンパク質(Galactitol-specific phosphotransferase enzyme IIB component)、
D-タガトース-1,6-ビスリン酸アルドラーゼサブユニット GatZ(D-tagatose-1,6-bisphosphate aldolase subunit GatZ)、
タガトース-6-リン酸キナーゼ(Tagatose-6-phosphate kinase)、
D-タガトース-1,6-ビスリン酸アルドラーゼサブユニット GatY(D-tagatose-1,6-bisphosphate aldolase subunit GatY)、
ガラクチトールパーミアーゼIIC構成タンパク質(Galactitol permease IIC component)、
GDP-マンノース-依存性 α-(1-2)-ホスファチジルイノシトール マンノシルトランスフェラーゼ(GDP-mannose-dependent alpha-(1-2)-phosphatidylinositol mannosyltransferase)、
L-キシルロース/3-ケト-L-グロン酸キナーゼ(L-xylulose/3-keto-L-gulonate kinase)、
2-デヒドロ-3-デオキシグルコノキナーゼ(2-dehydro-3-deoxygluconokinase)、
莢膜グルカン合成酵素(Capsular glucan synthase)、
3-オクタプレニル-4-ヒドロキシベンゾエートカルボキシ-リアーゼ パートナータンパク質(3-octaprenyl-4-hydroxybenzoate carboxy-lyase partner protein)、
2-オクタプレニルフェノールヒドロキシラーゼ(2-octaprenylphenol hydroxylase)、
フェノール酸デカルボキシラーゼ サブユニットC(Phenolic acid decarboxylase subunit C)、
オキサロ酢酸デカルボキシラーゼβ鎖(Oxaloacetate decarboxylase beta chain)、
アコニット酸ヒドラターゼ2(Aconitate hydratase 2)、
推定アルドラーゼ LsrF(Putative aldolase LsrF)、
推定アセチルトランスフェラーゼ(Putative acetyltransferase)、
プロパンジオール利用タンパク質 PduA(Propanediol utilization protein PduA)、
推定グリコシルトランスフェラーゼ EpsF(Putative glycosyltransferase EpsF)、
へミン結合ペリプラズムタンパク質 HmuT前駆体(Hemin-binding periplasmic protein HmuT precursor)、
タイコ酸排出ATP結合タンパク質 TagH(Teichoic acids export ATP-binding protein TagH)、
タイコ酸移行透過タンパク質 TagG(Teichoic acid translocation permease protein TagG)、
外膜タンパク質 TolC前駆体(Outer membrane protein TolC precursor)、
多剤輸送体 EmrE(Multidrug transporter EmrE)、
マグネシウム及びコバルト排出タンパク質 CorC(Magnesium and cobalt efflux protein CorC)、
内膜タンパク質 YibH(Inner membrane protein YibH)、
アスパラギン酸/アラニン交換輸送体(Aspartate/alanine antiporter)、
鉄エンテロバクチン受容体前駆体(Ferric enterobactin receptor precursor)、
シグナル伝達ヒスチジン-プロテインキナーゼ BarA(Signal transduction histidine-protein kinase BarA)、
ヘモリシン輸送タンパク質 ShlB前駆体(Hemolysin transporter protein ShlB precursor)、
オリゴペプチド輸送ATP結合タンパク質 OppD(Oligopeptide transport ATP-binding protein OppD)、
ヒ素ポンプ-駆動ATPアーゼ(Arsenical pump-driving ATPase)、
推定抗シグマ因子アンタゴニスト(Putative anti-sigma factor antagonist)、
推定膜タンパク質 YdfK(Putative membrane protein YdfK)、
推定ヘモグロビン及びヘモグロビン-へパトグロビン結合タンパク質2前駆体(Putative hemoglobin and hemoglobin-haptoglobin-binding protein 2 precursor)、
(2R)-3-スルホ乳酸デヒドロゲナーゼ(NADP(+))((2R)-3-sulfolactate dehydrogenase (NADP(+)))、
ぺプチダーゼE(Peptidase E)、
オリゴペプチダーゼA(Oligopeptidase A)、
ホスフィノトリシン N-アセチルトランスフェラーゼ(Phosphinothricin N-acetyltransferase)、
推定2-ヒドロキシ酸デヒドロゲナーゼ YoaD(Putative 2-hydroxyacid dehydrogenase YoaD)、
mRNAインターフェラーゼ RelE(mRNA interferase RelE)、
一本鎖DNA特異的エクソヌクレアーゼ RecJ(Single-stranded-DNA-specific exonuclease RecJ)、
チロシンリコンビナーゼ XerD_6(Tyrosine recombinase XerD_6)、
チロシンリコンビナーゼ XerD(Tyrosine recombinase XerD)、
グルシトールオペロンリプレッサー(Glucitol operon repressor)、
ギ酸塩ヒドロゲンリアーゼ転写活性因子(Formate hydrogenlyase transcriptional activator)、
HTH-タイプ転写制御因子 TdfR(HTH-type transcriptional regulator TdfR)、
HTH-タイプ転写制御因子 CatM(HTH-type transcriptional regulator CatM)、
転写制御タンパク質 tctD(Transcriptional regulatory protein tctD)、
HTH-タイプ転写抑制因子 AseR(HTH-type transcriptional repressor AseR)、
サイクリックdi-GMPホスホジエステラーゼ YahA(Cyclic di-GMP phosphodiesterase YahA)、
セリン-プロテインキナーゼ RsbW(Serine-protein kinase RsbW)、
繊維状赤血球凝集素(Filamentous hemagglutinin)、
ジヒドロプテロイン酸合成酵素(Dihydropteroate synthase)、
δ-アミノレブリン酸デヒドラターゼ(Delta-aminolevulinic acid dehydratase)、
好気呼吸制御タンパク質 ArcA(Aerobic respiration control protein ArcA)。
上述のTh1細胞誘導性細菌を、腸管内に定着させることによってTh1細胞の増殖又は活性化を誘導することができる。したがって、本発明は、Th1細胞の増殖又は活性化を誘導する組成物又はその方法を提供することができる。
上述のTh1細胞誘導性細菌は、腸管内に定着し、Th1細胞の増殖又は活性化を誘導することによって、クローン病及び潰瘍性大腸炎等のTh1細胞に起因する疾患を誘発する。そのため、前記Th1細胞誘導性細菌を標的として免疫応答を誘導することによって、腸管内から当該菌を除去すれば、前記疾患を治療、改善又は予防することができる。したがって、本発明は、Th1細胞に起因する疾患を治療、改善又は予防するための、ワクチン組成物又は前記細菌に対する免疫応答を誘導する方法を提供することができる。
上述のTh1細胞誘導性細菌は、腸管内に定着し、Th1細胞の増殖又は活性化を誘導し、また免疫作用を増強することによって、ひいてはクローン病及び潰瘍性大腸炎等のTh1細胞に起因する疾患を誘発する。そのため、前記細菌を腸管内より除去すれば、Th1細胞誘導が抑制され、また免疫作用が抑制されることによって、前記疾患の治療等に繋がる。また、図51に示す通り、Th1細胞の誘導及び免疫賦活化を誘導する前記細菌に由来する生理活性物質が、該生理活性物質に結合する物質(図中での「被験物質」に相当する物質)と結合することにより、その誘導作用が抑制されれば、Th1細胞誘導が抑制され、また免疫作用が抑制され、前記疾患の治療等に至れる。
本発明において、無菌マウスにヒトの唾液を摂取させることにより、腸管内でTh1細胞の増殖又は活性化を誘導する細菌を同定することができた。したがって、本発明は以下のスクリーニング方法を提供する。
本発明によれば、腸管内でTh1細胞の増殖又は活性化を誘導する細菌から、さらに誘導を担う生理活性物質をスクリーニングすることができる。すなわち、本発明は、以下のスクリーニング方法をも提供する。
上述の通り、本発明においては、腸管内でTh1細胞の増殖又は活性化を誘導する細菌を、マウスに経口にて摂取させることにより、該菌が腸管内に定着し、Th1細胞が誘導され、炎症が引き起こされることが、明らかになっている。
前述の通り、腸管内でTh1細胞の増殖又は活性化を誘導する細菌が腸管内に定着している非ヒト動物においては、腸管内でTh1細胞の増殖又は活性化を誘導されており、またそのため、Th1細胞に起因する疾患の非ヒトモデル動物として好適に用いることができる。したがって、本発明は、このモデル動物を用いた、以下のスクリーニング方法をも提供する。
本発明に関し、腸管内においては、図51に示す通り、Th1細胞誘導性細菌又は該菌に由来する生理活性物質は、腸管上皮細胞によって取り込まれて樹状細胞に譲渡される、または該生理活性物質は、直接樹状細胞に捕獲されることが想定される。さらに、前記生理活性物質を抗原として提示した樹状細胞が産生するサイトカインによって、ナイーブT細胞からTh1細胞への分化が誘導されることが想定される。
本発明によれば、腸管内でTh1細胞の増殖又は活性化を誘導する細菌から、さらに誘導を担う生理活性物質をスクリーニングすることができる。すなわち、本発明は、以下のスクリーニング方法をも提供する。
上述の通り、本発明によれば、インビトロにて、腸管内におけるTh1細胞の誘導を再現することができる。
前述の通り、腸管上皮細胞と末梢血単核細胞とを含む系、また更に腸管内でTh1細胞の増殖又は活性化を誘導する細菌を含む系は、Th1細胞の増殖又は活性化を評価するために、好適に用いることができる。したがって、本発明は、かかる系を用いた、以下のスクリーニング方法をも提供する。
上述の通り、本発明において、腸管内でTh1細胞の増殖又は活性化を誘導する細菌が、腸管内に定着することにより、Th1細胞が誘導され、Th1細胞に起因する疾患が誘発されることが明らかになった。そのため、前記細菌の存在を検出することにより、Th1細胞に起因する疾患を検査することが可能となる。
(a)前記特異的なヌクレオチド配列を挟み込むように設計された一対のプライマーであるポリヌクレオチド
(b)前記特異的なヌクレオチド配列を含むヌクレオチド配列にハイブリダイズするプライマー又はプローブであるポリヌクレオチド。
scaffold00004_68_Fプライマー(配列:AATCAAGGGCCCGAGTAAGT[配列番号:1])と、scaffold00004_68_Rプライマー(配列:CCAAACGCTACGCCATTTAT[配列番号:2])との組み合わせ
scaffold00004_298_Fプライマー(配列:AGCACTAGCGGCTGTGGTAT[配列番号:3])と、scaffold00004_298_Rプライマー(配列:ACTTACTCGGGCCCTTGATT[配列番号:4])との組み合わせ
scaffold00004_307_Fプライマー(配列:AATCAAGGGCCCGAGTAAGT[配列番号:5])と、scaffold00004_307_Rプライマー(配列:ATTCAGGGGCTGAAGGAGTT[配列番号:6])との組み合わせ。
C57BL/6マウス、BALB/cマウス及びIQIマウスは、SPF又はGF条件下にて維持されていたものを、三協ラボサービス(日本)、SLC(日本)又はクレア(日本)から購入した。
マウスの糞便を、10mM TrisHCl(pH8.0)含有20%グリセロール/PBSに、終濃度が10%(w/v)になるよう懸濁し、解析に供する迄は-80℃にて保存した。
(1)the 454primer A
[5’-CCATCTCATCCCTGCGTGTCTCCGACTCAG[配列番号:7](454アダプター配列)+バーコード(10bases)+AGRGTTTGATYMTGGCTCAG[配列番号:8]-3’(27Fmod)]
(2) the 454 primer B
[5’-CCTATCCCCTGTGTGCCTTGGCAGTCTCAG[配列番号:35](454アダプター配列)+TGCTGCCTCCCGTAGGAGT[配列番号:9]-3’(338R)]。
ヒト唾液サンプルは、H.S.Said et al.,DNA research:an international journal for rapid publication of reports on genes and genomes 21,15-25(2014)に記載のとおり、治験審査委員会の承認を受けた研究プロトコールに沿って、琉球大学医学部附属病院より得た。また、各被験者よりインフォームドコンセントを得ている。
CD#1の患者:IBD029,50歳、日本人男性,IOIBD スコア3(活動期)
CD#2の患者:IBD121,52歳、日本人男性,IOIBD スコア1(寛解期)
UC#1の患者:IBD096,23歳、日本人女性,UC-DAI 軽度
UC#2の患者:IBD118,65歳、日本人男性,UC-DAI 中等度
健常ドナー#1:S-AKO07,37歳、日本人男性
健常ドナー#2:S-AKO17,39歳、日本人女性。
(27F:5’-AGRGTTTGATYMTGGCTCAG-3’[配列番号:8],
1492R:5’-GGYTACCTTGTTACGACTT-3’[配列番号:10])
カルチャーコレクションにおいて配列が得られたサンプルは、それらの16SrRNA遺伝子配列が100%一致した場合に、「strains」に分類した。
SPF B6マウスを、イソフルランにて麻酔し、背臥位をとらせた。麻酔条件下、気管を約2cm縦に切開することによって正中線上で開き、無菌PBS又はKlebsiella懸濁液(1x106/10μL)を、無菌30ゲージニードルにて気管内に注入した。マウスは、細菌を接種してから7日後に屠殺して肺を回収し、リンパ球の単離及び組織解析に供した。
小腸及び大腸、肺、並びに口蓋を回収した。腸は縦方向に開き、全ての管腔内容物を除去するため、PBSにて洗浄した。全てのサンプルは、5mM EDTA含有ハンクス平衡塩溶液(HBSS)20mLにて、37℃に設定した振とう水浴中、20分間インキュベートし、上皮細胞を除去した。
腸はPBSにて洗浄し、2.5%グルタルアルデヒド含有50mMリン酸バッファー(pH7.2)にて固定し、1%四酸化オスミウム含有50mMリン酸バッファー(pH7.2)にて後固定した。サンプルは、エタノール系にて脱水させ、酢酸イソアミルに置換した。脱水後、サンプルを臨界点乾燥機(CPD 030;Leica Microsystems)に供し、白金にてコーティングし、走査型電子顕微鏡(SU-1510;Hitachi High-Technologies)を用い、5kV又は10kVにて観察した。全ての走査型顕微鏡による解析は、動物1個体あたり少なくとも10エリアを対象として行なった。なお、各グループは動物3~5個体にて構成される。
細菌の構造を可視化するため、金属接触凍結法及び凍結置換法を用いた。凍結装置は、冷凍剤として液体窒素を備えたtype VFZ-101(株式会社真空デバイス社製,茨城,日本)を用いた。凍結置換は2%OsO4含有アセトン、-80℃、120時間かけて行ない、徐々に室温に戻していった(12.5時間かけ-80℃から-55℃まで温度を上昇させ、その後-55℃を8時間維持した。10時間かけ-55℃から-25℃まで温度を上昇させ、その後-25℃を8時間維持した。そして、5時間かけ-25℃から0℃まで温度を上昇させた)。
4%酢酸ウラニル水溶液にて12分間、Reynolds’leadキレート溶液(E.S.Reynolds,The Journal of cell biology 17,208(Apr, 1963)参照)にて1分間、切片を染色した。そして、透過型電子顕微鏡(JEM-1400;Jeol,東京,日本)を用い、80kVにて観察した。
SPFマウス(WT B6,Il10-/-又はIfngr1-/-)を、菌強制摂取の前に、下記抗生物質を飲料水を介して4日間投与した。また、これら抗生物質を投与しないマウスも用意した。
抗生物質:アンピシリン(200mg/L)、タイロシン(500mg/L)、メトロニダゾール(500mg/L)、スペクチノマイシン(200mg/L)、バンコマイシン(200mg/L)。
Klebsiella(ompK36-3_F:5’-GCGACCAGACCTACATGCGT-3’[配列番号:11],ompK36-3_R:5’-AGTCGAAAGAGCCCGCGTC-3’[配列番号:12])、
Kp-2H7(sca4_298_F:5’-AGCACTAGCGGCTGTGGTAT-3’[配列番号:13],sca4_298_R:5’-ACTTACTCGGGCCCTTGATT-3’[配列番号:14])、
Ka-11E12(group_4037_F:5’-CTTCGCCTTCATCAGCTTCA-3’[配列番号:15],group_4037_R:5’-TCATCATTAACGCGGGTCAG-3’[配列番号:16])。
大腸組織を回収し、縦方向に切り開き、氷冷PBSにてよく洗浄し、上皮細胞(ECs)をガラススライドを用いてはがし、直ぐに液体窒素にて凍結させ、解析に供するまで-80℃にて保存した。
TRIzol試薬(Invitrogen社製)を用い、その説明書に従って、大腸のEC及びDCからトータルRNAを単離した。
Gapdh, 5’-CTCATGACCACAGTCCATGC-3’[配列番号:17] and 5’-CACATTGGGGGTAGGAACAC-3’[配列番号:18]
Gbp2, 5’-TGCTGGATCTTTGCTTTGGC-3’[配列番号:19] and 5’-AGTTAGCTCCGTCACATAGTGC-3’[配列番号:20]
Gbp6, 5’-AATGCCTTGAAGCTGATCCC-3’[配列番号:21] and 5’-GTTCTTTGTCATGCGTTGGC-3’[配列番号:22]
Ifi47, 5’-GGCTCATTGCTTCAGACTTTCC-3’[配列番号:23] and 5’-ACTGATCCATGGCAGTTACCAG-3’[配列番号:24]
Cxcl9, 5’-ATCATCTTCCTGGAGCAGTGTG-3’[配列番号:25] and 5’-TTGTTGCAATTGGGGCTTGG-3’[配列番号:26]
H2-Ab1, 5’-TTGGCCTTTTCATCCGTCAC-3’[配列番号:27] and 5’-ATTCGGAGCAGAGACATTCAGG-3’[配列番号:28]
H2-DMb1, 5’-TCTCCAGCGTTTGCAAAACG-3’[配列番号:29] and 5’-AAAGGTGTGGTTTGGGCTAC-3’[配列番号:30]
Ifi208, 5’-AGAACTTGCAGCTCGTGTTG-3’[配列番号:31] and 5’-TGGTTCTACTTCCCAAGCTTCC-3’[配列番号:32]
Ifng, 5’-ACGGCACAGTCATTGAAAGC-3’[配列番号:33] and 5’-ACCATCCTTTTGCCAGTTCC-3’[配列番号:34]。
Kp-2H7及びKa-11E12を対象とする蛍光in situハイブリダイゼーション(FISH)を行なうため、大腸組織をメタノール-カルノイ溶液にて固定し、パラフィンに包埋した。切片は、0.1M HClにて処理し、5’Alexa488-標識EUB338(5’-GCTGCCTCCCGTAGGAGT-3’)プローブとハイブリダイズさせ、ローダミン標識ヨーロッパハリエニシダアグルチニンI(UEA1、Vector Laboratories社製)にて染色した。全ての切片は、DAPIにて対比染色し、Fluoromount/Plus(Diagnostic BioSystems社製)に載せ、ライカ社製TCS SP5共焦点顕微鏡にて可視化した。
炎症性細胞浸潤(スコア:0~4)
粘膜肥厚(スコア:0~4)
杯細胞減少(スコア:0~4)
陰窩膿瘍(スコア:0~4)
構造破壊(スコア:0~4)。
最終的な組織学的スコアは、これらパラメーターのスコア総和により決定した。
肺胞(変化なし:0、浮腫:1、肺胞腔における炎症細胞の検出:2、肺膿腫を伴う肺胞の炎症性破壊)
細気管支(変化なし:0、壁における軽度の炎症:1、内腔のかさぶた(luminal slough)を伴う壁における重度の炎症:2、内腔のかさぶた及び気管支周囲炎を伴う重度の炎症:3)。
Klebsiella株のゲノムを、上記16SrRNA遺伝子パイロシークエンシングに記載の方法と同様の方法にて抽出した。そして、PacBioRSII及びイルミナMiSeqシークエンサーを用いたホールゲノムショットガン配列決定法にて、ゲノム配列を決定した。
各株のゲノム配列については、MLSTデータベースに対してアライメントをかけた(http://bigsdb.pasteur.fr/klebsiella/klebsiella.html)。そして、配列に基づくcapsular (K)タイピングを、wzi又はwzc遺伝子のシークエンシングに基づいて行なった。
細菌の運動分析は、14mLチューブに入れた0.25%含有半固形LB培地を用いて行なった。LBブロス培地にて培養した細菌を、半固形LB培地にストレートワイヤを用い、チューブの真ん中に、培地半分の深さまで突き刺し、接種した。37℃で24時間インキュベーションした後、培地中の至るところに放散及び漠然とした拡散が認めされた細菌を、運動性細菌と判定した。細菌の鞭毛は、HEK-Blue mTLR5細胞(InvivoGen社製)を用い、メーカープロトコールに従って検出した。
全ての統計解析は、GraphPad Prismソフトウェア(GraphPad Software,Inc.)又はJMPソフトウェアv.12(SAS Institute,Inc.)を用い、独立両側スチューデントt検定(パラメリック)、ウィルコクソンの順位和検定(ノンパラメリック)、並びに一元配置分散分析(ANOVA)及びそれに続くテューキーのポストホックテスト(3以上のグループ、パラメリック)を行なった。
本発明者らは、クローン病(CD)2患者の唾液サンプルを、C57BL/6(B6)無菌(GF)マウスに、強制的に経口投与してノトバイオートマウスを作製した。そして、各グループのマウスを、純粋隔離アイソレーターにて6週間飼育し、小腸及び結腸固有層(LP)の免疫細胞について調べた。
Gemella,Bifidobacterium,Streptococcus,Escherichia,Fusobacterium,Veillonella,Anaerococcus,Klebsiella。
535-538(2016) 参照)。
Kp-2H7が介するTh1細胞誘導が基礎となるメカニズムを探るため、様々なアプローチを試みた。
口腔由来の細菌とTh1細胞誘導との関連を更に確かめるために、2人の健常ドナー(He#1及びHe#2)、並びに活動期にある潰瘍性大腸炎(UC)の患者2人(UC#1及びUC#2)から、更なる唾液サンプルを得て、それらサンプルをGF WT B6マウスに経口投与した。
本発明者らは、次に、IBD及び他の疾患の患者において、Klebsiellaが豊富に存在しているかどうかを調べた。
<223> 人工的に合成されたプライマーの配列
Claims (49)
- 腸管内でTh1細胞の増殖又は活性化を誘導する細菌。
- 更に、Klebsiella pneumoniae又はKlebsiella aeromobilisに属することを特徴とする、請求項1に記載の細菌。
- 更に、下記タンパク質群から選択される少なくとも5つのタンパク質をコードする遺伝子を保有することを特徴とする、請求項1又は2に記載の細菌
タンパク質群:
マンノース-1-リン酸グアニルトランスフェラーゼ1、
マルチホスホリル転移タンパク質、
PTS系フルクトース特異的EIIABC構成タンパク質、
ホスホマンノムターゼ/ホスホグルコムターゼ、
マンノシルフルクトース-リン酸合成酵素、
3-オキソアシル[アシル輸送タンパク質]レダクターゼ FabG、
ラムノシル/マンノシルトランスフェラーゼ、
ガラクチトール-1-リン酸5-デヒドロゲナーゼ、
ガラクチトールパーミアーゼIIC構成タンパク質、
ガラクチトール特異的ホスホトランスフェラーゼ酵素IIB構成タンパク質、
D-タガトース-1,6-ビスリン酸アルドラーゼサブユニット GatZ、
タガトース-6-リン酸キナーゼ、
D-タガトース-1,6-ビスリン酸アルドラーゼサブユニット GatY、
ガラクチトールパーミアーゼIIC構成タンパク質、
GDP-マンノース-依存性 α-(1-2)-ホスファチジルイノシトール マンノシルトランスフェラーゼ、
L-キシルロース/3-ケト-L-グロン酸キナーゼ、
2-デヒドロ-3-デオキシグルコノキナーゼ、
莢膜グルカン合成酵素、
3-オクタプレニル-4-ヒドロキシベンゾエートカルボキシ-リアーゼ パートナータンパク質、
2-オクタプレニルフェノールヒドロキシラーゼ、
フェノール酸デカルボキシラーゼ サブユニットC、
オキサロ酢酸デカルボキシラーゼβ鎖、
アコニット酸ヒドラターゼ2、
推定アルドラーゼ LsrF、
推定アセチルトランスフェラーゼ、
プロパンジオール利用タンパク質 PduA、
推定グリコシルトランスフェラーゼ EpsF、
へミン結合ペリプラズムタンパク質 HmuT前駆体、
タイコ酸排出ATP結合タンパク質 TagH、
タイコ酸移行透過タンパク質 TagG、
外膜タンパク質 TolC前駆体、
多剤輸送体 EmrE、
マグネシウム及びコバルト排出タンパク質 CorC、
内膜タンパク質 YibH、
アスパラギン酸/アラニン交換輸送体、
鉄エンテロバクチン受容体前駆体、
シグナル伝達ヒスチジン-プロテインキナーゼ BarA、
ヘモリシン輸送タンパク質 ShlB前駆体、
オリゴペプチド輸送ATP結合タンパク質 OppD、
ヒ素ポンプ-駆動ATPアーゼ、
推定抗シグマ因子アンタゴニスト、
推定膜タンパク質 YdfK、
推定ヘモグロビン及びヘモグロビン-へパトグロビン結合タンパク質2前駆体、
(2R)-3-スルホ乳酸デヒドロゲナーゼ(NADP(+))、
ぺプチダーゼE、
オリゴペプチダーゼA、
ホスフィノトリシン N-アセチルトランスフェラーゼ、
推定2-ヒドロキシ酸デヒドロゲナーゼ YoaD、
mRNAインターフェラーゼ RelE、
一本鎖DNA特異的エクソヌクレアーゼ RecJ、
チロシンリコンビナーゼ XerD_6、
チロシンリコンビナーゼ XerD、
グルシトールオペロンリプレッサー、
ギ酸塩ヒドロゲンリアーゼ転写活性因子、
HTH-タイプ転写制御因子 TdfR、
HTH-タイプ転写制御因子 CatM、
転写制御タンパク質 tctD、
HTH-タイプ転写抑制因子 AseR、
サイクリックdi-GMPホスホジエステラーゼ YahA、
セリン-プロテインキナーゼ RsbW、
繊維状赤血球凝集素、
ジヒドロプテロイン酸合成酵素、
δ-アミノレブリン酸デヒドラターゼ、
好気呼吸制御タンパク質 ArcA。 - 更に、マンノース、フルクトース及びガラクトースのうちの少なくとも1の糖の代謝に関与する遺伝子を保有することを特徴とする、請求項1~3のうちのいずれか一項に記載の細菌。
- 請求項1~4のうちのいずれか一項に記載の細菌又は該細菌に由来する生理活性物質を有効成分として含む、免疫を賦活させるための組成物。
- 更に、Th1細胞の増殖又は活性化を誘導するための組成物であることを特徴とする、請求項5に記載の組成物。
- 請求項1~4のうちのいずれか一項に記載の細菌又は該細菌に由来する生理活性物質を、対象に摂取させ、該対象におけるTh1細胞の増殖又は活性化を誘導する方法。
- 請求項1~4のうちのいずれか一項に記載の細菌又は該細菌に由来する生理活性物質を、対象に摂取させ、該対象における免疫を賦活する方法。
- 請求項1~4のうちのいずれか一項に記載の細菌又は該細菌に特異的な抗原を有効成分として含む、ワクチン組成物。
- 請求項1~4のうちのいずれか一項に記載の細菌又は該細菌に特異的な抗原を、対象に摂取させ、該対象における前記細菌に対する免疫応答を誘導する方法。
- 請求項1~4のうちのいずれか一項に記載の細菌に対して抗菌活性を有する物質又は請求項1~4のうちのいずれか一項に記載の細菌に由来する生理活性物質に結合する物質を、有効成分として含む、免疫を抑制するための組成物。
- 更に、Th1細胞の増殖又は活性化を抑制するための組成物であることを特徴とする、請求項11に記載の組成物。
- 更に、Th1細胞に起因する疾患を治療、改善又は予防するための組成物であることを特徴とする、請求項11又は12に記載の組成物。
- 請求項1~4のうちのいずれか一項に記載の細菌に対して抗菌活性を有する物質又は請求項1~4のうちのいずれか一項に記載の細菌に由来する生理活性物質に結合する物質を、対象に摂取させ、該対象におけるTh1細胞の増殖又は活性化を抑制する方法。
- 請求項1~4のうちのいずれか一項に記載の細菌に対して抗菌活性を有する物質又は請求項1~4のうちのいずれか一項に記載の細菌に由来する生理活性物質に結合する物質を、対象に摂取させ、該対象における免疫を抑制する方法。
- 請求項1~4のうちのいずれか一項に記載の細菌に対して抗菌活性を有する物質又は請求項1~4のうちのいずれか一項に記載の細菌に由来する生理活性物質に結合する物質を、対象に摂取させ、該対象におけるTh1細胞に起因する疾患を治療、改善又は予防する方法。
- 請求項1~4のうちのいずれか一項に記載の細菌が、腸管内に定着している、非ヒト動物。
- 更に、Th1細胞に起因する疾患の非ヒトモデル動物であることを特徴とする、請求項17に記載の非ヒト動物。
- 請求項17又は18に記載の非ヒト動物の製造方法であって、
腸管内でTh1細胞の増殖又は活性化を誘導する細菌を、非ヒト動物に摂取させ、該細菌を該動物の腸管内に定着させる工程を含む、方法。 - 請求項1~4のうちのいずれか一項に記載の細菌と、腸管上皮細胞と、末梢血単核細胞とを含む、Th1細胞の増殖又は活性化を評価するためのキット。
- 腸管上皮細胞と、末梢血単核細胞とを含む、Th1細胞の増殖又は活性化を評価するためのキット。
- 腸管内でTh1細胞の増殖又は活性化を誘導する細菌をスクリーニングする方法であって、
被験試料を非ヒト無菌動物に摂取させる工程と、
該非ヒト無菌動物の腸管内におけるTh1細胞の数又は活性を検出する工程と、
前記工程にてTh1細胞の増殖又は活性化が検出された非ヒト無菌動物の腸管内試料から細菌を単離する工程とを、含む方法。 - 腸管内でTh1細胞の増殖又は活性化を誘導する生理活性物質をスクリーニングする方法であって、
腸管内でTh1細胞の増殖又は活性化を誘導する細菌に由来する生理活性物質を、非ヒト無菌動物に摂取させる工程と、
該非ヒト無菌動物の腸管内におけるTh1細胞の数又は活性を検出する工程と、
前記工程にてTh1細胞の増殖又は活性化が検出された場合に、前記生理活性物質を、腸管内でTh1細胞の増殖又は活性化を誘導する生理活性物質であると判定する工程とを、含む方法。 - 腸管内でTh1細胞の増殖又は活性化を誘導する細菌をスクリーニングする方法であって、
腸管上皮細胞と末梢血単核細胞とを含む系において、該腸管上皮細胞に被験細菌を添加する工程と、
該系におけるTh1細胞の数又は活性を検出する工程と、
前記工程にてTh1細胞の増殖又は活性化が検出された場合には、前記被験細菌を、腸管内でTh1細胞の増殖又は活性化を誘導する細菌であると判定する工程とを、含む方法。 - 腸管内でTh1細胞の増殖又は活性化を誘導する生理活性物質をスクリーニングする方法であって、
腸管上皮細胞と末梢血単核細胞とを含む系において、該腸管上皮細胞に、腸管内でTh1細胞の増殖又は活性化を誘導する細菌に由来する生理活性物質を添加させる工程と、
該系におけるTh1細胞の数又は活性を検出する工程と、
前記工程にてTh1細胞の増殖又は活性化が検出された場合には、前記生理活性物質を、腸管内でTh1細胞の増殖又は活性化を誘導する生理活性物質であると判定する工程とを、含む方法。 - 腸管内でTh1細胞の増殖又は活性化を誘導する物質のスクリーニング方法であって、
腸管上皮細胞と末梢血単核細胞とを含む系において、該腸管上皮細胞に、腸管内でTh1細胞の増殖又は活性化を誘導する細菌と、被験物質とを添加する工程と、
該系におけるTh1細胞の数又は活性を検出する工程と、
前記工程にて検出されたTh1細胞の数又は活性が、前記被験物質を添加しなかった場合におけるそれよりも増加している場合に、前記被験化合物は、腸管内でTh1細胞の増殖又は活性化を誘導する物質であると判定する工程とを、含む方法。 - 腸管内でTh1細胞の増殖又は活性化を誘導する物質のスクリーニング方法であって、
請求項17に記載の非ヒト動物に、被験物質を摂取させる工程と、
該非ヒト動物の腸管内におけるTh1細胞の数又は活性を検出する工程と、
前記工程にて検出されたTh1細胞の数又は活性が、前記被験物質を摂取させなかった場合におけるそれよりも増加している場合に、前記被験物質は、腸管内でTh1細胞の増殖又は活性化を誘導する物質であると判定する工程とを、含む方法。 - 請求項22~27のうちのいずれか一項に記載のスクリーニング方法によって得られる、細菌、生理活性物質又は物質を、有効成分として含む、免疫を賦活するための組成物。
- 更に、Th1細胞の増殖又は活性化を誘導するための組成物であることを特徴とする、請求項28に記載の組成物。
- 請求項22若しくは24に記載のスクリーニング方法によって得られる細菌又は該細菌に特異的な抗原を有効成分として含む、ワクチン組成物。
- Th1細胞に起因する疾患を誘発又は憎悪させる活性を有する物質を、スクリーニングするための方法であって、
請求項18に記載の非ヒト動物に、被験物質を摂取させる工程と、
該非ヒト動物におけるTh1細胞に起因する疾患の病変の程度を検出する工程と、
前記工程にて検出された病変の程度が、前記被験物質を摂取させなかった場合におけるそれよりも増加している場合に、前記被験物質は、Th1細胞に起因する疾患を誘発又は憎悪させる活性を有する物質であると判定する工程とを、含む方法。 - 請求項31に記載のスクリーニング方法によって得られる物質を、有効成分として含む、Th1細胞に起因する疾患を誘発又は憎悪させるための組成物。
- 腸管内でTh1細胞の増殖又は活性化を抑制する細菌をスクリーニングする方法であって、
被験試料を非ヒト無菌動物に摂取させる工程と、
該非ヒト無菌動物の腸管内におけるTh1細胞の数又は活性を検出する工程と、
前記工程にてTh1細胞の増殖又は活性化の抑制が検出された非ヒト無菌動物の腸管内試料から細菌を単離する工程とを、含む方法。 - 腸管内でTh1細胞の増殖又は活性化を抑制する生理活性物質をスクリーニングする方法であって、
腸管内でTh1細胞の増殖又は活性化を抑制する細菌に由来する生理活性物質を、非ヒト無菌動物に摂取させる工程と、
該非ヒト無菌動物の腸管内におけるTh1細胞の数又は活性を検出する工程と、
前記工程にてTh1細胞の増殖又は活性化の抑制が検出された場合に、前記生理活性物質を、腸管内でTh1細胞の増殖又は活性化を抑制する生理活性物質であると判定する工程とを、含む方法。 - 腸管内でTh1細胞の増殖又は活性化を抑制する細菌をスクリーニングする方法であって、
腸管上皮細胞と末梢血単核細胞とを含む系において、該腸管上皮細胞に被験細菌を添加する工程と、
該系におけるTh1細胞の数又は活性を検出する工程と、
前記工程にてTh1細胞の増殖又は活性化の抑制が検出された場合には、前記被験細菌を、腸管内でTh1細胞の増殖又は活性化を抑制する細菌であると判定する工程とを、含む方法。 - 腸管内でTh1細胞の増殖又は活性化を抑制する生理活性物質をスクリーニングする方法であって、
腸管上皮細胞と末梢血単核細胞とを含む系において、該腸管上皮細胞に、腸管内でTh1細胞の増殖又は活性化を抑制する細菌に由来する生理活性物質を添加する工程と、
該系におけるTh1細胞の数又は活性を検出する工程と、
前記工程にてTh1細胞の増殖又は活性化の抑制が検出された場合には、前記生理活性物質を、腸管内でTh1細胞の増殖又は活性化を抑制する生理活性物質であると判定する工程とを、含む方法。 - 腸管内でTh1細胞の増殖又は活性化を抑制する物質のスクリーニング方法であって、
腸管上皮細胞と末梢血単核細胞とを含む系において、該腸管上皮細胞に、腸管内でTh1細胞の増殖又は活性化を誘導する細菌と、被験物質とを添加する工程と、
該系におけるTh1細胞の数又は活性を検出する工程と、
前記工程にて検出されたTh1細胞の数又は活性が、前記被験化合物を添加しなかった場合におけるそれよりも低減している場合に、前記被験物質は、腸管内でTh1細胞の増殖又は活性化を抑制する物質であると判定する工程とを、含む方法。 - 腸管内でTh1細胞の増殖又は活性化を抑制する物質のスクリーニング方法であって、
請求項17に記載の非ヒト動物に、被験物質を摂取させる工程と、
該非ヒト動物の腸管内におけるTh1細胞の数又は活性を検出する工程と、
前記工程にて検出されたTh1細胞の数又は活性が、前記被験物質を摂取させなかった場合におけるそれよりも低減している場合に、前記被験物質は、腸管内でTh1細胞の増殖又は活性化を抑制する物質であると判定する工程とを、含む方法。 - 請求項33~38のうちのいずれか一項に記載のスクリーニング方法によって得られる、細菌、生理活性物質又は物質を、有効成分として含む、免疫を抑制するための組成物。
- 更に、Th1細胞の増殖又は活性化を抑制するための組成物であることを特徴とする、請求項39に記載の組成物。
- 更に、Th1細胞に起因する疾患を治療、改善又は予防するための組成物であることを特徴とする、請求項39又は40に記載の組成物。
- Th1細胞に起因する疾患を治療、改善又は予防する活性を有する物質をスクリーニングするための方法であって、
請求項18に記載の非ヒト動物に、被験物質を摂取させる工程と、
該非ヒト動物におけるTh1細胞に起因する疾患の病変の程度を検出する工程と、
前記工程にて検出された病変の程度が、前記被験物質を摂取させなかった場合におけるそれよりも低減している場合に、前記被験物質は、Th1細胞に起因する疾患を治療、改善又は予防する活性を有する物質であると判定する工程とを、含む方法。 - 請求項42に記載のスクリーニング方法によって得られる物質を、有効成分として含む、Th1細胞に起因する疾患を治療、改善又は予防するための組成物。
- 請求項1~4のうちのいずれか一項に記載の細菌を特異的に認識する抗体を含む、Th1細胞に起因する疾患を検査するための組成物。
- 請求項1~4のうちのいずれか一項に記載の細菌に特異的なヌクレオチド配列を検出するためのポリヌクレオチドを含む、Th1細胞に起因する疾患を検査するための組成物。
- 請求項1~4のうちのいずれか一項に記載の細菌に由来する生理活性物質。
- 請求項1~4のうちのいずれか一項に記載の細菌に特異的な抗原。
- 請求項1~4のうちのいずれか一項に記載の細菌を特異的に認識する抗体。
- 請求項1~4のうちのいずれか一項に記載の細菌に特異的なヌクレオチド配列を検出するためのポリヌクレオチド。
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WO2019017389A1 (ja) | 2017-07-18 | 2019-01-24 | 学校法人慶應義塾 | Th1細胞誘導性細菌に対する抗菌組成物 |
US20190046591A1 (en) * | 2011-12-01 | 2019-02-14 | The University Of Tokyo | Human-derived bacteria that induce proliferation or accumulation of regulatory t cells |
US10322150B2 (en) | 2010-06-04 | 2019-06-18 | The University Of Tokyo | Composition for inducing proliferation or accumulation of regulatory T cells |
WO2020179868A1 (ja) | 2019-03-07 | 2020-09-10 | 学校法人慶應義塾 | 薬剤耐性細菌又は炎症惹起性細菌に対する抗菌組成物 |
CN114369146A (zh) * | 2022-01-14 | 2022-04-19 | 上海交通大学医学院附属仁济医院 | 一种阿克曼氏菌Amuc_2172蛋白及其制备方法和用途 |
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US10588925B2 (en) | 2010-06-04 | 2020-03-17 | The University Of Tokyo | Composition for inducing proliferation or accumulation of regulatory T cells |
US10322150B2 (en) | 2010-06-04 | 2019-06-18 | The University Of Tokyo | Composition for inducing proliferation or accumulation of regulatory T cells |
US10328108B2 (en) | 2010-06-04 | 2019-06-25 | The University Of Tokyo | Composition for inducing proliferation or accumulation of regulatory T cells |
US10555978B2 (en) | 2010-06-04 | 2020-02-11 | The University Of Tokyo | Composition for inducing proliferation or accumulation of regulatory T cells |
US11090343B2 (en) | 2010-06-04 | 2021-08-17 | The University Of Tokyo | Composition for inducing proliferation or accumulation of regulatory T cells |
US11547732B2 (en) | 2011-12-01 | 2023-01-10 | The University Of Tokyo | Human-derived bacteria that induce proliferation or accumulation of regulatory T cells |
US10342832B2 (en) | 2011-12-01 | 2019-07-09 | The University Of Tokyo | Human-derived bacteria that induce proliferation or accumulation of |
US20190046591A1 (en) * | 2011-12-01 | 2019-02-14 | The University Of Tokyo | Human-derived bacteria that induce proliferation or accumulation of regulatory t cells |
US10624933B2 (en) | 2011-12-01 | 2020-04-21 | The University Of Tokyo | Human-derived bacteria that induce proliferation or accumulation of regulatory T cells |
US10835559B2 (en) * | 2011-12-01 | 2020-11-17 | The University Of Tokyo | Human-derived bacteria that induce proliferation or accumulation of regulatory T cells |
WO2019017389A1 (ja) | 2017-07-18 | 2019-01-24 | 学校法人慶應義塾 | Th1細胞誘導性細菌に対する抗菌組成物 |
US11633433B2 (en) | 2017-07-18 | 2023-04-25 | Keio University | Anti-bacterial composition against TH1 cell-inducing bacteria |
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CN114369146A (zh) * | 2022-01-14 | 2022-04-19 | 上海交通大学医学院附属仁济医院 | 一种阿克曼氏菌Amuc_2172蛋白及其制备方法和用途 |
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EP3546566A1 (en) | 2019-10-02 |
US20230293656A1 (en) | 2023-09-21 |
JPWO2018084172A1 (ja) | 2019-09-19 |
EP3546566A4 (en) | 2020-05-27 |
AU2017354716A1 (en) | 2019-05-02 |
CA3041277A1 (en) | 2018-05-11 |
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CN110352236A (zh) | 2019-10-18 |
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