US20190338374A1 - Klebsiella pneumoniae strain that induces inflammation in the liver - Google Patents
Klebsiella pneumoniae strain that induces inflammation in the liver Download PDFInfo
- Publication number
- US20190338374A1 US20190338374A1 US16/389,454 US201916389454A US2019338374A1 US 20190338374 A1 US20190338374 A1 US 20190338374A1 US 201916389454 A US201916389454 A US 201916389454A US 2019338374 A1 US2019338374 A1 US 2019338374A1
- Authority
- US
- United States
- Prior art keywords
- klebsiella pneumoniae
- strain
- sclerosing cholangitis
- primary sclerosing
- pneumoniae strain
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
- 241000588747 Klebsiella pneumoniae Species 0.000 title claims abstract description 92
- 210000004185 liver Anatomy 0.000 title claims abstract description 46
- 206010061218 Inflammation Diseases 0.000 title claims abstract description 8
- 230000004054 inflammatory process Effects 0.000 title claims abstract description 8
- 208000010157 sclerosing cholangitis Diseases 0.000 claims abstract description 57
- 206010008609 Cholangitis sclerosing Diseases 0.000 claims abstract description 56
- 201000000742 primary sclerosing cholangitis Diseases 0.000 claims abstract description 54
- 206010009900 Colitis ulcerative Diseases 0.000 claims abstract description 41
- 201000006704 Ulcerative Colitis Diseases 0.000 claims abstract description 41
- 238000011161 development Methods 0.000 claims abstract description 16
- 230000001939 inductive effect Effects 0.000 claims abstract description 9
- 241000194030 Enterococcus gallinarum Species 0.000 claims description 37
- 241000588770 Proteus mirabilis Species 0.000 claims description 37
- 239000011148 porous material Substances 0.000 claims description 29
- 238000003745 diagnosis Methods 0.000 claims description 22
- 210000004347 intestinal mucosa Anatomy 0.000 claims description 21
- 210000000068 Th17 cell Anatomy 0.000 claims description 20
- 230000001580 bacterial effect Effects 0.000 claims description 18
- MUSRXJZXWIHVSH-UHFFFAOYSA-N 2,4,6-trimethyl-1,4-dihydropyridine Chemical compound CC1C=C(C)NC(C)=C1 MUSRXJZXWIHVSH-UHFFFAOYSA-N 0.000 claims description 11
- 238000010172 mouse model Methods 0.000 claims description 11
- 108010046179 Type VI Secretion Systems Proteins 0.000 claims description 10
- 238000004519 manufacturing process Methods 0.000 claims description 8
- 238000000034 method Methods 0.000 claims description 8
- 239000002773 nucleotide Substances 0.000 claims description 8
- 125000003729 nucleotide group Chemical group 0.000 claims description 8
- 244000005700 microbiome Species 0.000 abstract description 2
- 241000699670 Mus sp. Species 0.000 description 55
- 230000002550 fecal effect Effects 0.000 description 39
- 239000000523 sample Substances 0.000 description 33
- 241000699666 Mus <mouse, genus> Species 0.000 description 26
- 239000000203 mixture Substances 0.000 description 26
- 230000000968 intestinal effect Effects 0.000 description 17
- 210000004027 cell Anatomy 0.000 description 16
- 108020004414 DNA Proteins 0.000 description 14
- 210000002966 serum Anatomy 0.000 description 13
- 241000894006 Bacteria Species 0.000 description 12
- 238000004458 analytical method Methods 0.000 description 11
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 11
- 239000002953 phosphate buffered saline Substances 0.000 description 11
- 239000000243 solution Substances 0.000 description 11
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 10
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 10
- 238000000684 flow cytometry Methods 0.000 description 10
- 208000003167 cholangitis Diseases 0.000 description 9
- 238000008050 Total Bilirubin Reagent Methods 0.000 description 8
- 210000001072 colon Anatomy 0.000 description 8
- 239000002609 medium Substances 0.000 description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 7
- 238000011156 evaluation Methods 0.000 description 7
- 208000006454 hepatitis Diseases 0.000 description 7
- 230000006698 induction Effects 0.000 description 7
- 210000002429 large intestine Anatomy 0.000 description 7
- 210000001165 lymph node Anatomy 0.000 description 7
- 238000005259 measurement Methods 0.000 description 7
- 210000000013 bile duct Anatomy 0.000 description 6
- 238000003501 co-culture Methods 0.000 description 6
- 206010009887 colitis Diseases 0.000 description 6
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 6
- 239000002158 endotoxin Substances 0.000 description 6
- 210000002220 organoid Anatomy 0.000 description 6
- 239000000725 suspension Substances 0.000 description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 5
- 229940125426 RORyt inverse agonist Drugs 0.000 description 5
- 201000010099 disease Diseases 0.000 description 5
- 230000014509 gene expression Effects 0.000 description 5
- 239000012188 paraffin wax Substances 0.000 description 5
- DHRLEVQXOMLTIM-UHFFFAOYSA-N phosphoric acid;trioxomolybdenum Chemical compound O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.OP(O)(O)=O DHRLEVQXOMLTIM-UHFFFAOYSA-N 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- 206010070545 Bacterial translocation Diseases 0.000 description 4
- 208000004232 Enteritis Diseases 0.000 description 4
- 208000017006 IgG4-related sclerosing cholangitis Diseases 0.000 description 4
- KPKZJLCSROULON-QKGLWVMZSA-N Phalloidin Chemical compound N1C(=O)[C@@H]([C@@H](O)C)NC(=O)[C@H](C)NC(=O)[C@H](C[C@@](C)(O)CO)NC(=O)[C@H](C2)NC(=O)[C@H](C)NC(=O)[C@@H]3C[C@H](O)CN3C(=O)[C@@H]1CSC1=C2C2=CC=CC=C2N1 KPKZJLCSROULON-QKGLWVMZSA-N 0.000 description 4
- 208000016977 Secondary sclerosing cholangitis Diseases 0.000 description 4
- 102000054727 Serum Amyloid A Human genes 0.000 description 4
- 108700028909 Serum Amyloid A Proteins 0.000 description 4
- 230000007375 bacterial translocation Effects 0.000 description 4
- 231100000283 hepatitis Toxicity 0.000 description 4
- 230000001744 histochemical effect Effects 0.000 description 4
- 229920006008 lipopolysaccharide Polymers 0.000 description 4
- 210000005087 mononuclear cell Anatomy 0.000 description 4
- 210000000056 organ Anatomy 0.000 description 4
- 239000002356 single layer Substances 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- 210000000952 spleen Anatomy 0.000 description 4
- 238000010186 staining Methods 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 210000001519 tissue Anatomy 0.000 description 4
- 238000002054 transplantation Methods 0.000 description 4
- 108020004465 16S ribosomal RNA Proteins 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 208000031481 Pathologic Constriction Diseases 0.000 description 3
- 210000000447 Th1 cell Anatomy 0.000 description 3
- 230000006907 apoptotic process Effects 0.000 description 3
- 238000001574 biopsy Methods 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 230000001684 chronic effect Effects 0.000 description 3
- 238000010835 comparative analysis Methods 0.000 description 3
- 235000005911 diet Nutrition 0.000 description 3
- 230000037213 diet Effects 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000002073 fluorescence micrograph Methods 0.000 description 3
- 210000002490 intestinal epithelial cell Anatomy 0.000 description 3
- 208000019423 liver disease Diseases 0.000 description 3
- 210000004400 mucous membrane Anatomy 0.000 description 3
- 230000000306 recurrent effect Effects 0.000 description 3
- 208000024891 symptom Diseases 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 238000012070 whole genome sequencing analysis Methods 0.000 description 3
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 2
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 2
- 108091093088 Amplicon Proteins 0.000 description 2
- 208000008439 Biliary Liver Cirrhosis Diseases 0.000 description 2
- 238000011740 C57BL/6 mouse Methods 0.000 description 2
- 102000003952 Caspase 3 Human genes 0.000 description 2
- 108090000397 Caspase 3 Proteins 0.000 description 2
- 101100447432 Danio rerio gapdh-2 gene Proteins 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 2
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 description 2
- 101150112014 Gapdh gene Proteins 0.000 description 2
- 208000032843 Hemorrhage Diseases 0.000 description 2
- 101000825954 Homo sapiens R-spondin-1 Proteins 0.000 description 2
- 239000012480 LAL reagent Substances 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 241000566145 Otus Species 0.000 description 2
- 108010009711 Phalloidine Proteins 0.000 description 2
- 208000012654 Primary biliary cholangitis Diseases 0.000 description 2
- 206010037143 Pseudopolyposis Diseases 0.000 description 2
- 102100022762 R-spondin-1 Human genes 0.000 description 2
- 208000025865 Ulcer Diseases 0.000 description 2
- 230000002159 abnormal effect Effects 0.000 description 2
- 239000012574 advanced DMEM Substances 0.000 description 2
- 210000003445 biliary tract Anatomy 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 238000013189 cholangiography Methods 0.000 description 2
- 230000000112 colonic effect Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 210000000981 epithelium Anatomy 0.000 description 2
- 230000003628 erosive effect Effects 0.000 description 2
- 210000002603 extrahepatic bile duct Anatomy 0.000 description 2
- 210000003608 fece Anatomy 0.000 description 2
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 2
- 239000007850 fluorescent dye Substances 0.000 description 2
- 238000012268 genome sequencing Methods 0.000 description 2
- 210000003405 ileum Anatomy 0.000 description 2
- 238000001802 infusion Methods 0.000 description 2
- 210000005027 intestinal barrier Anatomy 0.000 description 2
- 230000007358 intestinal barrier function Effects 0.000 description 2
- 210000003228 intrahepatic bile duct Anatomy 0.000 description 2
- 230000009545 invasion Effects 0.000 description 2
- 239000010410 layer Substances 0.000 description 2
- 230000003902 lesion Effects 0.000 description 2
- 210000005228 liver tissue Anatomy 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 238000001000 micrograph Methods 0.000 description 2
- 102000045246 noggin Human genes 0.000 description 2
- 108700007229 noggin Proteins 0.000 description 2
- 238000010899 nucleation Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 239000003642 reactive oxygen metabolite Substances 0.000 description 2
- 210000000664 rectum Anatomy 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 238000013424 sirius red staining Methods 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 231100000397 ulcer Toxicity 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- NHBKXEKEPDILRR-UHFFFAOYSA-N 2,3-bis(butanoylsulfanyl)propyl butanoate Chemical compound CCCC(=O)OCC(SC(=O)CCC)CSC(=O)CCC NHBKXEKEPDILRR-UHFFFAOYSA-N 0.000 description 1
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 1
- 208000030507 AIDS Diseases 0.000 description 1
- 102000007469 Actins Human genes 0.000 description 1
- 108010085238 Actins Proteins 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- HJCMDXDYPOUFDY-WHFBIAKZSA-N Ala-Gln Chemical compound C[C@H](N)C(=O)N[C@H](C(O)=O)CCC(N)=O HJCMDXDYPOUFDY-WHFBIAKZSA-N 0.000 description 1
- IGAZHQIYONOHQN-UHFFFAOYSA-N Alexa Fluor 555 Chemical compound C=12C=CC(=N)C(S(O)(=O)=O)=C2OC2=C(S(O)(=O)=O)C(N)=CC=C2C=1C1=CC=C(C(O)=O)C=C1C(O)=O IGAZHQIYONOHQN-UHFFFAOYSA-N 0.000 description 1
- 241000234282 Allium Species 0.000 description 1
- 235000002732 Allium cepa var. cepa Nutrition 0.000 description 1
- 206010001985 Amoebic colitis Diseases 0.000 description 1
- 206010051268 Anastomotic stenosis Diseases 0.000 description 1
- 206010003694 Atrophy Diseases 0.000 description 1
- 206010003827 Autoimmune hepatitis Diseases 0.000 description 1
- 208000031729 Bacteremia Diseases 0.000 description 1
- 208000017283 Bile Duct disease Diseases 0.000 description 1
- 206010056375 Bile duct obstruction Diseases 0.000 description 1
- 208000033222 Biliary cirrhosis primary Diseases 0.000 description 1
- 241000589876 Campylobacter Species 0.000 description 1
- 208000029655 Caroli Disease Diseases 0.000 description 1
- 208000008964 Chemical and Drug Induced Liver Injury Diseases 0.000 description 1
- 241000606161 Chlamydia Species 0.000 description 1
- 241000725101 Clea Species 0.000 description 1
- 241000193163 Clostridioides difficile Species 0.000 description 1
- 206010009895 Colitis ischaemic Diseases 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 108020004635 Complementary DNA Proteins 0.000 description 1
- 208000027205 Congenital disease Diseases 0.000 description 1
- 208000011231 Crohn disease Diseases 0.000 description 1
- 201000003883 Cystic fibrosis Diseases 0.000 description 1
- 206010011831 Cytomegalovirus infection Diseases 0.000 description 1
- 206010013003 Dilatation intrahepatic duct congenital Diseases 0.000 description 1
- 206010072268 Drug-induced liver injury Diseases 0.000 description 1
- 208000027244 Dysbiosis Diseases 0.000 description 1
- 241000792859 Enema Species 0.000 description 1
- 206010014950 Eosinophilia Diseases 0.000 description 1
- 102000009024 Epidermal Growth Factor Human genes 0.000 description 1
- 241001646719 Escherichia coli O157:H7 Species 0.000 description 1
- 206010017914 Gastroenteritis salmonella Diseases 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- 239000007995 HEPES buffer Substances 0.000 description 1
- 206010019635 Hepatic artery embolism Diseases 0.000 description 1
- 206010019728 Hepatitis alcoholic Diseases 0.000 description 1
- 206010019799 Hepatitis viral Diseases 0.000 description 1
- 208000017604 Hodgkin disease Diseases 0.000 description 1
- 208000010747 Hodgkins lymphoma Diseases 0.000 description 1
- 101001046686 Homo sapiens Integrin alpha-M Proteins 0.000 description 1
- 101000917858 Homo sapiens Low affinity immunoglobulin gamma Fc region receptor III-A Proteins 0.000 description 1
- 101000917839 Homo sapiens Low affinity immunoglobulin gamma Fc region receptor III-B Proteins 0.000 description 1
- 101150012417 IL1B gene Proteins 0.000 description 1
- 208000021330 IgG4-related disease Diseases 0.000 description 1
- 208000037142 IgG4-related systemic disease Diseases 0.000 description 1
- 208000004187 Immunoglobulin G4-Related Disease Diseases 0.000 description 1
- 208000022559 Inflammatory bowel disease Diseases 0.000 description 1
- 102100022338 Integrin alpha-M Human genes 0.000 description 1
- PWKSKIMOESPYIA-BYPYZUCNSA-N L-N-acetyl-Cysteine Chemical compound CC(=O)N[C@@H](CS)C(O)=O PWKSKIMOESPYIA-BYPYZUCNSA-N 0.000 description 1
- 102100029185 Low affinity immunoglobulin gamma Fc region receptor III-B Human genes 0.000 description 1
- 108010053229 Lysyl endopeptidase Proteins 0.000 description 1
- 239000006154 MacConkey agar Substances 0.000 description 1
- 241000736262 Microbiota Species 0.000 description 1
- 208000000996 Mirizzi syndrome Diseases 0.000 description 1
- 206010028140 Mucous stools Diseases 0.000 description 1
- 102000016943 Muramidase Human genes 0.000 description 1
- 108010014251 Muramidase Proteins 0.000 description 1
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 1
- 108091007491 NSP3 Papain-like protease domains Proteins 0.000 description 1
- 239000004677 Nylon Substances 0.000 description 1
- 239000008118 PEG 6000 Substances 0.000 description 1
- 108020002230 Pancreatic Ribonuclease Proteins 0.000 description 1
- 102000005891 Pancreatic ribonuclease Human genes 0.000 description 1
- 208000030852 Parasitic disease Diseases 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 208000005374 Poisoning Diseases 0.000 description 1
- 229920002584 Polyethylene Glycol 6000 Polymers 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 101710098940 Pro-epidermal growth factor Proteins 0.000 description 1
- 238000011530 RNeasy Mini Kit Methods 0.000 description 1
- 108091008778 RORγ2 Proteins 0.000 description 1
- 206010054828 Rectal lesion Diseases 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- 208000004732 Systemic Vasculitis Diseases 0.000 description 1
- 201000008736 Systemic mastocytosis Diseases 0.000 description 1
- 239000007984 Tris EDTA buffer Substances 0.000 description 1
- 208000024248 Vascular System injury Diseases 0.000 description 1
- 208000012339 Vascular injury Diseases 0.000 description 1
- 206010047115 Vasculitis Diseases 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 229960004308 acetylcysteine Drugs 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 208000002353 alcoholic hepatitis Diseases 0.000 description 1
- 206010002022 amyloidosis Diseases 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 229940041181 antineoplastic drug Drugs 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 201000010313 ascending cholangitis Diseases 0.000 description 1
- 238000003149 assay kit Methods 0.000 description 1
- 230000037444 atrophy Effects 0.000 description 1
- 238000011888 autopsy Methods 0.000 description 1
- 239000007621 bhi medium Substances 0.000 description 1
- 230000000740 bleeding effect Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 208000027503 bloody stool Diseases 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- KQNZDYYTLMIZCT-KQPMLPITSA-N brefeldin A Chemical compound O[C@@H]1\C=C\C(=O)O[C@@H](C)CCC\C=C\[C@@H]2C[C@H](O)C[C@H]21 KQNZDYYTLMIZCT-KQPMLPITSA-N 0.000 description 1
- JUMGSHROWPPKFX-UHFFFAOYSA-N brefeldin-A Natural products CC1CCCC=CC2(C)CC(O)CC2(C)C(O)C=CC(=O)O1 JUMGSHROWPPKFX-UHFFFAOYSA-N 0.000 description 1
- 238000010804 cDNA synthesis Methods 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 230000005754 cellular signaling Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 208000006990 cholangiocarcinoma Diseases 0.000 description 1
- 230000001587 cholestatic effect Effects 0.000 description 1
- 208000019425 cirrhosis of liver Diseases 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 238000010219 correlation analysis Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 238000011257 definitive treatment Methods 0.000 description 1
- 230000008034 disappearance Effects 0.000 description 1
- 238000011978 dissolution method Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 201000008865 drug-induced hepatitis Diseases 0.000 description 1
- 230000007140 dysbiosis Effects 0.000 description 1
- 239000007920 enema Substances 0.000 description 1
- 229940095399 enema Drugs 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 230000002327 eosinophilic effect Effects 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 230000003325 follicular Effects 0.000 description 1
- 108010043649 gastrin I Proteins 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 210000004907 gland Anatomy 0.000 description 1
- 102000006602 glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 1
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 1
- 210000002175 goblet cell Anatomy 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 208000035861 hematochezia Diseases 0.000 description 1
- 230000002440 hepatic effect Effects 0.000 description 1
- 230000002962 histologic effect Effects 0.000 description 1
- 230000003118 histopathologic effect Effects 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 206010020718 hyperplasia Diseases 0.000 description 1
- 230000009390 immune abnormality Effects 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 230000008595 infiltration Effects 0.000 description 1
- 238000001764 infiltration Methods 0.000 description 1
- 210000004969 inflammatory cell Anatomy 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 208000028774 intestinal disease Diseases 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 238000010212 intracellular staining Methods 0.000 description 1
- 201000008222 ischemic colitis Diseases 0.000 description 1
- 208000023589 ischemic disease Diseases 0.000 description 1
- 238000012317 liver biopsy Methods 0.000 description 1
- 208000018191 liver inflammation Diseases 0.000 description 1
- 239000004325 lysozyme Substances 0.000 description 1
- 229960000274 lysozyme Drugs 0.000 description 1
- 235000010335 lysozyme Nutrition 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 108010082117 matrigel Proteins 0.000 description 1
- 210000004379 membrane Anatomy 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- WSFSSNUMVMOOMR-NJFSPNSNSA-N methanone Chemical compound O=[14CH2] WSFSSNUMVMOOMR-NJFSPNSNSA-N 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 239000012120 mounting media Substances 0.000 description 1
- 208000008338 non-alcoholic fatty liver disease Diseases 0.000 description 1
- 206010053219 non-alcoholic steatohepatitis Diseases 0.000 description 1
- 229920001778 nylon Polymers 0.000 description 1
- 230000000414 obstructive effect Effects 0.000 description 1
- 238000003305 oral gavage Methods 0.000 description 1
- 229910000489 osmium tetroxide Inorganic materials 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000007918 pathogenicity Effects 0.000 description 1
- 230000007310 pathophysiology Effects 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 230000010412 perfusion Effects 0.000 description 1
- 230000002085 persistent effect Effects 0.000 description 1
- 231100000572 poisoning Toxicity 0.000 description 1
- 230000000607 poisoning effect Effects 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 210000003240 portal vein Anatomy 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 229940126409 proton pump inhibitor Drugs 0.000 description 1
- 239000000612 proton pump inhibitor Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 230000007115 recruitment Effects 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 201000000306 sarcoidosis Diseases 0.000 description 1
- 238000001878 scanning electron micrograph Methods 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000000405 serological effect Effects 0.000 description 1
- 238000004904 shortening Methods 0.000 description 1
- 206010040882 skin lesion Diseases 0.000 description 1
- 231100000444 skin lesion Toxicity 0.000 description 1
- 238000004544 sputter deposition Methods 0.000 description 1
- 229910001220 stainless steel Inorganic materials 0.000 description 1
- 239000010935 stainless steel Substances 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 201000008827 tuberculosis Diseases 0.000 description 1
- 230000002792 vascular Effects 0.000 description 1
- 201000001862 viral hepatitis Diseases 0.000 description 1
- 239000011534 wash buffer Substances 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Images
Classifications
-
- C12R1/22—
-
- 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
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
- A01K67/027—New breeds of vertebrates
-
- 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
- A61K39/02—Bacterial antigens
- A61K39/025—Enterobacteriales, e.g. Enterobacter
-
- 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
- A61K39/02—Bacterial antigens
- A61K39/025—Enterobacteriales, e.g. Enterobacter
- A61K39/0266—Klebsiella
-
- 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
- A61K39/02—Bacterial antigens
- A61K39/09—Lactobacillales, e.g. aerococcus, enterococcus, lactobacillus, lactococcus, streptococcus
-
- 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
- A61K39/02—Bacterial antigens
- A61K39/116—Polyvalent bacterial antigens
-
- 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
- C12N1/20—Bacteria; Culture media therefor
-
- C12R1/37—
-
- C12R1/46—
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; 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; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2207/00—Modified animals
- A01K2207/20—Animals treated with compounds which are neither proteins nor nucleic acids
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2207/00—Modified animals
- A01K2207/25—Animals on a special diet
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; 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; CARE OF BIRDS, FISHES, INSECTS; 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
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; 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
- A01K2267/0387—Animal model for diseases of the immune system
-
- 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
-
- 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
- C12R2001/37—Proteus
-
- 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
- C12R2001/46—Streptococcus ; Enterococcus; Lactococcus
Definitions
- the present invention relates to a Klebsiella pneumoniae strain inducing inflammation in the liver.
- Non-Patent Literatures 1 to 3 Considering the difference in clinical symptoms of ulcerative colitis complicated with primary sclerosing cholangitis and typical ulcerative colitis not complicated with primary sclerosing cholangitis, it is suggested that abnormality in the intestinal bacterial flora (dysbiosis) may play an important role in the development of primary sclerosing cholangitis associated with ulcerative colitis.
- FIG. 4 a includes graphs showing the results of counting the cell number of the Klebsiella pneumoniae (KP), Proteus mirabilis (PM), and Enterococcus gallinarum (EG) contained in fecal samples of Hc, Psc/Uc, and Uc.
- FIG. 4 b includes graphs showing correlations between the proportion of Th17 cells or Th1 cells in the liver of PSCUC and the number of Klebsiella pneumoniae cells in the fecal samples thereof;
- An aspect of the present invention relates to a Klebsiella pneumoniae strain inducing inflammation in the liver.
- the inflammation of livers includes not only inflammation of livers but also inflammation of intra- and extra-hepatic bile ducts, and examples thereof include hepatitis and cholangitis.
- examples of hepatitis include viral hepatitis, drug-induced hepatitis, alcoholic hepatitis, non-alcoholic steatohepatitis, and autoimmune hepatitis.
- the Klebsiella pneumoniae strain of the present invention preferably has a DNA consisting of the nucleotide sequence registered in the National Center for Biotechnology Information (NCBI) under Assembly Name: ASM385182v1, and the Klebsiella pneumoniae strain is more preferably one whose deposit number is NITE BP-02879.
- NCBI National Center for Biotechnology Information
- ulcerative colitis can be clearly diagnosed when findings grossly and histologically characteristic to ulcerative colitis are observed by excision surgery or autopsy.
- Another aspect of the present invention relates to use of the Klebsiella pneumoniae strain only or use of the Klebsiella pneumoniae strain, a Proteus mirabilis strain, and an Enterococcus gallinarum strain for predicting the development of primary sclerosing cholangitis.
- Glyceraldehyde-3-phosphate dehydrogenase Mm03302249_g1
- Livers were collected from the DDC-treated humanized gnotobiotic mice and were fixed in 10% formalin and embedded in paraffin according to a usual method to produce paraffin blocks. Sections were cut from the paraffin blocks and were stained with hematoxylin-eosin and Masson-trichrome or stained with Sirius red to prepare specimen samples. The results of microscopic observation of the specimen samples are shown in FIG. 3 b . The upper part of FIG. 3 b shows the results of Sirius red staining, and the lower part of FIG. 3 b shows the results of co-staining with hematoxylin-eosin and Masson-trichrome. In the liver of the humanized gnotobiotic mice administered the fecal sample derived from Psc/Uc, hepatic disorder due to fiber formation in the liver was observed.
- Livers were collected from the DCC-treated mice and were perfused with PBS from the portal vein. After the perfusion, the livers were chopped and passed through a 100- ⁇ m nylon mesh to obtain cells of the livers.
- the intracellular stained cells were treated with Ionomycine (500 ng/mL) or Golgistop (10 ⁇ g/mL, manufactured by BD Biosciences) in the presence of lipopolysaccharide (derived from Escherichia coli B5, manufactured by Sigma-Aldrich Co. LLC) or PMA (50 ng/mL, manufactured by Sigma-Aldrich Co. LLC) and brefeldin A (10 ⁇ g/mL, manufactured by BD Biosciences).
- Ionomycine 500 ng/mL
- Golgistop 10 ⁇ g/mL, manufactured by BD Biosciences
- lipopolysaccharide derived from Escherichia coli B5, manufactured by Sigma-Aldrich Co. LLC
- PMA 50 ng/mL, manufactured by Sigma-Aldrich Co. LLC
- brefeldin A 10 ⁇ g/mL, manufactured by BD Biosciences.
- Th17 cell induction was recognized in the KP administration groups, in particular, in the 3-mix administration group, compared to those in GF mice (GF) and SPF mice (SPF) not administered the strains.
- Healthy human colon organoids were embedded in Matrigel (Corning) and were three-dimensionally cultured in Advanced DMEM/F12 culture solution (containing penicillin/streptomycin, 10 mM HEPES, 2 mM GlutaMAX, 1 ⁇ B27 (manufactured by Life Technologies), 1 mM N-acetylcysteine (manufactured by FUJIFILM Wako Pure Chemical Corporation), 10 nM Gastrin I (manufactured by Sigma-Aldrich Co.
- Advanced DMEM/F12 culture solution containing penicillin/streptomycin, 10 mM HEPES, 2 mM GlutaMAX, 1 ⁇ B27 (manufactured by Life Technologies), 1 mM N-acetylcysteine (manufactured by FUJIFILM Wako Pure Chemical Corporation), 10 nM Gastrin I (manufactured by Sigma-Aldrich Co.
- CM Afamin-Wnt3a
- R-spondin 1 R-CM (10% v/v)
- N Noggin
Abstract
To identify a microorganism causing the development of primary sclerosing cholangitis associated with ulcerative colitis. A Klebsiella pneumoniae strain inducing inflammation in the liver.
Description
- The present invention relates to a Klebsiella pneumoniae strain inducing inflammation in the liver.
- Primary sclerosing cholangitis (PSC) is an idiopathic chronic cholestatic chronic liver disease and causes end-stage hepatic cirrhosis through the progression of biliary stricture and collapse of the biliary tree. To date, no definitive treatment other than liver transplantation for this symptom has been established, and a further understanding of the pathophysiology is needed for treating this incurable disease.
- Since portal bacteremia and high-level endotoxin in cholangiocytes have been observed in patients with primary sclerosing cholangitis, it is considered that bacterial translocation, in which intestinal bacterial flora passes through the damaged intestinal barrier, plays an important role in liver inflammation. However, regardless of the presence of many evidences of bacterial translocation in patients with primary sclerosing cholangitis, the mechanisms that trigger collapse of intestinal barrier and bacterial translocation are still unknown.
- It is known that more than half of the patients with primary sclerosing cholangitis suffer from ulcerative colitis, and it is suggested that the bacterial translocation induced by chronic colitis has a risk of causing hepatitis. In contrast, only a very small number of patients with ulcerative colitis have primary sclerosing cholangitis. Furthermore, the ulcerative colitis patients having primary sclerosing cholangitis exhibit discontinuous segmental colitis (right sided colitis). This is remarkably contrast to continuous rectal lesions in typical ulcerative colitis not complicated with primary sclerosing cholangitis. Accordingly, only having colitis as the underlying disease is insufficient for the onset of primary sclerosing cholangitis complicated with ulcerative colitis, and it is considered that other disease factors are involved in the onset.
- In recent years, microbial analysis has reported that the intestinal bacterial flora of ulcerative colitis patients having primary sclerosing cholangitis is different from those of healthy subjects and typical ulcerative colitis patients not having primary sclerosing cholangitis (Non-Patent
Literatures 1 to 3). Considering the difference in clinical symptoms of ulcerative colitis complicated with primary sclerosing cholangitis and typical ulcerative colitis not complicated with primary sclerosing cholangitis, it is suggested that abnormality in the intestinal bacterial flora (dysbiosis) may play an important role in the development of primary sclerosing cholangitis associated with ulcerative colitis. -
- [Non-Patent Literature 1]
- Sabino, J., et al., Primary sclerosing cholangitis is characterised by intestinal dysbiosis independent from IBD, Gut 65, 1681-1689, (2016)
- [Non-Patent Literature 2]
- Kummen, M., et al., The gut microbial profile in patients with primary sclerosing cholangitis is distinct from patients with ulcerative colitis without biliary disease and healthy controls, Gut 66, 611-619, (2017)
- [Non-Patent Literature 3]
- Iwasawa, K., et al., Characterisation of the faecal microbiota in Japanese patients with paediatric-onset primary sclerosing cholangitis, Gut 66, 1344-1346, (2017)
- However, there is not yet clear evidence showing that dysbiosis causes the development of primary sclerosing cholangitis associated with ulcerative colitis, and it is also unclear whether a certain microbial species contributes to the development of primary sclerosing cholangitis associated with ulcerative colitis. Accordingly, there is a strong demand for identification of a microorganism that causes the development of primary sclerosing cholangitis associated with ulcerative colitis.
- The present inventors have intensively studied in view of the above problems and, as a result, have identified a Klebsiella pneumoniae strain, a Proteus mirabilis strain, and an Enterococcus gallinarum strain that cause the development of primary sclerosing cholangitis associated with ulcerative colitis, and the present invention has been accomplished. That is, the present invention, for example, relates to the following [1] to [14]:
- [1] A Klebsiella pneumoniae strain inducing inflammation in the liver;
- [2] The Klebsiella pneumoniae strain according to [1], inducing a Th17 cell in the liver;
- [3] The Klebsiella pneumoniae strain according to [1] or [2], having an ability to form a pore on the large intestinal epithelium;
- [4] The Klebsiella pneumoniae strain according to any one of [1] to [3], having a
type 6 secretion system; - [5] The Klebsiella pneumoniae strain according to any one of [1] to [4], derived from a patient suffering from both primary sclerosing cholangitis and ulcerative colitis;
- [6] The Klebsiella pneumoniae strain according to any one of [1] to [5], comprising a DNA consisting of the nucleotide sequence registered in the National Center for Biotechnology Information (NCBI) under Assembly Name: ASM385182v1;
- [7] The Klebsiella pneumoniae strain according to any one of [1] to [6], whose deposit number is NITE BP-02879;
- [8] The Klebsiella pneumoniae strain according to any one of [1] to [5], comprising a DNA consisting of the nucleotide sequence registered in the National Center for Biotechnology Information (NCBI) under Assembly Name: ASM386511v1;
- [9] A Proteus mirabilis strain whose accession number is NITE ABP-02923;
- [10] An Enterococcus gallinarum strain whose accession number is NITE ABP-02922;
- [11] Use of the Klebsiella pneumoniae strain according to any one of [1] to [8] for prediction or diagnosis of the development of primary sclerosing cholangitis;
- [12] Use of the Klebsiella pneumoniae strain according to any one of [1] to [8], a Proteus mirabilis strain, and an Enterococcus gallinarum strain for prediction or diagnosis of the development of primary sclerosing cholangitis;
- [13] The use for prediction or diagnosis of the development of primary sclerosing cholangitis according to [12], wherein the Proteus mirabilis strain is one whose accession number is NITE ABP-02923; and the Enterococcus gallinarum strain is one whose accession number is NITE ABP-02922; and
- [14] A method for producing a mouse model suffering from both primary sclerosing cholangitis and ulcerative colitis, the method comprising:
- administering a bacterial solution containing the Klebsiella pneumoniae strain according to any one of [1] to [8] to a mouse; and
- administering 3,5-dicarbetoxy-1,4-dihydrocollidine to a mouse.
- According to the present invention, a Klebsiella pneumoniae strain, a Proteus mirabilis strain, and an Enterococcus gallinarum strain can be used for prediction of the development of primary sclerosing cholangitis. In addition, a mouse model suffering from both primary sclerosing cholangitis and ulcerative colitis can be used for establishing a method for treating primary sclerosing cholangitis.
-
FIG. 1a is a graph showing the results of unweighted UniFrac analysis of bacterial flora present in fecal samples of healthy subjects (Hc 1 to 4), patients suffering from both primary sclerosing cholangitis and ulcerative colitis (Psc/Uc 1 to 5), and patients suffering from ulcerative colitis only (Uc 1 to 4) and in fecal samples of humanized gnotobiotic mice administered the fecal samples of these patients. FIG. 1 b is a graph showing the results of principal coordinates analysis based on the UniFrac analysis of the human fecal samples and the humanized gnotobiotic mouse fecal samples; -
FIGS. 2a to 2c are graphs showing the results of measurement of gene expression levels of serum amyloid protein A (Saa 1 to 3) and IL-1β (Il1b) in the livers (2A), colons (2B), and spleens (2C) of GF mice (GF), humanized gnotobiotic mice administered a fecal sample of a healthy subject (HC), and humanized gnotobiotic mice administered a fecal sample of a patient suffering from both primary sclerosing cholangitis and ulcerative colitis (PSCUC). * : P<0.05, **: P<0.01, ***: P<0.001, ****: P<0.0001 (The same applies to the following figures); -
FIG. 3a includes graphs showing the results of measurement of serum alkaline phosphatase (ALP, left) and serum total bilirubin (TB, right) of GF, HC, and PSCUC.FIG. 3b includes photomicrographs of samples of specimens prepared by Sirius red staining (above) and hematoxylin-eosin staining and Masson-trichrome staining (below) of liver tissue sections of GF, HC, and PSCUC. The scale bars indicate 100 μm.FIG. 3c includes graphs showing the results of flow cytometric analysis of the cells in the livers of GF, HC, and PSCUC; -
FIG. 4a includes graphs showing the results of counting the cell number of the Klebsiella pneumoniae (KP), Proteus mirabilis (PM), and Enterococcus gallinarum (EG) contained in fecal samples of Hc, Psc/Uc, and Uc.FIG. 4b includes graphs showing correlations between the proportion of Th17 cells or Th1 cells in the liver of PSCUC and the number of Klebsiella pneumoniae cells in the fecal samples thereof; -
FIG. 5a includes graphs showing the results of flow cytometric analysis of the cells in the livers, colons, and mesenteric lymph nodes of humanized gnotobiotic mice administered KP only (KP), PM and EG (PM+EG), and three strains of KP, PM, and EG (3-mix), respectively, in fecal samples derived from Psc/Uc, GF mice (GF), and SPF mice (SPF).FIG. 5b includes fluorescence micrographs of the ilea of the SPF mouse and the humanized gnotobiotic mice (KP gnoto, PM+EG gnoto, and 3-mix gnoto). The scale bars indicate 50 μm; -
FIG. 6a includes scanning electron micrographs of large intestinal epithelia co-cultured, in a monolayer organoid co-culture system of the large intestine, with an enterohemorrhagic Escherichia coli strain O-157:H7, a KP strain (KP JCM1662) not having an ability to form a pore on the large intestinal epithelium, and KP strains (KP-P1 and KP-P5) isolated from mesenteric lymph nodes of mice administered fecal sample derived from Psc/Uc. The scale bars indicate 20 μm.FIG. 6b is a graph showing the number of pores formed per unit area (mm2) of each of the large intestinal epithelia.FIG. 6c includes fluorescence micrographs of the co-cultured large intestinal epithelia triple-stained with an anti-cleaved caspase-3 antibody, phalloidin, and Hoechst 33324. The scale bars indicate 100 μm; -
FIG. 7 is a chart showing the results of comparative analysis after whole genome sequencing of KP strains (ATCC 700603, JCM1664, JCM1662, and JCM1663) not having an ability to form a pore on the large intestinal epithelium and KP strains (JCM20694, JCM20034, JCM20348, ATCC BAA2552, ATCC 700721, KP-P1, KP-P5, ATCC BAA1705, and JCM20507) having an ability to form a pore on the large intestinal epithelium was performed; -
FIG. 8a is a schematic diagram illustrating an experimental scheme.FIG. 8b includes fluorescence micrographs of the livers of a humanized gnotobiotic mouse (3-mix gnoto) prepared by administering KP-P1, PM, and EG to a GF mouse and of a humanized gnotobiotic mouse (m3-mix gnoto) prepared by administering JCM1662, PM, and EG to a mouse. The scale bars indicate 20 μm.FIG. 8c is a graph showing the measurement results of serum lipopolysaccharide (LPS) of each mouse.FIG. 8d is a graph showing the results of flow cytometric analysis of the cells in the liver of each mouse; -
FIG. 9a is a schematic diagram illustrating an experimental scheme.FIG. 9b includes graphs showing the results of flow cytometric analysis of the cells in the livers of humanized gnotobiotic mice (3-mix gnoto) treated with 3,5-dicarbetoxy-1,4-dihydrocollidine (DDC) and RORγt inverse agonist (RORIA) or water as a control.FIG. 9c includes graphs showing the measurement results of serum ALP (left) and serum TB (right) of the humanized gnotobiotic mice; and -
FIG. 10 includes micrographs of hematoxylin-eosin and Masson-trichrome stained liver sections of humanized gnotobiotic mice (3-mix gnoto) treated with DDC and RORIA. The scale bars indicate 100 μm. - Embodiments of the present invention will now be described with reference to the drawings, but the scope of the present invention is not limited to the disclosed embodiments.
- The present invention will now be described in detail.
- <Klebsiella pneumoniae Strain>
- An aspect of the present invention relates to a Klebsiella pneumoniae strain inducing inflammation in the liver. The inflammation of livers includes not only inflammation of livers but also inflammation of intra- and extra-hepatic bile ducts, and examples thereof include hepatitis and cholangitis. Examples of hepatitis include viral hepatitis, drug-induced hepatitis, alcoholic hepatitis, non-alcoholic steatohepatitis, and autoimmune hepatitis. Examples of cholangitis include sclerosing cholangitis (such as primary sclerosing cholangitis, IgG4-related sclerosing cholangitis, and secondary sclerosing cholangitis), primary biliary cholangitis (primary biliary cirrhosis), ascending cholangitis, secondary sclerosing cholangitis, and recurrent pyogenic cholangitis.
- The Klebsiella pneumoniae strain of the present invention is preferably a strain inducing Th17 cells in the liver. The induction of Th17 cells can be verified by, for example, treating mononuclear cells of the liver with a fluorescence labeled anti-CD4 antibody and an anti-IL-17 antibody and verifying a significant increase in IL-17-producing CD4 positive helper T (Th17) cells by flow cytometric analysis.
- The Klebsiella pneumoniae strain of the present invention preferably has the ability to form pores which is capable of forming pores on the large intestinal epithelia. The pores may have any shape and size allowing enteric bacteria to leak through the pores. For example, the diameter may be 0.1 to 20 μm, more preferably 0.5 to 15 μm, in observation of large intestinal epithelium with a scanning electron microscope. The leakage of enteric bacteria through the pores can be verified by, for example, hybridizing the DNA of the enteric bacteria with a fluorescent probe, staining the large intestinal epithelial cells with a fluorescent dye that emits fluorescence having a color different from that of the fluorescence of the probe, and observing the section of the large intestinal epithelium with a fluorescence microscope. Although the details of the mechanism of forming pores are unclear, it is inferred that the Klebsiella pneumoniae strain of the present invention comes into direct contact with large intestinal epithelium to induce apoptosis, resulting in the formation of pores.
- The Klebsiella pneumoniae strain of the present invention preferably has a
type 6 secretion system (T6SS). Thetype 6 secretion system can be verified by, for example, the presence of a gene in comparative analysis of whole genome sequencing of the strain. - The Klebsiella pneumoniae strain of the present invention is preferably derived from a patient suffering from both primary sclerosing cholangitis and ulcerative colitis described below. The strain can be isolated from a fecal sample of the patient by a known method using a generic growth medium. Examples of the medium include a brain heart infusion (BHI) medium, a MacConkey agar medium, and a VRBG medium.
- The Klebsiella pneumoniae strain of the present invention preferably has a DNA consisting of the nucleotide sequence registered in the National Center for Biotechnology Information (NCBI) under Assembly Name: ASM385182v1, and the Klebsiella pneumoniae strain is more preferably one whose deposit number is NITE BP-02879.
- The Klebsiella pneumoniae strain of the present invention preferably has a DNA consisting of the nucleotide sequence registered in the National Center for Biotechnology Information (NCBI) under Assembly Name: ASM386511v1.
- Primary sclerosing cholangitis can be diagnosed in accordance with known clinical guidelines. For example, as the diagnosis items, when “bile duct image” (A) and “increase in alkaline phosphatase level” (B) are defined as major items; “complication of inflammatory bowel disease” (a) and “liver tissue image (fibrous cholangitis/onion skin lesion)” (b) are defined as minor items; and the bile duct image (A) is classified into “recognition of findings of bile duct image characteristic to primary sclerosing cholangitis” (A1) and “no recognition of findings of bile duct image characteristic to primary sclerosing cholangitis” (A2), only definite diagnosis and probable diagnosis in the following Table 1 can be treated as primary sclerosing cholangitis.
-
TABLE 1 Major item Minor item (A1) (A2) (B) (a) (b) Diagnosis ∘ — ∘ — — Definite diagnosis ∘ — — ∘ — Definite diagnosis ∘ — — — ∘ Definite diagnosis — ∘ ∘ ∘ ∘ Definite diagnosis ∘ — — — — Probable diagnosis — ∘ ∘ ∘ — Probable diagnosis — ∘ ∘ — ∘ Probable diagnosis — ∘ — ∘ ∘ Probable diagnosis — ∘ — ∘ — Possible diagnosis — ∘ — — ∘ Possible diagnosis - The alkaline phosphatase level can be measured by a known method. For example, it can be measured in accordance with the common standard method proposed by the Japan Society of Clinical Chemistry (JSCC). When the measured value is, for example, 2 to 3 times as high as the reference value, the value can be determined as an abnormal value.
- The bile duct image can be judged to be (A1) or (A2) mentioned above based on, for example, findings of diffuse wall irregularities and strictures associated with inflammation of intra- and extra-hepatic bile ducts characteristic to primary sclerosing cholangitis by performing an endoscopic retrograde cholangiography (ERC) or magnetic resonance cholangiopancreatography (MRCP) test.
- The above-mentioned diagnosis of primary sclerosing cholangitis needs to exclude malignant tumors such as cholangiocarcinoma, IgG4-related sclerosing cholangitis, and secondary sclerosing cholangitis. The IgG4-related sclerosing cholangitis can be diagnosed based on, for example, combination of four criteria ((1) finding of characteristic bile duct image, (2) increase in serum IgG4 level, (3) complication of IgG4-related disease of an organ other than biliary tract, and (4) characteristic histopathological findings) in Clinical diagnostic criteria of IgG4-related sclerosing cholangitis, 2012 (Journal of Hepato-Biliary-Pancreatic Sciences, vol. 19, pp. 536-542).
- Examples of the secondary sclerosing cholangitis include congenital diseases, such as Caroli's disease and cystic fibrosis; chronic obstructive diseases, such as choledocholith, biliary stricture, Mirizzi syndrome, anastomotic stenosis after liver transplantation, and tumors; infectious diseases, such as bacterial cholangitis, recurrent pyogenic cholangitis, parasitic infection, and cytomegalovirus infection; poisoning diseases, such as erroneous intrabiliary injection of alcohol, formaldehyde, or hypertonic saline; immune abnormalities, such as eosinophilic cholangitis and those with AIDS; ischemic diseases, such as those related to vascular injury, post-traumatic sclerosing cholangitis, hepatic artery embolism after liver transplantation, rejection after liver transplantation, or coronary arterial anticancer drug infusion; and invasive lesions, such as systemic vasculitis, amyloidosis, sarcoidosis, systemic mastocytosis, eosinophilia, Hodgkin's disease, and xanthogranulomatous cholangitis.
- Ulcerative colitis can be diagnosed in accordance with known clinical guidelines. For example, ulcerative colitis can be clearly diagnosed when the following item (B-1) or (B-2) is satisfied, in addition to the items (A) and (C), and the diseases (D) are excluded.
- (A) Clinical symptom: Persistent or recurrent mucous or bloody stool or medical history thereof is observed.
- (B-1) Endoscopic examination: (1) The mucous membrane is diffusely affected, the visible vascular pattern disappears, and coarse or fine granular form is observed. Furthermore, friability and hemorrhage-prone properties (contact bleeding) cause attachment of mucous and bloody purulent secretion; or (2) multiple erosion, ulcer, or pseudopolyposis is observed. (3) In general, lesions are observed continuously from the rectum.
- (B-2) Enema X-ray examination: (1) Diffuse change like a coarse or fine granular form of the surface of the mucosal membrane, (2) multiple erosion or ulcer, and (3) pseudopolyposis are observed. In addition, disappearance of Haustra coli (lead pipe appearance) or narrowing or shortening of the intestine is observed.
- (C) Biopsy histologic examination: In the active phase, diffuse inflammatory cell infiltration, cryptic tumor, and a severe decrease in goblet cells are observed in the entire mucosal layer. Since all of them are nonspecific findings, comprehensive judgment is performed. In the remission phase, abnormal arrangement of glands (meander, bifurcation) and atrophy remain. These changes are generally observed continuously from the rectum to the oral side.
- (D) Infectious enteritis, such as bacterial and Clostridium difficile enteritis, amebic colitis, Salmonella enteritis, Campylobacter enteritis, colonic tuberculosis, and Chlamydia enteritis; Crohn's disease; radiation irradiated colitis; drug-induced colitis; lymphoid follicular hyperplasia; ischemic colitis; intestinal Behcet, and so on.
- In addition to the above definite diagnosis cases, when the items (B-1) or (B-2) and (C) are satisfied multiple times, ulcerative colitis can be clearly diagnosed when findings grossly and histologically characteristic to ulcerative colitis are observed by excision surgery or autopsy.
- Another aspect of the present invention relates to use of the Klebsiella pneumoniae strain only or use of the Klebsiella pneumoniae strain, a Proteus mirabilis strain, and an Enterococcus gallinarum strain for predicting the development of primary sclerosing cholangitis.
- As described above, for example, a Klebsiella pneumoniae strain derived from a patient suffering from both primary sclerosing cholangitis and ulcerative colitis is characterized by (1) having a
type 6 secretion system, (2) having an ability to form a pore on large intestinal epithelia, and (3) inducing Th17 cells in the liver. This suggests that in a patient suffering from ulcerative colitis, the Klebsiella pneumoniae strain having atype 6 secretion system forms pores on the large intestinal epithelium to leak enteric bacteria, such as a Proteus mirabilis strain and an Enterococcus gallinarum strain, and induce Th17 cells in the liver, resulting in complication of primary sclerosing cholangitis. - Accordingly, if genome sequencing has revealed that the Klebsiella pneumoniae strain in a fecal sample of a patient suffering from ulcerative colitis has a
type 6 secretion system, the development of primary sclerosing cholangitis can be predicted. In addition, if formation of pores is recognized on the large intestinal epithelium when a biopsy tissue sample of the large intestine is subsequently observed with a scanning electron microscope, the prediction accuracy is increased. Furthermore, if an increase in Th17 cells in the liver is recognized in, for example, flow cytometry of a biopsy sample of the liver, the prediction accuracy is further increased. - In addition, a fecal sample of a patient is cultured, and the presence or absence of a Proteus mirabilis strain or an Enterococcus gallinarum strain, in addition to the Klebsiella pneumoniae strain, can be used as a predictor. The Proteus mirabilis strain is preferably a Proteus mirabilis strain whose accession number is NITE ABP-02923 and the Enterococcus gallinarum strain is preferably an Enterococcus gallinarum strain whose accession number is NITE ABP-02922.
- <Method for Producing Mouse Model Suffering from Both Primary Sclerosing Cholangitis and Ulcerative Colitis>
- Another aspect of the present invention relates to a method for producing a mouse model suffering from both primary sclerosing cholangitis and ulcerative colitis. Specifically, the method for producing a mouse model suffering from both primary sclerosing cholangitis and ulcerative colitis includes a step of administering a bacterial solution containing the Klebsiella pneumoniae strain described in the paragraph <Klebsiella pneumoniae strain> described above to a mouse and a step of administering 3,5-dicarbetoxy-1,4-dihydrocollidine to a mouse. Although the step of administering a bacterial solution containing the Klebsiella pneumoniae strain to a mouse and the step of administering 3,5-dicarbetoxy-1,4-dihydrocollidine to a mouse may be performed in any order, it is preferred to perform the step of administering a bacterial solution containing the Klebsiella pneumoniae strain to a mouse and then administering 3,5-dicarbetoxy-1,4-dihydrocollidine to the mouse.
- Although the original mouse to be used for producing the mouse model may be any mouse that can suffer from both primary sclerosing cholangitis and ulcerative colitis, a germ-free mouse is preferred. Examples of the germ-free mouse include mice produced from strains such as BALB/c, C57BL/6, and ICR.
- In order to produce the mouse model, for example, a suspension is prepared by suspending fecal samples collected from primary sclerosing cholangitis and ulcerative colitis patients in, for example, a phosphate-buffered saline (PBS) solution, and the suspension is orally administered to original mice to produce humanized gnotobiotic mice. Subsequently, the humanized gnotobiotic mice 19 to 23 days after the administration can be given free access to a diet containing 0.01% to 0.1% 3,5-dicarbetoxy-1,4-dihydrocollidine (DDC) for 12 to 16 days to produce a mouse model suffering from both primary sclerosing cholangitis and ulcerative colitis.
- Another aspect of the present invention relates to the Proteus mirabilis strain whose accession number is NITE ABP-02923.
- Another aspect of the present invention relates to the Enterococcus gallinarum strain whose accession number is NITE ABP-02922.
- Although the embodiments of the present invention have been described in detail, they are merely examples, and the present invention is not limited thereto. The scope of the present invention should be interpreted by the claims and includes all modifications within the meaning and scope equivalent to the claims.
- The present invention will now be further specifically described based on Examples, but is not limited to these Examples.
- Subjects were 14 patients suffering from both primary sclerosing cholangitis and ulcerative colitis (hereinafter referred to as “Psc/Uc” in some cases), 8 patients suffering from ulcerative colitis only (hereinafter referred to as “Uc” in some cases), and 10 healthy subjects (hereinafter referred to as “Hc” in some cases). Primary sclerosing cholangitis was diagnosed based on clinical guidelines and findings by cholangiography and liver biopsy. Ulcerative colitis was diagnosed by combination of endoscopic examination, histopathologic examination, and radiographic and serological examinations. Fecal samples were collected using feces collection tubes and were suspended in a solution containing 40% glycerol and an equivalent amount (w/v) of PBS, and the resulting suspensions were rapidly frozen and were stored at −80° C. until use.
- The frozen stock of each fecal sample of the above (1) was thawed and suspended in 6 volumes of PBS, and the suspension was passed through a 70-μm cell strainer to prepare a suspension for oral administration. 200 μL of the suspension for oral administration was orally administered to 6 to 8-week old male germ-free mice (GF mice, available from Sankyo Laboratories) using a stainless steel oral gavage needle to produce humanized gnotobiotic mice. Fecal samples were collected from the humanized
gnotobiotic mice 3 to 4 weeks after the administration. - DNAs of the bacteria in the human fecal samples in the above (1) and the humanized gnotobiotic mice fecal samples in the above (2) were isolated by an enzymatic dissolution method using lysozyme (manufactured by Sigma-Aldrich Co. LLC) and achromopeptidase (manufactured by FUJIFILM Wako Pure Chemical Corporation). The resulting DNA samples were treated with Ribonuclease A (manufactured by FUJIFILM Wako Pure Chemical Corporation) for purification and were then precipitated with 20% polyethylene glycol solution (PEG 6000, 2.5 M sodium chloride aqueous solution). Each precipitate was collected by centrifugation and was then washed with 75% ethanol and dissolved in a Tris-EDTA buffer.
- (4) 16S rRNA Metagenomic Analysis
- The hypervariable region V3-V4 of 16S rRNA gene of each of the DNAs obtained in the above (3) was amplified with TaKaRa Ex Taq® Hot Start Version (manufactured by Takara Bio Inc.), and the amplicon was purified with AMPure XP (manufactured by Beckman Coulter, Inc.). A mixture sample was prepared from the resulting DNA, and sequencing was performed with Miseq Reagent Kit V3 and Miseq Sequencer (manufactured by Illumina, Inc.) in accordance with the product manual. The sequence analysis was performed with QIIME software package ver. 1.9.1. Paired-end sequences were joined using a fastq-join tool in the EA-Utils software package. From the quality filter-passed sequences, 15,000 high-quality sequences were chosen for each sample. Chimera sequences were detected by the USEARCH, and the primer sequences were removed using cutadapt. Assignment of OTUs was performed using the UCLUST algorism with a sequence similarity of 96%. The assignment of OTUs was performed using the GLSEARCH program by similarity searching against the 16S (RDP version 10.27 and
CORE update 2 Sep. 2012) and the NCBI genome database. The data were rarefied to 10,000 sequences per sample, as determined by the rarefaction curves, and the relative abundances of the bacteria were determined. The unweighted UniFrac analysis was performed in accordance with the method described in the document (Tsuda, A., et al., Influence of Proton-Pump Inhibitors on the Luminal Microbiota in the Gastrointestinal Tract, Clin. Transl. Gastroenterol., 6, e89 (2015)). The results are shown inFIGS. 1a and 1b . The results demonstrated that the main bacterial flora was conserved in human and humanized gnotobiotic mice. - The liver, colon, and spleen of the humanized gnotobiotic mouse produced in Example 1 were homogenized, and total RNA of each organ was extracted using RNeasy Mini Kit (manufactured by QIAGEN N.V.). Complementary DNA was synthesized from 1 μg of the resulting total RNA by reverse transcription, and each target gene was amplified by PCR using AmpliTaq Gold Fast PCR MasterMix (manufactured by Applied Biosystems, Inc.) and the following designed primers (manufactured by Thermo Fisher Scientific Inc.).
- Glyceraldehyde-3-phosphate dehydrogenase (Gapdh): Mm03302249_g1
- Saa1: Mm00656927_g1
- Saa2: Mm04208126_mH
- Saa3: Mm00441203_m1
- IL-1β: Mm01336189_g1
- The amplicons were quantitatively measured by real-time PCR using TaqMan Universal Master Mix and StepOne Plus systems (manufactured by Applied Biosystems, Inc.). In each sample, the target gene expression level was standardized using Gapdh. The results are shown in
FIGS. 2a to 2c . The results demonstrated that in the liver (FIG. 2a ) and the colon (FIG. 2b ) of humanized gnotobiotic mice (PSCUC in the graphs) administered a fecal sample derived from Psc/Uc, the gene expression of serum amyloid protein A and IL-1β was increased and that bacteria involved in hepatic and large intestinal diseases were present in the intestinal bacterial flora of Psc/Uc. - GF mice and the humanized gnotobiotic mice produced in Example 1 (2) by orally administering feces of Hc or Psc/
Uc 21 days after the administration were given free access to a diet containing 0.05% 3,5-dicarbetoxy-1,4-dihydrocollidine (DDC) for 14 days, and serum alkaline phosphatase (ALP) and total bilirubin (TB) were measured by an LDH-UV kinetic method (manufactured by SRL, Inc.). The results are shown inFIG. 3a . The humanized gnotobiotic mice administered the fecal sample derived from Psc/Uc (PSCUC in the graphs) showed high expression levels of both ALP (left inFIG. 1a ) and TB (right inFIG. 1a ), compared to those in GF mice (GF in the graphs) and the humanized gnotobiotic mice administered the fecal sample derived from Hc (HC in the graphs). - Livers were collected from the DDC-treated humanized gnotobiotic mice and were fixed in 10% formalin and embedded in paraffin according to a usual method to produce paraffin blocks. Sections were cut from the paraffin blocks and were stained with hematoxylin-eosin and Masson-trichrome or stained with Sirius red to prepare specimen samples. The results of microscopic observation of the specimen samples are shown in
FIG. 3b . The upper part ofFIG. 3b shows the results of Sirius red staining, and the lower part ofFIG. 3b shows the results of co-staining with hematoxylin-eosin and Masson-trichrome. In the liver of the humanized gnotobiotic mice administered the fecal sample derived from Psc/Uc, hepatic disorder due to fiber formation in the liver was observed. - Livers were collected from the DCC-treated mice and were perfused with PBS from the portal vein. After the perfusion, the livers were chopped and passed through a 100-μm nylon mesh to obtain cells of the livers.
- The resulting cells were suspended in 40% Percoll solution and then overlaid in 75% Percoll fraction, followed by density gradient centrifugal separation at 840×g for 20 minutes at room temperature. Mononuclear cells were collected from the intermediate layer. The resulting mononuclear cells were washed and were suspended in FACS buffer. The mononuclear cells were subjected to blocking treatment using an anti-Fc antibody (CD16/32, manufactured by BD Pharmingen) and were then subjected to intracellular staining with an anti-CD4 antibody (APC-cy7, BV510) and an anti-CD11b antibody (APC-cy7). The intracellular stained cells were treated with Ionomycine (500 ng/mL) or Golgistop (10 μg/mL, manufactured by BD Biosciences) in the presence of lipopolysaccharide (derived from Escherichia coli B5, manufactured by Sigma-Aldrich Co. LLC) or PMA (50 ng/mL, manufactured by Sigma-Aldrich Co. LLC) and brefeldin A (10 μg/mL, manufactured by BD Biosciences).
- Subsequently, an anti-IFN-γ antibody, an anti-TNF-α antibody, an anti-IL-1β antibody, and an anti-IL-17 antibody (manufactured by BD Pharmingen) were added thereto, followed by co-culture at 4° C. for 20 minutes. The cells after the culture were washed with PBS and were measured with a cell sorter (“FACS CantoII,” manufactured by Becton, Dickinson and Company) and analyzed using FlowJo software (manufactured by Tree Star, Inc.). The results are shown in
FIG. 3c . The results demonstrated that in DDC-treated mice administered a fecal sample derived from Psc/Uc, the development of hepatic disorder and recruitment of Th17 cells and IL-1β+ CD11b+ macrophages were observed, and a mouse model suffering from both primary sclerosing cholangitis and ulcerative colitis was provided. - In order to identify the bacteria that induce hepatitis, the livers, mesenteric lymph nodes, and spleens were collected from humanized gnotobiotic mice orally administered a fecal sample of Hc or Psc/Uc and SPF mice (6- to 8-week old C57BL/6 mice, available from CLEA Japan, Inc.) as a control group on the 21st day from the administration, and the bacteria were cultured on agar plates. As a result, no colonies were observed in the liver and spleen of any of the mice. In the mesenteric lymph nodes, colonies were observed in only that from the humanized gnotobiotic mice administered a fecal sample derived from Psc/Uc. Bacterial flora analysis of 16S rRNA of the colonies demonstrated that the bacteria of the colonies were Klebsiella pneumoniae (KP), Proteus mirabilis (PM), and Enterococcus gallinarum (EG).
- Fecal samples derived from Hc, Psc/Uc, and Uc were anaerobically cultured in BHI medium (manufactured by Becton, Dickinson and Company), and the KP, PM, and EG cells were counted. The results are shown in
FIG. 4a . It was demonstrated that in the fecal sample derived from Psc/Uc, KP was remarkably present, and PM and EG were also present. The KP included a KP strain (deposit number: NITE BP-02879) having a DNA consisting of the nucleotide sequence registered in the National Center for Biotechnology Information (NCBI) under Assembly Name: ASM385182v1 and a KP strain having a DNA consisting of the nucleotide sequence registered in the National Center for Biotechnology Information (NCBI) under Assembly Name: ASM386511v1. The PM included a strain whose accession number is NITE ABP-02923, and the EG included a strain whose accession number is NITE ABP-02922. - As in Example 2 (3), correlations between the proportion of Th17 cells or Th1 cells in the liver of the humanized gnotobiotic mice administered the fecal sample derived from Psc/Uc and the number of KP cells in the fecal sample were verified by flow cytometric analysis using an anti-CD3e antibody (FITC), an anti-CD4 antibody (APC-cy7, BV510), an anti-IFN-γ antibody, and an anti-IL-17 antibody, and the significance was verified by a Spearman rank-order correlation test. The results are shown in
FIG. 4b . A correlation was found between KP and Th17 cells (left inFIG. 4b ), but no correlation was found between KP and Th1 cells (right inFIG. 4b ). The results demonstrated that KP derived from Psc/Uc contributed to induction of Th17 cells in the liver. - In order to investigate contribution of the above-mentioned three strains to Th17 cell induction, 1×108 CFU/200 μL of KP, PM, and EG were orally administered to GF mice by (1) KP alone (KP), (2) combination of PM and EG (PM+EG), or (3) combination of three strains (3-mix) each twice per week. On the 21st day from the administration, as in Example 2 (3), the liver, colon, and mesenteric lymph node were analyzed by flow cytometry using an anti-CD4 antibody (APC-cy7, BV510), an anti-IL-17 antibody, and an anti-RORγt antibody (manufactured by BD Pharmingen). The results are shown in
FIG. 5a . In all organs of the liver (left inFIG. 5a ), the colon (center inFIG. 5a ), and the mesenteric lymph node (right inFIG. 5a ), Th17 cell induction was recognized in the KP administration groups, in particular, in the 3-mix administration group, compared to those in GF mice (GF) and SPF mice (SPF) not administered the strains. - Ileum tissues including fecal materials of humanized gnotobiotic mice prepared by administering KP alone, PM and EG, and three strains of KP, PM, and EG to GF mice (KP, PM+EG gnoto, and 3-mix gnoto) were fixed in Carnoy's solution for 3 hours and embedded in paraffin to produce paraffin blocks. Tissue sections were cut from the blocks and were hybridized at 50° C. overnight using EUB338 probe (Alexa555 label), hybridizing with the DNA of the above-mentioned strains, prepared such that the final concentration in hybridization buffer (20 mM Tris-HCl (pH 7.4), 0.9 M NaCl, 0.1% SDS, 20% formamide) was 10 μg/mL.
- The tissue sections were washed with washing buffer (20 mM Tris-HCl (pH 7.4), 0.9 M NaCl) for 10 minutes and with PBS for 10 minutes three times and were then stained with Phalloidin-iFluor (manufactured by Abcam plc.). After washing with PBS for 10 minutes three times, the sections were mounted in Prolong anti-fade mounting media with DAPI (manufactured by Life Technologies). Microscopic observation was performed with a BIO-REVO BZ-9000 fluorescence microscope (manufactured by Keyence Corporation). The results are shown in
FIG. 5b . It was observed that the bacteria stained in red in the micrographs invaded the large intestinal epithelium mucous membrane in the 3-mix gnoto and KP gnoto, and some of the bacteria further invaded the large intestinal epithelium. In particular, the invasion was remarkable in the 3-mix gnoto. In contrast, the invasion was only slight in the PM+EG gnoto and was not observed in the control SPF. - Healthy human colon organoids were embedded in Matrigel (Corning) and were three-dimensionally cultured in Advanced DMEM/F12 culture solution (containing penicillin/streptomycin, 10 mM HEPES, 2 mM GlutaMAX, 1×B27 (manufactured by Life Technologies), 1 mM N-acetylcysteine (manufactured by FUJIFILM Wako Pure Chemical Corporation), 10 nM Gastrin I (manufactured by Sigma-Aldrich Co. LLC), 50 ng/mL human recombinant EGF, 0.5 μM A83-01, 3 μM SB202190, 50% Afamin-Wnt3a (Afm-W) complex condition medium (CM, v/v), R-spondin 1 (R)-CM (10% v/v), and Noggin (N)-CM (10% v/v)).
- The three-dimensionally cultured human colon organoids were cultured for at least one day in a medium containing 10 μM Y-27632 and containing Afm-Wnt, R-
spondin 1, Noggin, EGF, A83-01, and SB202190 and were then separated and seeded in a ThinCert 24-well plate (manufactured by Greiner Bio-One International GmbH) coated with 10% Cellmatrix type I-C (manufactured by Nitta Gelatin Inc.) and having a pore size of 0.4 μm. 2 to 3 days after the seeding, the medium was replaced by a condition medium not containing Afm-Wnt and SB202190Y-27632. The colonic epithelium was washed with Advanced DMEM/F12 culture solution before seeding of the strain, and antibiotic-free DM was added thereto to produce a monolayer organoid co-culture system. - KP strains (KP-P1 and KP-P5) isolated from the mesenteric lymph nodes of mice administered a fecal sample derived from Psc/Uc, enterohemorrhagic Escherichia coli O-157:H7, and a KP strain (KP JCM1662) obtained from Riken BioResource Research Center were added to the monolayer organoid co-culture systems each at a concentration of 1×105 CFU, followed by culturing for 8 hours. As a control, PBS was used. The co-cultured large intestinal epithelium was collected and pre-fixed in 5% glutaraldehyde (manufactured by TCI Chemicals) dissolved in PBS at 4° C. overnight. The pre-fixed sample was post-fixed in 1% osmium tetraoxide dissolved in PBS for 1 hour. The post-fixed sample was dehydrated with ethanol and coated by gold sputtering and was then observed with a scanning electron microscope (“VHX-D510 scanning electron microscope,” manufactured by Keyence Corporation)” in a high-vacuum mode. The results are shown in
FIG. 6 a. - In addition, the pores having a pore size of 10 μm or more on the large intestine were counted by observation of four independent positions of the large intestinal epithelium. The experiment was repeated 3 to 6 times. The results are shown in
FIG. 6b . The results demonstrated that the KP strains (KP-P1 and KP-P5) of mice receiving a fecal sample derived from Psc/Uc formed pores on the large intestinal epithelium, but the JCM1662 strain formed no pores. In addition, a same test using other KP strains demonstrated that seven KP strains (JCM20034, ATCC BAA1705, ATCC BAA2552, ATCC700721, JCM20348, JCM20694, and JCM20507) formed pores on the large intestinal epithelium, but four strains (JCM1662, JCM1663, JCM1664, and ATCC700603) formed no pores on the large intestinal epithelium. - Furthermore, the co-cultured large intestinal epithelial cells were triple-stained with an anti-cleaved caspase-3 antibody (manufactured by Cell Signaling Technology, Inc.), a filamentous actin stain phalloidin (manufactured by Thermo Fisher Scientific Inc.), and a DNA stain Hoechst 33324 (manufactured by Thermo Fisher Scientific Inc.), and apoptosis cells were observed with a confocal laser microscope (SP5, manufactured by Leica). The results are shown in
FIG. 6c . It is inferred from the results that KP-P1 comes into direct contact with the large intestinal epithelium and induces apoptosis. - The whole genome sequencing of the KP strain having or not having an ability to form a pore on the large intestinal epithelium was performed by whole-genome shotgun sequencing using PacBio RSII and Illumina MiSeq sequencers, and comparative analysis was performed. The results are shown in
FIG. 7 . The results demonstrated that in 97 orthologous genes, the KP strain having an ability to form a pore on the large intestinal epithelia includes genes involved in atype 6 secretion system (T6SS) and reactive oxygen species (ROS) decomposition. - Humanized gnotobiotic mice (3-mix gnoto) were produced by administering to GF mice three strains: PM, EG, and KP-P1 which is a KP strain forming pores on the large intestinal epithelia, and humanized gnotobiotic mice (m3-mix gnoto) were produced by administering to mice three strains: PM, EG, and KP JCM1662 which is a KP strain not forming pores on the large intestinal epithelia. On the 21st day from the administration, the ileum was collected from each mouse and was subjected to histochemical observation in the same manner as in Example 5 (2). The results are shown in
FIG. 8b . The results demonstrated that 3-mix gnoto invaded the large intestine mucous membrane and the epithelial tissue, whereas m3-mix gnoto only slightly invaded the large intestine mucous membrane. - Blood was collected from GF mice, SPF mice, 3-mix gnoto, and m3-mix gnoto, and samples were prepared using ToxinSensor Chromogenic Limulus Amebocyte Lysate (LAL) Endotoxin Assay Kit (manufactured by GenScript Biotech Corporation) in accordance with the manual of the product. The absorbance at 540 nm was measured using FilterMax F3 Multi-Mode Microplate Reader (manufactured by Molecular Devices, LLC.). The results are shown in
FIG. 8c . The results demonstrated that the serum LPS level in 3-mix significantly increased compared to those in the GF mice, SPF mice, and m3-mix groups. - The livers of GF mice, 3-mix gnoto, and m3-mix gnoto were analyzed by flow cytometry using an anti-CD4 antibody (APC-cy7, BV510), an anti-IL-17 antibody, and an anti-RORγt antibody (manufactured by BD Pharmingen) in the same manner as in Example 2 (3). The results are shown in
FIG. 8d . The results demonstrated that 3-mix gnoto significantly induced Th17 cells compared to GF mice and m3-mix gnoto. - 3-mix gnoto was fed on a diet containing an RORγt inverse agonist (RORγt IA) or water as a control and DDC every day for 2 weeks 14 days after the administration of the strain (
FIG. 9a ). Subsequently, the liver of each mouse was subjected to flow cytometric analysis using an anti-CD4 antibody (APC-cy7, BV510), an anti-IL-17 antibody, an anti-IFN-γ antibody, and an anti-RORγt antibody (manufactured by BD Pharmingen) in the same manner as in Example 2 (3). The results are shown inFIG. 9 b. - In addition, serum alkaline phosphatase and serum total bilirubin were measured. The results are shown in
FIG. 9c in the same manner as in Example 3 (1). Furthermore, sections of the liver were stained with hematoxylin-eosin and Masson-trichrome and were subjected to microscopic observation in the same manner as in Example 3 (2). The results are shown inFIG. 10 . - It is inferred from these results that Th17 cells induced by strains in the liver play a pathogenetic role in the development of bile duct disorder induced by DDC.
- The contents of Japanese Patent Application No. 2018-082192 (application date: Apr. 23, 2018), including the specification, claims, drawings, and abstract, are incorporated herein by reference in its entirety.
Claims (14)
1. A Klebsiella pneumoniae strain inducing inflammation in the liver.
2. The Klebsiella pneumoniae strain according to claim 1 , Inducing a Th17 cell in the liver.
3. The Klebsiella pneumoniae strain according to claim 1 , having an ability to form a pore on the large intestinal epithelium.
4. The Klebsiella pneumoniae strain according to claim 1 , having a type 6 secretion system.
5. The Klebsiella pneumoniae strain according to claim 1 , derived from a patient suffering from both primary sclerosing cholangitis and ulcerative colitis.
6. The Klebsiella pneumoniae strain according to claim 1 , comprising a DNA consisting of a nucleotide sequence registered in the National Center for Biotechnology Information (NCBI) under Assembly Name: ASM385182v1.
7. The Klebsiella pneumoniae strain according to claim 1 , whose deposit number is NITE BP-02879.
8. The Klebsiella pneumoniae strain according to claim 1 , comprising a DNA consisting of a nucleotide sequence registered in the National Center for Biotechnology Information (NCBI) under Assembly Name: ASM386511v1.
9. A Proteus mirabilis strain whose accession number is NITE BP-02923.
10. An Enterococcus gallinarum strain whose accession number is NITE BP-02922.
11. Use of a Klebsiella pneumoniae strain according to claim 1 for prediction or diagnosis of the development of primary sclerosing cholangitis.
12. Use of a Klebsiella pneumoniae strain according to claim 1 , a Proteus mirabilis strain, and an Enterococcus gallinarum strain for prediction or diagnosis of the development of primary sclerosing cholangitis.
13. The use for prediction or diagnosis of the development of primary sclerosing cholangitis according to claim 12 , wherein the Proteus mirabilis strain is one whose accession number is NITE BP-02923; and the Enterococcus gallinarum strain is one whose accession number is NITE BP-02922.
14. A method for producing a mouse model suffering from both primary sclerosing cholangitis and ulcerative colitis, the method comprising:
administering a bacterial solution containing a Klebsiella pneumoniae strain according to claim 1 to a mouse; and
administering 3,5-dicarbetoxy-1,4-dihydrocollidine to a mouse.
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US17/130,557 US11859174B2 (en) | 2018-04-23 | 2020-12-22 | Klebsiella pneumoniae strain inducing inflammation in liver |
US17/130,570 US11845926B2 (en) | 2018-04-23 | 2020-12-22 | Klebsiella pneumoniae strain inducing inflammation in liver |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2018-082192 | 2018-04-23 | ||
JP2018082192 | 2018-04-23 |
Related Child Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US17/130,557 Division US11859174B2 (en) | 2018-04-23 | 2020-12-22 | Klebsiella pneumoniae strain inducing inflammation in liver |
US17/130,570 Continuation US11845926B2 (en) | 2018-04-23 | 2020-12-22 | Klebsiella pneumoniae strain inducing inflammation in liver |
Publications (1)
Publication Number | Publication Date |
---|---|
US20190338374A1 true US20190338374A1 (en) | 2019-11-07 |
Family
ID=68384676
Family Applications (3)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US16/389,454 Abandoned US20190338374A1 (en) | 2018-04-23 | 2019-04-19 | Klebsiella pneumoniae strain that induces inflammation in the liver |
US17/130,570 Active 2039-09-08 US11845926B2 (en) | 2018-04-23 | 2020-12-22 | Klebsiella pneumoniae strain inducing inflammation in liver |
US17/130,557 Active 2039-11-29 US11859174B2 (en) | 2018-04-23 | 2020-12-22 | Klebsiella pneumoniae strain inducing inflammation in liver |
Family Applications After (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US17/130,570 Active 2039-09-08 US11845926B2 (en) | 2018-04-23 | 2020-12-22 | Klebsiella pneumoniae strain inducing inflammation in liver |
US17/130,557 Active 2039-11-29 US11859174B2 (en) | 2018-04-23 | 2020-12-22 | Klebsiella pneumoniae strain inducing inflammation in liver |
Country Status (2)
Country | Link |
---|---|
US (3) | US20190338374A1 (en) |
JP (1) | JP7360140B2 (en) |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN114502722A (en) * | 2019-08-01 | 2022-05-13 | 延世大学校产学协力团 | Culture medium composition for enhancing activity of Wnt protein |
-
2019
- 2019-04-19 US US16/389,454 patent/US20190338374A1/en not_active Abandoned
- 2019-04-22 JP JP2019080978A patent/JP7360140B2/en active Active
-
2020
- 2020-12-22 US US17/130,570 patent/US11845926B2/en active Active
- 2020-12-22 US US17/130,557 patent/US11859174B2/en active Active
Also Published As
Publication number | Publication date |
---|---|
JP7360140B2 (en) | 2023-10-12 |
JP2019187416A (en) | 2019-10-31 |
US20210123109A1 (en) | 2021-04-29 |
US11859174B2 (en) | 2024-01-02 |
US11845926B2 (en) | 2023-12-19 |
US20210123108A1 (en) | 2021-04-29 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Nakamoto et al. | Gut pathobionts underlie intestinal barrier dysfunction and liver T helper 17 cell immune response in primary sclerosing cholangitis | |
Zegarra-Ruiz et al. | A diet-sensitive commensal lactobacillus strain mediates TLR7-dependent systemic autoimmunity | |
Ha et al. | Translocation of viable gut microbiota to mesenteric adipose drives formation of creeping fat in humans | |
Gur et al. | LGR5 expressing skin fibroblasts define a major cellular hub perturbed in scleroderma | |
Abedini et al. | Urinary single-cell profiling captures the cellular diversity of the kidney | |
Semenyuk et al. | Analysis of bacterial communities during Clostridium difficile infection in the mouse | |
Darfeuille-Michaud et al. | Presence of adherent Escherichia coli strains in ileal mucosa of patients with Crohn's disease | |
Halloran et al. | Molecular assessment of rejection and injury in lung transplant biopsies | |
US11859174B2 (en) | Klebsiella pneumoniae strain inducing inflammation in liver | |
Akbarpour et al. | Residual endotoxin induces primary graft dysfunction through ischemia/reperfusion-primed alveolar macrophages | |
Melhem et al. | Epithelial GPR35 protects from Citrobacter rodentium infection by preserving goblet cells and mucosal barrier integrity | |
Cui et al. | Intestinal barrier breakdown and mucosal microbiota disturbance in neuromyelitis optical spectrum disorders | |
EP2603604B1 (en) | Method and kit for the diagnosis and/or prognosis of tolerance in liver transplantation | |
Daniels et al. | Hepatitis in common variable immunodeficiency | |
Shen et al. | Expansion of macrophage and liver sinusoidal endothelial cell subpopulations during non-alcoholic steatohepatitis progression | |
Suarez et al. | Dysregulation of the engulfment pathway in the gut fuels Inflammatory Bowel Disease | |
KR20230147033A (en) | Enriched bioactive kidney cell population, its characteristics and uses | |
Motz et al. | Sirolimus-eluting airway stent reduces profibrotic Th17 cells and inhibits laryngotracheal stenosis | |
Ahmad | Metal, magnet or transplant: options in primary sclerosing cholangitis with stricture | |
WO2018082037A1 (en) | Method for monitoring xenotransplantation immune rejection by using porcine specific free dna, and primers | |
EP3895716A1 (en) | Fmt performance prediction test to guide and optimize therapeutic management of gvhd patients | |
JP7304053B2 (en) | Method for determining risk of developing liver cancer, and kit for determining risk of developing liver cancer | |
Levin | Pathogenesis of IgA nephropathy and diabetic kidney disease: linking molecular profile to morphological and clinical picture | |
Ray | The role of the renin angiotensin system and its principal receptor AT1 in the pathogenesis of intestinal fibrosis in inflammatory bowel disease | |
Zhu et al. | Folic acid modified TPGS nanoparticles alleviate non-alcoholic steatohepatitis via ER stress sensor XBP1s mediated intestinal barrier damage and gut dysbiosis |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AS | Assignment |
Owner name: KEIO UNIVERSITY, JAPAN Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:NAKAMOTO, NOBUHIRO;SASAKI, NOBUO;AOKI, RYO;AND OTHERS;REEL/FRAME:049715/0115 Effective date: 20190708 |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: NON FINAL ACTION MAILED |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: RESPONSE TO NON-FINAL OFFICE ACTION ENTERED AND FORWARDED TO EXAMINER |
|
STCB | Information on status: application discontinuation |
Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION |