WO2019041597A1 - Utilisation de candida dans la préparation d'un produit pour la prévention et le traitement de maladies du système immunitaire - Google Patents

Utilisation de candida dans la préparation d'un produit pour la prévention et le traitement de maladies du système immunitaire Download PDF

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WO2019041597A1
WO2019041597A1 PCT/CN2017/113539 CN2017113539W WO2019041597A1 WO 2019041597 A1 WO2019041597 A1 WO 2019041597A1 CN 2017113539 W CN2017113539 W CN 2017113539W WO 2019041597 A1 WO2019041597 A1 WO 2019041597A1
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candida
enteric
intestinal
product
dendritic cells
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PCT/CN2017/113539
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Chinese (zh)
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石彦
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清华大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/06—Fungi, e.g. yeasts
    • A61K36/062—Ascomycota
    • A61K36/064—Saccharomycetales, e.g. baker's yeast
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
    • C12N1/165—Yeast isolates
    • 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
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/72—Candida
    • C12R2001/74—Candida tropicalis

Definitions

  • the invention belongs to the field of biotechnology and relates to the application of a Candida tropicalis in preparing products for preventing and treating diseases of the immune system.
  • lymph nodes are the origin of immune responses, how symbiotic bacteria regulate these widely distributed immune organs has become an important issue.
  • lymph nodes are the origin of immune responses, how symbiotic bacteria regulate these widely distributed immune organs has become an important issue.
  • the link between these commensal bacteria and the development of the peripheral immune system has not been clear so far.
  • lymphoid tissue-inducing cells LTi
  • T and B cell regions the number of cells contained continues to increase.
  • the infiltrated lymphocytes form clear T and B cell regions, almost identical to adult mouse lymph nodes.
  • the development of lymph nodes in sterile animals completely ceased after birth. The above developmental problems bring about defects in the immune response.
  • the sterile mice were not immune to the infection after being infected by the intestinal pathogen Shigella flexneri.
  • the symptoms of Salmonella infection in sterile mice are also more severe.
  • As the main site of the immune response it is conceivable that defects in the structure of the lymph nodes may cause disorders in the immune response. So how does intestinal colony initiate lymph node development after birth?
  • novel use provided by the present invention is specifically: the application of the lipid extract of Candida enteric or Candida enteric in any of the following (A)-(D):
  • a method for preventing and/or treating an immune disorder-related disease is to prevent and/or treat an immune disorder-related disease using a lipid extract of Candida enteric or Entericococcus.
  • a method of regulating the function of the peripheral immune system is to use a lipid extract of Candida enteric or Candida enteric to regulate peripheral immune system function.
  • a method of driving intestinal-derived dendritic cells to lymph nodes is to use a lipid extract of Candida enteric or Candida enteric to drive intestinal-derived dendritic cells to lymph nodes.
  • a method of promoting lymph node development and/or promoting lymph node function is to use a lipid extract of Candida enteric or Entericococcus to promote lymph node development and/or to promote lymph node function.
  • the present invention also claims a product having any of the following functions: preventing and/or treating immune disorder-related diseases, modulating peripheral immune system function, driving intestinal-derived dendritic cells to lymph node metastasis, promoting lymph node development and/or Or promote lymph nodes to function.
  • Candida albicans are specifically Candida tropicalis.
  • all of the intestinal-derived dendritic cells described above are specifically intestinal-derived dendritic cells expressing CD103, CD11b, and retinal dehydrogenase.
  • all of the lipid extracts of the aforementioned Candida of the genus Candida can be obtained by a method comprising the steps of: placing the intestinal candida in a volume ratio of 2:1 of chloroform and methanol. The mixture was sonicated, and after centrifugation, the methanol was washed with water, and the chloroform was blown dry with nitrogen to obtain the lipid extract of Candida enteric.
  • all of the aforementioned immune disorder-related diseases may be autoimmune diseases or hyperimmune inflammatory reactions. Specifically, such as rheumatism, lupus erythematosus, enteritis, multiple sclerosis or ankylosing spondylitis.
  • all of the aforementioned driving intestinal-derived dendritic cells to lymph nodes may specifically drive the transfer of the intestinal-derived dendritic cells to mesenteric lymph nodes and/or non-intestinal peripheral lymph nodes.
  • Figure 1 shows lymphocytes isolated from lymph nodes of adult normal mice. After labeling, normal (SPF) or sterile (GF) mice are injected through the tail vein.
  • SPF normal
  • GF sterile mice
  • Figure 2 shows the absence of RALDH positive DC cells in adult sterile mouse lymph nodes.
  • Figure 3 shows that intestinal microbes regulate lymph node development through retinal dehydrogenase RALDH + dendritic cells.
  • Figure 4 is an analysis of the source of CD103 + CD11b + RALDH + DC-like cells.
  • Figure 5 shows that Candida tropicalis drives intestinal CD103 + CD11b + RALDH + dendritic cells to lymph node metastasis.
  • RALDH + CD103 + at the ordinate indicates CD103 + CD11b + RALDH + dendritic cells.
  • Figure 6 shows that the lipid extract of Candida tropicalis can drive BMDC to lymph node metastasis.
  • Example 1 Candida tropicalis and its lipid extracts drive intestinal DC cells to lymph node metastasis
  • lymphocytes to lymph nodes are defective in sterile mice.
  • Lymphocytes isolated from lymph nodes of adult normal C57BL/6 mice were labeled with CFSE and injected into normal (SPF) or sterile (GF) C57BL/6 mice via the tail vein (dose was 2 x per mouse) 10 6 cells) After 24 hours, various lymph nodes were isolated, subjected to fluorescence imaging after sectioning, or after cell suspension was obtained and analyzed by flow cytometry.
  • FIG. 1 Left: Infusion of lymphocytes into the inguinal lymph nodes (iLN), mesenteric lymph nodes (mLN) and spleen (spl), and analysis of lymphocyte subtypes to iLN reflux (bottom left).
  • HEV high endothelial venules
  • lymph nodes of normal (SPF) and sterile (GF) C57BL/6 mice were compared for staining, and the presence or absence of RALDH-positive DC cells was observed.
  • RALDH + dendritic cells and RALDH - dendritic cells were isolated from SPF-class C57BL/6 mouse lymph nodes, and sterile (GF) C57BL/6 mice (injected at a dose of 2 ⁇ 10 5 cells) were injected through the tail vein. Seven days later, lymph nodes were taken for sectioning and flow cytometry analysis.
  • lymph nodes When these cells isolated from normal mice are injected into sterile mice through the tail vein, they promote the development of the latter lymph nodes and a large number of lymphocytes enter the lymph nodes.
  • These dendritic cells highly express retinal dehydrogenase and can express a substance called peripheral lymphotropic substance on the vascular epithelium at the entrance of the lymph node. It can also be regarded as a marker for address localization. T and B cells form lymph nodes by recognizing peripheral lymphotropins into lymph nodes. This process is particularly evident when the mouse is born. However, in adult rats, there are still a small number of such dendritic cells in the lymph nodes, which maintain the long-term homeostasis of the lymph nodes.
  • dendritic cells such as lymph nodes disappear, causing structural damage.
  • these dendritic cells are derived from the gut and are of the same type as the unconventional dendritic cells (expressing CD103, CD11b and retinal dehydrogenase) in the intestinal lamina propria. .
  • the specific method is as follows: neonatal C57BL/6 mice were intragastrically administered with FITC Dextran (FITC-dextran2000KD (Sigma) 0.3 mg/g body weight), and 6 hours later, the CD11b and CD103 single positive in the peripheral lymph nodes were detected by flow cytometry, and the double negative ( The ratio of DN) to double positive (DP) dendritic cells.
  • FITC-dextran2000KD Sigma
  • Candida tropicalis drives intestinal CD103 + CD11b + RALDH + dendritic cells to lymph node metastasis
  • FIG. 5 The result is shown in Figure 5.
  • Upper left Adult SPF mice were mixed with antibiotics or the antifungal fluconazole in drinking water, CD103 + CD11b + RALDH + dendrites in mesenteric lymph nodes (mLN) and Peyer's patches (PP) after 3 weeks. The percentage of cells.
  • Upper right Adult normal mice were inguinal lymph nodes (iLN) and mesenteric lymph nodes (mLN) after 24 hours of intragastric administration with cultured Candida tropicalis, Saccharomyces cerevisiae and Trichosporon. Percentage of CD103 + CD11b + RALDH + dendritic cells.
  • Bottom left Similar to the upper right, the result of newborn mice.
  • Bottom right Similar to the results of upper right and lower left, adult sterile mice. It can be seen that Candida (C. tropicalis) in intestinal microbes drives the transfer of CD103 + CD11b + RALDH + dendritic cells to lymph nodes.
  • the lipid extract of Candida tropicalis can drive intestinal CD103 + CD11b + RALDH + dendritic cells to lymph node metastasis
  • the ribonucleic acids, proteins and lipids of various fungi and bacteria are obtained by a separate extraction method. After these isolates were injected into mice, only Candida tropicalis lipids could drive the transfer of dendritic cells to lymph nodes. details as follows:
  • the fungi and bacterial lipids were extracted by chloroform-methanol extraction, wherein the fungi were Candida tropicalis, Saccharomyces cerevisiae and Trichosporon, and the bacteria were E coli.
  • the fungus or bacteria were sonicated in chloroform-methanol (2:1 by volume), centrifuged, washed with methanol, chloroform, and dried under low temperature nitrogen to obtain a lipid extract.
  • the lipid extracts of the fungi and bacteria obtained above were separately subjected to liquid chromatography analysis, as follows: chromatography column: diameter 1.5 cm, length 30 cm; filler silica gel (200-300 mesh, particle size 45-75 ⁇ m, pore size 40-70A) , specific surface area of 400-600 m 2 /g, pore volume of 0.60-0.85 ml / g), atmospheric pressure filling.
  • chromatography column diameter 1.5 cm, length 30 cm
  • filler silica gel 200-300 mesh, particle size 45-75 ⁇ m, pore size 40-70A
  • specific surface area 400-600 m 2 /g
  • atmospheric pressure filling The lipid extracted from 5 g (wet weight) fungus or bacteria was loaded with 100% chloroform (30 ml), and then the volume ratio of chloroform:methanol was 10:0, 8:2, 6:4, 5:5, 3:7.
  • a segmented component is obtained.
  • Each segment obtained by liquid phase analysis is dissolved in the same volume of DMSO as the unsegmented total lipid, and Transwell experiment is carried out (lipid is added to the lower chamber, lipid The ratio of the culture medium to the 1:1000 assay for the activity of each segment to attract the migration of mouse bone marrow-derived dendritic cells (BMDC) (the skilled person knows that the chemotaxis of BMDC is similar to that of CD103 + CD11b + RALDH + dendritic cells, The reason for using BMDC instead of CD103 + CD11b + RALDH + dendritic cells is that it is difficult to obtain sufficient cell numbers for large-scale analysis. Furthermore, the latter has poor activity in culture and is not suitable for long-term use. experiment).
  • BMDC mouse bone marrow-derived dendritic cells
  • A Liquid chromatography spectrum of various fungal lipids
  • B Several passages in tropical yeast can induce the migration of dendritic cells in vitro, with the ninth segment being the strongest. It can be seen that the relevant activity in the lipids in the fungus can be concentrated and purified by the liquid phase.
  • Candida albicans such as Candida tropicalis and its lipid extracts can drive intestinal-derived dendritic cells (expressing CD103, CD11b, and retinal dehydrogenase) to lymph nodes. Therefore, in practical applications, it is expected to regulate the number and metabolism of Candida albicans such as Candida tropicalis. And the method of regulating the strength and type of the body's immune response and the number and function of each immune cell subtype by means of oral intake and injection of lipid extracts of such fungi, thereby achieving treatment and/or prevention of immune disorder-related diseases.
  • immune disorder-related diseases such as rheumatism, lupus erythematosus, enteritis, multiple sclerosis, and the purpose of enhancing immune function (such as immunotherapy and vaccination).

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Abstract

L'invention concerne une utilisation de Candida intestinal ou d'un extrait lipidique de celui-ci dans la préparation d'un produit pour prévenir et traiter une maladie du système immunitaire. L'invention concerne également un produit contenant Candid intestinal ou un extrait lipidique de celui-ci en tant que principe actif.
PCT/CN2017/113539 2017-08-31 2017-11-29 Utilisation de candida dans la préparation d'un produit pour la prévention et le traitement de maladies du système immunitaire WO2019041597A1 (fr)

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CN201710774122.XA CN109419820B (zh) 2017-08-31 2017-08-31 热带假丝酵母菌在制备防治免疫系统疾病产品中的应用
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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106036080A (zh) * 2016-06-08 2016-10-26 中国农业科学院饲料研究所 一种含热带假丝酵母菌的甲烷调控剂及其应用

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106036080A (zh) * 2016-06-08 2016-10-26 中国农业科学院饲料研究所 一种含热带假丝酵母菌的甲烷调控剂及其应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
"Candida Tropicalis Makes a Natural Flavoring-Yeast Extract", FOOD RESEARCH AND DEVELOPMENT, vol. 20, no. 4, 31 December 1998 (1998-12-31), pages 13 - 15, ISSN: 1005-6521 *
ZONGDE ZHANG ET AL.: "Peripheral Lymphoid Volume Expansion and Maintenance Are Controlled by Gut Microbiota via RALDH+ Dendritic Cells", IMMUNITY, vol. 44, 16 February 2016 (2016-02-16), pages 330 - 342, XP029428472, ISSN: 1074-7613, DOI: doi:10.1016/j.immuni.2016.01.004 *

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