IE86045B1 - Probiotic bifidobacteria strains - Google Patents
Probiotic bifidobacteria strainsInfo
- Publication number
- IE86045B1 IE86045B1 IE2008/0245A IE20080245A IE86045B1 IE 86045 B1 IE86045 B1 IE 86045B1 IE 2008/0245 A IE2008/0245 A IE 2008/0245A IE 20080245 A IE20080245 A IE 20080245A IE 86045 B1 IE86045 B1 IE 86045B1
- Authority
- IE
- Ireland
- Prior art keywords
- formulation
- strain
- bzfidobacterium
- inflammatory
- treatment
- Prior art date
Links
- 230000000529 probiotic Effects 0.000 title claims description 48
- 239000006041 probiotic Substances 0.000 title claims description 48
- 235000018291 probiotics Nutrition 0.000 title claims description 48
- 241000186000 Bifidobacterium Species 0.000 title claims description 31
- 230000000069 prophylaxis Effects 0.000 claims abstract description 22
- 230000002757 inflammatory Effects 0.000 claims abstract description 20
- 206010021972 Inflammatory bowel disease Diseases 0.000 claims abstract description 10
- 230000002496 gastric Effects 0.000 claims abstract description 7
- 239000000203 mixture Substances 0.000 claims description 45
- 238000009472 formulation Methods 0.000 claims description 37
- 201000010099 disease Diseases 0.000 claims description 21
- 102000004127 Cytokines Human genes 0.000 claims description 16
- 108090000695 Cytokines Proteins 0.000 claims description 16
- 230000001580 bacterial Effects 0.000 claims description 16
- 239000003795 chemical substances by application Substances 0.000 claims description 16
- 230000003110 anti-inflammatory Effects 0.000 claims description 13
- 230000000770 pro-inflamatory Effects 0.000 claims description 12
- 206010061218 Inflammation Diseases 0.000 claims description 11
- 206010012735 Diarrhoea Diseases 0.000 claims description 10
- 230000004054 inflammatory process Effects 0.000 claims description 10
- 201000008286 diarrhea Diseases 0.000 claims description 9
- 239000003814 drug Substances 0.000 claims description 9
- 208000006673 Asthma Diseases 0.000 claims description 8
- 206010020751 Hypersensitivity Diseases 0.000 claims description 8
- 235000013406 prebiotics Nutrition 0.000 claims description 8
- 201000005794 allergic hypersensitivity disease Diseases 0.000 claims description 7
- 239000000969 carrier Substances 0.000 claims description 7
- 150000001875 compounds Chemical class 0.000 claims description 7
- 201000009910 diseases by infectious agent Diseases 0.000 claims description 6
- 102000004169 proteins and genes Human genes 0.000 claims description 6
- 108090000623 proteins and genes Proteins 0.000 claims description 6
- 210000004080 Milk Anatomy 0.000 claims description 5
- 230000000172 allergic Effects 0.000 claims description 5
- 201000008937 atopic dermatitis Diseases 0.000 claims description 5
- 229940079593 drugs Drugs 0.000 claims description 5
- 239000000463 material Substances 0.000 claims description 5
- 235000013336 milk Nutrition 0.000 claims description 5
- 239000008267 milk Substances 0.000 claims description 5
- 239000000843 powder Substances 0.000 claims description 5
- 230000002458 infectious Effects 0.000 claims description 4
- 235000013618 yogurt Nutrition 0.000 claims description 4
- 206010003816 Autoimmune disease Diseases 0.000 claims description 3
- 241000193403 Clostridium Species 0.000 claims description 3
- 206010009887 Colitis Diseases 0.000 claims description 3
- 208000002551 Irritable Bowel Syndrome Diseases 0.000 claims description 3
- 206010038683 Respiratory disease Diseases 0.000 claims description 3
- 230000000240 adjuvant Effects 0.000 claims description 3
- 239000002671 adjuvant Substances 0.000 claims description 3
- 239000002775 capsule Substances 0.000 claims description 3
- 239000003937 drug carrier Substances 0.000 claims description 3
- 235000013305 food Nutrition 0.000 claims description 3
- 239000003826 tablet Substances 0.000 claims description 3
- 206010006334 Breathing abnormality Diseases 0.000 claims description 2
- 206010009900 Colitis ulcerative Diseases 0.000 claims description 2
- 206010011401 Crohn's disease Diseases 0.000 claims description 2
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 claims description 2
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 claims description 2
- 208000002389 Pouchitis Diseases 0.000 claims description 2
- 241000702670 Rotavirus Species 0.000 claims description 2
- 229940088594 Vitamin Drugs 0.000 claims description 2
- 235000020167 acidified milk Nutrition 0.000 claims description 2
- 235000013361 beverage Nutrition 0.000 claims description 2
- 150000001720 carbohydrates Chemical class 0.000 claims description 2
- 235000013351 cheese Nutrition 0.000 claims description 2
- 239000012141 concentrate Substances 0.000 claims description 2
- 230000001524 infective Effects 0.000 claims description 2
- 150000002632 lipids Chemical class 0.000 claims description 2
- 239000002366 mineral element Substances 0.000 claims description 2
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 2
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 2
- 239000011573 trace mineral Substances 0.000 claims description 2
- 235000013619 trace mineral Nutrition 0.000 claims description 2
- 201000006704 ulcerative colitis Diseases 0.000 claims description 2
- 239000011782 vitamin Substances 0.000 claims description 2
- 235000013343 vitamin Nutrition 0.000 claims description 2
- 150000003722 vitamin derivatives Chemical class 0.000 claims description 2
- 229930003231 vitamins Natural products 0.000 claims description 2
- 230000002519 immonomodulatory Effects 0.000 abstract description 11
- 210000004027 cells Anatomy 0.000 description 57
- 229940092253 Ovalbumin Drugs 0.000 description 40
- 108010058846 Ovalbumin Proteins 0.000 description 40
- 241000894006 Bacteria Species 0.000 description 33
- 210000001035 Gastrointestinal Tract Anatomy 0.000 description 23
- 241001608472 Bifidobacterium longum Species 0.000 description 19
- 239000000902 placebo Substances 0.000 description 17
- 229940068196 placebo Drugs 0.000 description 17
- 241001465754 Metazoa Species 0.000 description 15
- 102100006815 IL2RA Human genes 0.000 description 14
- 101700082799 IL2RA Proteins 0.000 description 14
- 101700015336 ISG20 Proteins 0.000 description 14
- 230000000694 effects Effects 0.000 description 13
- 229920002424 Internal transcribed spacer Polymers 0.000 description 11
- 230000001717 pathogenic Effects 0.000 description 11
- 230000012010 growth Effects 0.000 description 10
- 229920003013 deoxyribonucleic acid Polymers 0.000 description 9
- 230000001900 immune effect Effects 0.000 description 9
- 239000002609 media Substances 0.000 description 9
- 244000005700 microbiome Species 0.000 description 9
- 238000002360 preparation method Methods 0.000 description 9
- 102100019461 CD28 Human genes 0.000 description 8
- 101700033362 CD28 Proteins 0.000 description 8
- 108090000174 Interleukin-10 Proteins 0.000 description 8
- 102000003814 Interleukin-10 Human genes 0.000 description 8
- 229940076144 Interleukin-10 Drugs 0.000 description 8
- 230000028709 inflammatory response Effects 0.000 description 8
- 230000000968 intestinal Effects 0.000 description 8
- 239000000047 product Substances 0.000 description 8
- 230000004043 responsiveness Effects 0.000 description 8
- 239000000427 antigen Substances 0.000 description 7
- 108091007172 antigens Proteins 0.000 description 7
- 102000038129 antigens Human genes 0.000 description 7
- 230000003115 biocidal Effects 0.000 description 7
- 201000011510 cancer Diseases 0.000 description 7
- 239000000306 component Substances 0.000 description 7
- 239000002158 endotoxin Substances 0.000 description 7
- 239000012530 fluid Substances 0.000 description 7
- 238000000338 in vitro Methods 0.000 description 7
- 200000000018 inflammatory disease Diseases 0.000 description 7
- 230000004044 response Effects 0.000 description 7
- 101700064140 FOXP3 Proteins 0.000 description 6
- BJHIKXHVCXFQLS-UYFOZJQFSA-N Fructose Natural products OC[C@@H](O)[C@@H](O)[C@H](O)C(=O)CO BJHIKXHVCXFQLS-UYFOZJQFSA-N 0.000 description 6
- 210000000987 Immune System Anatomy 0.000 description 6
- 108010002350 Interleukin-2 Proteins 0.000 description 6
- 102000000588 Interleukin-2 Human genes 0.000 description 6
- 210000001616 Monocytes Anatomy 0.000 description 6
- 229920001850 Nucleic acid sequence Polymers 0.000 description 6
- 210000003819 Peripheral blood mononuclear cell Anatomy 0.000 description 6
- 230000004075 alteration Effects 0.000 description 6
- 108090001123 antibodies Proteins 0.000 description 6
- 102000004965 antibodies Human genes 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 6
- 230000028993 immune response Effects 0.000 description 6
- 230000001965 increased Effects 0.000 description 6
- 108090000978 Interleukin-4 Proteins 0.000 description 5
- 210000001744 T-Lymphocytes Anatomy 0.000 description 5
- 230000016396 cytokine production Effects 0.000 description 5
- 238000011161 development Methods 0.000 description 5
- 230000018109 developmental process Effects 0.000 description 5
- 238000004519 manufacturing process Methods 0.000 description 5
- 230000002829 reduced Effects 0.000 description 5
- 210000003289 regulatory T cell Anatomy 0.000 description 5
- 241000894007 species Species 0.000 description 5
- 210000001519 tissues Anatomy 0.000 description 5
- 210000000941 Bile Anatomy 0.000 description 4
- 108010002616 Interleukin-5 Proteins 0.000 description 4
- GUBGYTABKSRVRQ-YOLKTULGSA-N Maltose Natural products O([C@@H]1[C@H](O)[C@@H](O)[C@H](O)O[C@H]1CO)[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 GUBGYTABKSRVRQ-YOLKTULGSA-N 0.000 description 4
- PYMYPHUHKUWMLA-LMVFSUKVSA-N Ribose Natural products OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 description 4
- 210000000952 Spleen Anatomy 0.000 description 4
- 239000011324 bead Substances 0.000 description 4
- 238000000684 flow cytometry Methods 0.000 description 4
- 230000000051 modifying Effects 0.000 description 4
- 210000000056 organs Anatomy 0.000 description 4
- 230000002633 protecting Effects 0.000 description 4
- 235000018102 proteins Nutrition 0.000 description 4
- 230000001105 regulatory Effects 0.000 description 4
- 230000000638 stimulation Effects 0.000 description 4
- -1 troches Substances 0.000 description 4
- 229920001817 Agar Polymers 0.000 description 3
- 229940009291 Bifidobacterium longum Drugs 0.000 description 3
- 229940105657 CATALASE Drugs 0.000 description 3
- 102100008428 CCL2 Human genes 0.000 description 3
- 101700006000 CCL2 Proteins 0.000 description 3
- 210000001072 Colon Anatomy 0.000 description 3
- GZCGUPFRVQAUEE-KCDKBNATSA-N D-(+)-Galactose Natural products OC[C@@H](O)[C@H](O)[C@H](O)[C@@H](O)C=O GZCGUPFRVQAUEE-KCDKBNATSA-N 0.000 description 3
- WQZGKKKJIJFFOK-GASJEMHNSA-N D-Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 3
- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 description 3
- 102000016938 EC 1.11.1.6 Human genes 0.000 description 3
- 108010053835 EC 1.11.1.6 Proteins 0.000 description 3
- 210000003979 Eosinophils Anatomy 0.000 description 3
- 241000588724 Escherichia coli Species 0.000 description 3
- 210000003608 Feces Anatomy 0.000 description 3
- 239000005715 Fructose Substances 0.000 description 3
- 101700086956 IFNG Proteins 0.000 description 3
- 102100016020 IFNG Human genes 0.000 description 3
- 102000004889 Interleukin-6 Human genes 0.000 description 3
- 108090001005 Interleukin-6 Proteins 0.000 description 3
- 229960002329 Methacholine Drugs 0.000 description 3
- JQXXHWHPUNPDRT-ZNQWNCHJSA-N O([C@](C1=O)(C)O/C=C/[C@@H]([C@H]([C@@H](OC(C)=O)[C@H](C)[C@H](O)[C@H](C)[C@@H](O)[C@@H](C)\C=C\C=C(C)/C(=O)Nc2c(O)c3c(O)c4C)C)OC)c4c1c3c(O)c2C=NN1CCN(C)CC1 Chemical compound O([C@](C1=O)(C)O/C=C/[C@@H]([C@H]([C@@H](OC(C)=O)[C@H](C)[C@H](O)[C@H](C)[C@@H](O)[C@@H](C)\C=C\C=C(C)/C(=O)Nc2c(O)c3c(O)c4C)C)OC)c4c1c3c(O)c2C=NN1CCN(C)CC1 JQXXHWHPUNPDRT-ZNQWNCHJSA-N 0.000 description 3
- 206010039073 Rheumatoid arthritis Diseases 0.000 description 3
- 229940081190 Rifampin Drugs 0.000 description 3
- 210000002784 Stomach Anatomy 0.000 description 3
- 241000194026 Streptococcus gordonii Species 0.000 description 3
- 102100009534 TNF Human genes 0.000 description 3
- 101710040537 TNF Proteins 0.000 description 3
- 102000009270 Tumour necrosis factor alpha Human genes 0.000 description 3
- 108050000101 Tumour necrosis factor alpha Proteins 0.000 description 3
- 239000008272 agar Substances 0.000 description 3
- 230000000845 anti-microbial Effects 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 230000002401 inhibitory effect Effects 0.000 description 3
- 238000003780 insertion Methods 0.000 description 3
- 238000002955 isolation Methods 0.000 description 3
- NZWOPGCLSHLLPA-UHFFFAOYSA-N methacholine Chemical compound C[N+](C)(C)CC(C)OC(C)=O NZWOPGCLSHLLPA-UHFFFAOYSA-N 0.000 description 3
- 238000000034 method Methods 0.000 description 3
- 238000010172 mouse model Methods 0.000 description 3
- 230000035772 mutation Effects 0.000 description 3
- 239000002773 nucleotide Substances 0.000 description 3
- 230000035755 proliferation Effects 0.000 description 3
- 229960001225 rifampicin Drugs 0.000 description 3
- 230000028327 secretion Effects 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 230000002269 spontaneous Effects 0.000 description 3
- 108010044241 tetanus toxin fragment C Proteins 0.000 description 3
- 238000002560 therapeutic procedure Methods 0.000 description 3
- 238000002255 vaccination Methods 0.000 description 3
- 229960005486 vaccines Drugs 0.000 description 3
- BHQCQFFYRZLCQQ-UMZBRFQRSA-N 4-[(3R,5S,7R,12S)-3,7,12-trihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]pentanoic acid Chemical class C([C@H]1C[C@H]2O)[C@H](O)CCC1(C)C1C2C2CCC(C(CCC(O)=O)C)C2(C)[C@@H](O)C1 BHQCQFFYRZLCQQ-UMZBRFQRSA-N 0.000 description 2
- 229940064005 Antibiotic throat preparations Drugs 0.000 description 2
- 229940083879 Antibiotics FOR TREATMENT OF HEMORRHOIDS AND ANAL FISSURES FOR TOPICAL USE Drugs 0.000 description 2
- 229940042052 Antibiotics for systemic use Drugs 0.000 description 2
- 229940042786 Antitubercular Antibiotics Drugs 0.000 description 2
- 210000003719 B-Lymphocytes Anatomy 0.000 description 2
- 229940093761 Bile Salts Drugs 0.000 description 2
- 206010006895 Cachexia Diseases 0.000 description 2
- 229940015062 Campylobacter jejuni Drugs 0.000 description 2
- 241000589875 Campylobacter jejuni Species 0.000 description 2
- 229940041514 Candida albicans extract Drugs 0.000 description 2
- 102000009016 Cholera Toxin Human genes 0.000 description 2
- 108010049048 Cholera Toxin Proteins 0.000 description 2
- 102000020504 Collagenase family Human genes 0.000 description 2
- 108060005980 Collagenase family Proteins 0.000 description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N D-sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 2
- 229940110715 ENZYMES FOR TREATMENT OF WOUNDS AND ULCERS Drugs 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 229940093922 Gynecological Antibiotics Drugs 0.000 description 2
- 241000282412 Homo Species 0.000 description 2
- 210000004969 Inflammatory Cells Anatomy 0.000 description 2
- 108010053490 Infliximab Proteins 0.000 description 2
- GUBGYTABKSRVRQ-UUNJERMWSA-N Lactose Natural products O([C@@H]1[C@H](O)[C@H](O)[C@H](O)O[C@@H]1CO)[C@H]1[C@@H](O)[C@@H](O)[C@H](O)[C@H](CO)O1 GUBGYTABKSRVRQ-UUNJERMWSA-N 0.000 description 2
- 210000000265 Leukocytes Anatomy 0.000 description 2
- 210000004072 Lung Anatomy 0.000 description 2
- 210000004698 Lymphocytes Anatomy 0.000 description 2
- 210000002540 Macrophages Anatomy 0.000 description 2
- 229940045184 Malt extract Drugs 0.000 description 2
- MUPFEKGTMRGPLJ-ZQSKZDJDSA-N Raffinose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO[C@@H]2[C@@H]([C@@H](O)[C@@H](O)[C@@H](CO)O2)O)O1 MUPFEKGTMRGPLJ-ZQSKZDJDSA-N 0.000 description 2
- 241000293869 Salmonella enterica subsp. enterica serovar Typhimurium Species 0.000 description 2
- 206010070834 Sensitisation Diseases 0.000 description 2
- 210000002966 Serum Anatomy 0.000 description 2
- 241000580858 Simian-Human immunodeficiency virus Species 0.000 description 2
- CZMRCDWAGMRECN-GDQSFJPYSA-N Sucrose Natural products O([C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](CO)O1)[C@@]1(CO)[C@H](O)[C@@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-GDQSFJPYSA-N 0.000 description 2
- 229940024982 Topical Antifungal Antibiotics Drugs 0.000 description 2
- SRBFZHDQGSBBOR-SQOUGZDYSA-N Xylose Natural products O[C@@H]1CO[C@@H](O)[C@@H](O)[C@@H]1O SRBFZHDQGSBBOR-SQOUGZDYSA-N 0.000 description 2
- 239000004480 active ingredient Substances 0.000 description 2
- 238000007792 addition Methods 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 230000030741 antigen processing and presentation Effects 0.000 description 2
- 239000004599 antimicrobial Substances 0.000 description 2
- 150000001479 arabinose derivatives Chemical class 0.000 description 2
- 244000052616 bacterial pathogens Species 0.000 description 2
- 239000003833 bile salt Substances 0.000 description 2
- 230000000975 bioactive Effects 0.000 description 2
- 238000004166 bioassay Methods 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 229960002424 collagenase Drugs 0.000 description 2
- 230000001351 cycling Effects 0.000 description 2
- 230000002354 daily Effects 0.000 description 2
- 230000002708 enhancing Effects 0.000 description 2
- 230000004077 genetic alteration Effects 0.000 description 2
- 231100000118 genetic alteration Toxicity 0.000 description 2
- 238000010353 genetic engineering Methods 0.000 description 2
- 210000002865 immune cell Anatomy 0.000 description 2
- 238000002649 immunization Methods 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 229960000598 infliximab Drugs 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 229940079866 intestinal antibiotics Drugs 0.000 description 2
- 239000008101 lactose Substances 0.000 description 2
- GUBGYTABKSRVRQ-XLOQQCSPSA-N lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 210000004877 mucosa Anatomy 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- 229940005935 ophthalmologic Antibiotics Drugs 0.000 description 2
- 244000052769 pathogens Species 0.000 description 2
- 108010004621 phosphoketolase Proteins 0.000 description 2
- 230000001681 protective Effects 0.000 description 2
- 230000008313 sensitization Effects 0.000 description 2
- 230000001235 sensitizing Effects 0.000 description 2
- 210000000813 small intestine Anatomy 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- 230000004083 survival Effects 0.000 description 2
- 230000001225 therapeutic Effects 0.000 description 2
- 102000003995 transcription factors Human genes 0.000 description 2
- 108090000464 transcription factors Proteins 0.000 description 2
- 238000002054 transplantation Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 230000004580 weight loss Effects 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- WQZGKKKJIJFFOK-PHYPRBDBSA-N α-D-galactose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-PHYPRBDBSA-N 0.000 description 2
- WQZGKKKJIJFFOK-VFUOTHLCSA-N β-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 2
- WOBHKFSMXKNTIM-UHFFFAOYSA-N 2-hydroxyethyl 2-methylacrylate Chemical compound CC(=C)C(=O)OCCO WOBHKFSMXKNTIM-UHFFFAOYSA-N 0.000 description 1
- 206010000496 Acne Diseases 0.000 description 1
- 208000002874 Acne Vulgaris Diseases 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 206010001488 Aggression Diseases 0.000 description 1
- 208000007502 Anemia Diseases 0.000 description 1
- 210000000628 Antibody-Producing Cells Anatomy 0.000 description 1
- 206010003011 Appendicitis Diseases 0.000 description 1
- 206010003246 Arthritis Diseases 0.000 description 1
- 210000000649 B-Lymphocyte Subsets Anatomy 0.000 description 1
- 206010060945 Bacterial infection Diseases 0.000 description 1
- 241000606125 Bacteroides Species 0.000 description 1
- 210000004369 Blood Anatomy 0.000 description 1
- 210000004204 Blood Vessels Anatomy 0.000 description 1
- 210000000481 Breast Anatomy 0.000 description 1
- 102100003268 CD14 Human genes 0.000 description 1
- 101700027514 CD14 Proteins 0.000 description 1
- 210000003169 Central Nervous System Anatomy 0.000 description 1
- KDLRVYVGXIQJDK-AWPVFWJPSA-N Clindamycin Chemical compound CN1C[C@H](CCC)C[C@H]1C(=O)N[C@H]([C@H](C)Cl)[C@@H]1[C@H](O)[C@H](O)[C@@H](O)[C@@H](SC)O1 KDLRVYVGXIQJDK-AWPVFWJPSA-N 0.000 description 1
- 241001112696 Clostridia Species 0.000 description 1
- 241001112695 Clostridiales Species 0.000 description 1
- 206010009839 Coeliac disease Diseases 0.000 description 1
- 102000008186 Collagen Human genes 0.000 description 1
- 108010035532 Collagen Proteins 0.000 description 1
- 210000002808 Connective Tissue Anatomy 0.000 description 1
- 208000004981 Coronary Disease Diseases 0.000 description 1
- JYGXADMDTFJGBT-VWUMJDOOSA-N Cortisol Chemical compound O=C1CC[C@]2(C)[C@H]3[C@@H](O)C[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 JYGXADMDTFJGBT-VWUMJDOOSA-N 0.000 description 1
- 230000008836 DNA modification Effects 0.000 description 1
- 206010061428 Decreased appetite Diseases 0.000 description 1
- 206010012601 Diabetes mellitus Diseases 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 208000000999 Encephalomyelitis, Autoimmune, Experimental Diseases 0.000 description 1
- 206010014698 Endocrine disease Diseases 0.000 description 1
- ZMMJGEGLRURXTF-UHFFFAOYSA-N Ethidium bromide Chemical compound [Br-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 ZMMJGEGLRURXTF-UHFFFAOYSA-N 0.000 description 1
- 210000002744 Extracellular Matrix Anatomy 0.000 description 1
- 102100000368 F8 Human genes 0.000 description 1
- 101700070229 F8 Proteins 0.000 description 1
- 102100011855 FOXP3 Human genes 0.000 description 1
- 206010017533 Fungal infection Diseases 0.000 description 1
- 240000007842 Glycine max Species 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 208000009329 Graft vs Host Disease Diseases 0.000 description 1
- 101710029273 HEMA1 Proteins 0.000 description 1
- 208000005721 HIV Infections Diseases 0.000 description 1
- 229940088597 Hormone Drugs 0.000 description 1
- 206010061598 Immunodeficiency Diseases 0.000 description 1
- 206010022114 Injury Diseases 0.000 description 1
- 108010002352 Interleukin-1 Proteins 0.000 description 1
- 102000000589 Interleukin-1 Human genes 0.000 description 1
- 206010061255 Ischaemia Diseases 0.000 description 1
- XUJNEKJLAYXESH-REOHCLBHSA-N L-cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 1
- 229940039696 Lactobacillus Drugs 0.000 description 1
- 241000186660 Lactobacillus Species 0.000 description 1
- 240000004403 Lactobacillus casei Species 0.000 description 1
- 229940017800 Lactobacillus casei Drugs 0.000 description 1
- 235000013958 Lactobacillus casei Nutrition 0.000 description 1
- 210000001165 Lymph Nodes Anatomy 0.000 description 1
- MINDHVHHQZYEEK-HBBNESRFSA-N MUPIROCIN Chemical compound C[C@H](O)[C@H](C)[C@@H]1O[C@H]1C[C@@H]1[C@@H](O)[C@@H](O)[C@H](C\C(C)=C\C(=O)OCCCCCCCCC(O)=O)OC1 MINDHVHHQZYEEK-HBBNESRFSA-N 0.000 description 1
- 208000002720 Malnutrition Diseases 0.000 description 1
- 206010061289 Metastatic neoplasm Diseases 0.000 description 1
- 229960000282 Metronidazole Drugs 0.000 description 1
- 206010028116 Mucosal inflammation Diseases 0.000 description 1
- 210000004400 Mucous Membrane Anatomy 0.000 description 1
- 229960003128 Mupirocin Drugs 0.000 description 1
- 206010029151 Nephropathy Diseases 0.000 description 1
- 208000001132 Osteoporosis Diseases 0.000 description 1
- 101710043203 P23p89 Proteins 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 210000001986 Peyer's Patches Anatomy 0.000 description 1
- 210000002381 Plasma Anatomy 0.000 description 1
- 229940082622 Prostaglandin cardiac therapy preparations Drugs 0.000 description 1
- 229940077717 Prostaglandin drugs for peptic ulcer and gastro-oesophageal reflux disease (GORD) Drugs 0.000 description 1
- 206010037660 Pyrexia Diseases 0.000 description 1
- MUPFEKGTMRGPLJ-RMMQSMQOSA-N Raffinose Natural products O(C[C@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@@H](O[C@@]2(CO)[C@H](O)[C@@H](O)[C@@H](CO)O2)O1)[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 MUPFEKGTMRGPLJ-RMMQSMQOSA-N 0.000 description 1
- 108010033725 Recombinant Proteins Proteins 0.000 description 1
- 102000007312 Recombinant Proteins Human genes 0.000 description 1
- 206010038428 Renal disease Diseases 0.000 description 1
- 206010040490 Sexually transmitted disease Diseases 0.000 description 1
- 244000057717 Streptococcus lactis Species 0.000 description 1
- 235000014897 Streptococcus lactis Nutrition 0.000 description 1
- 210000000662 T-Lymphocyte Subsets Anatomy 0.000 description 1
- 210000001541 Thymus Gland Anatomy 0.000 description 1
- 231100000765 Toxin Toxicity 0.000 description 1
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 1
- 206010067584 Type 1 diabetes mellitus Diseases 0.000 description 1
- 206010068760 Ulcers Diseases 0.000 description 1
- 206010046694 Urogenital disease Diseases 0.000 description 1
- MYPYJXKWCTUITO-LYRMYLQWSA-N VANCOMYCIN Chemical compound O([C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=C2C=C3C=C1OC1=CC=C(C=C1Cl)[C@@H](O)[C@H](C(N[C@@H](CC(N)=O)C(=O)N[C@H]3C(=O)N[C@H]1C(=O)N[C@H](C(N[C@@H](C3=CC(O)=CC(O)=C3C=3C(O)=CC=C1C=3)C(O)=O)=O)[C@H](O)C1=CC=C(C(=C1)Cl)O2)=O)NC(=O)[C@@H](CC(C)C)NC)[C@H]1C[C@](C)(N)[C@H](O)[C@H](C)O1 MYPYJXKWCTUITO-LYRMYLQWSA-N 0.000 description 1
- 229960003165 Vancomycin Drugs 0.000 description 1
- 108010059993 Vancomycin Proteins 0.000 description 1
- 210000003462 Veins Anatomy 0.000 description 1
- 206010047461 Viral infection Diseases 0.000 description 1
- 208000001756 Virus Disease Diseases 0.000 description 1
- 229960003487 Xylose Drugs 0.000 description 1
- 230000001594 aberrant Effects 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000003213 activating Effects 0.000 description 1
- 239000011149 active material Substances 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000001464 adherent Effects 0.000 description 1
- 230000001058 adult Effects 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 238000009632 agar plate Methods 0.000 description 1
- 150000001413 amino acids Chemical group 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 230000003042 antagnostic Effects 0.000 description 1
- 230000003466 anti-cipated Effects 0.000 description 1
- 230000002924 anti-infective Effects 0.000 description 1
- 239000002260 anti-inflammatory agent Substances 0.000 description 1
- 229940121363 anti-inflammatory agents Drugs 0.000 description 1
- 230000000890 antigenic Effects 0.000 description 1
- 230000002238 attenuated Effects 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 239000006161 blood agar Substances 0.000 description 1
- 230000023555 blood coagulation Effects 0.000 description 1
- 230000000711 cancerogenic Effects 0.000 description 1
- 231100000315 carcinogenic Toxicity 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 230000001413 cellular Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000005591 charge neutralization Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000001684 chronic Effects 0.000 description 1
- 229960002227 clindamycin Drugs 0.000 description 1
- 230000015271 coagulation Effects 0.000 description 1
- 238000005345 coagulation Methods 0.000 description 1
- 229920001436 collagen Polymers 0.000 description 1
- 229960005188 collagen Drugs 0.000 description 1
- 238000004891 communication Methods 0.000 description 1
- 235000008504 concentrate Nutrition 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 201000008739 coronary artery disease Diseases 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 238000000432 density-gradient centrifugation Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 230000000378 dietary Effects 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 230000003292 diminished Effects 0.000 description 1
- 230000029578 entry into host Effects 0.000 description 1
- 210000002919 epithelial cells Anatomy 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000001815 facial Effects 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 235000012041 food component Nutrition 0.000 description 1
- 239000005428 food component Substances 0.000 description 1
- 230000037406 food intake Effects 0.000 description 1
- 238000005755 formation reaction Methods 0.000 description 1
- 125000002485 formyl group Chemical group [H]C(*)=O 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 230000002068 genetic Effects 0.000 description 1
- 230000005017 genetic modification Effects 0.000 description 1
- 235000013617 genetically modified food Nutrition 0.000 description 1
- 230000001738 genotoxic Effects 0.000 description 1
- 231100000024 genotoxic Toxicity 0.000 description 1
- 150000004676 glycans Polymers 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 239000001963 growth media Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000013632 homeostatic process Effects 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 201000001820 human immunodeficiency virus infectious disease Diseases 0.000 description 1
- 229960000890 hydrocortisone Drugs 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 230000003308 immunostimulating Effects 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 230000002452 interceptive Effects 0.000 description 1
- 230000031261 interleukin-10 production Effects 0.000 description 1
- 239000000543 intermediate Substances 0.000 description 1
- 230000002147 killing Effects 0.000 description 1
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 230000001665 lethal Effects 0.000 description 1
- 231100000518 lethal Toxicity 0.000 description 1
- 150000002617 leukotrienes Chemical class 0.000 description 1
- 201000009673 liver disease Diseases 0.000 description 1
- 230000003211 malignant Effects 0.000 description 1
- 230000001071 malnutrition Effects 0.000 description 1
- 235000000824 malnutrition Nutrition 0.000 description 1
- VAOCPAMSLUNLGC-UHFFFAOYSA-N metronidazole Chemical compound CC1=NC=C([N+]([O-])=O)N1CCO VAOCPAMSLUNLGC-UHFFFAOYSA-N 0.000 description 1
- 230000000813 microbial Effects 0.000 description 1
- 230000002906 microbiologic Effects 0.000 description 1
- 239000003094 microcapsule Substances 0.000 description 1
- 244000005706 microflora Species 0.000 description 1
- 239000006151 minimal media Substances 0.000 description 1
- 230000004899 motility Effects 0.000 description 1
- 201000006417 multiple sclerosis Diseases 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- 230000001264 neutralization Effects 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 201000008875 nutrition disease Diseases 0.000 description 1
- 229920001542 oligosaccharide Polymers 0.000 description 1
- 150000002482 oligosaccharides Polymers 0.000 description 1
- MYMOFIZGZYHOMD-UHFFFAOYSA-N oxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229940094443 oxytocics Prostaglandins Drugs 0.000 description 1
- 230000000242 pagocytic Effects 0.000 description 1
- 239000003973 paint Substances 0.000 description 1
- 244000045947 parasites Species 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 201000008838 periodontal disease Diseases 0.000 description 1
- 230000002093 peripheral Effects 0.000 description 1
- 230000008855 peristalsis Effects 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 239000002953 phosphate buffered saline Substances 0.000 description 1
- 239000006187 pill Substances 0.000 description 1
- 229920000023 polynucleotide Polymers 0.000 description 1
- 239000002157 polynucleotide Substances 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 150000004804 polysaccharides Polymers 0.000 description 1
- 238000010837 poor prognosis Methods 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 231100000586 procarcinogen Toxicity 0.000 description 1
- 230000000644 propagated Effects 0.000 description 1
- 150000003180 prostaglandins Chemical class 0.000 description 1
- 201000004681 psoriasis Diseases 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000036647 reaction Effects 0.000 description 1
- 239000012048 reactive intermediate Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000025053 regulation of cell proliferation Effects 0.000 description 1
- 230000024833 regulation of cytokine production Effects 0.000 description 1
- 230000000241 respiratory Effects 0.000 description 1
- 230000003248 secreting Effects 0.000 description 1
- 239000006152 selective media Substances 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 150000003431 steroids Chemical class 0.000 description 1
- 230000004936 stimulating Effects 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000000829 suppository Substances 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000002459 sustained Effects 0.000 description 1
- 235000019722 synbiotics Nutrition 0.000 description 1
- 201000010874 syndrome Diseases 0.000 description 1
- 239000006188 syrup Substances 0.000 description 1
- 235000020357 syrup Nutrition 0.000 description 1
- 230000017423 tissue regeneration Effects 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 230000001131 transforming Effects 0.000 description 1
- 239000006150 trypticase soy agar Substances 0.000 description 1
- 239000007195 tryptone soya broth Substances 0.000 description 1
- 231100000397 ulcer Toxicity 0.000 description 1
- 241001515965 unidentified phage Species 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- 230000017613 viral reproduction Effects 0.000 description 1
- 230000003442 weekly Effects 0.000 description 1
- 230000029663 wound healing Effects 0.000 description 1
- 230000002034 xenobiotic Effects 0.000 description 1
- 239000002676 xenobiotic agent Substances 0.000 description 1
Abstract
ABSTRACT Bifidobacteri um strain AH1206 or mutants or variants thereof are immunomodulatory following oral consumption and are useful in the prophylaxis and/or treatment of inflammatory activity for example undesireable gastrointestinal inflammatory activity such as inflammatory bowel disease.
Description
Probiotic Bifidobacterium strains Introduction The invention relates to a Bzfidobacterium strain and its use as a probiotic bacteria in particular as an immunomodulatory biotherapeutic agent.
The defense mechanisms to protect the human gastrointestinal tract from colonization by intestinal bacteria are highly complex and involve both immunological and non-immunological aspects (1). Innate defense mechanisms include the low pH of the stomach, bile salts, peristalsis, mucin layers and anti-microbial compounds such as Iysozyme (2). immunological mechanisms include specialized lymphoid aggregates, underlying M cells, called peyers patches which are distributed throughout the small intestine and colon (3). Luminal antigens presented at these sites result in stimulation of appropriate T and B cell subsets with establishment of cytokine networks and secretion of antibodies into the gastrointestinal tract (4). In addition, antigen presentation may occur via epithelial cells to intraepithelial lymphocytes and to the underlying lamina propria immune cells (5). Therefore, the host invests substantially in immunological defense of the gastrointestinal tract, However, as the gastrointestinal mucosa is the largest surface at which the host interacts with the external environment, specific control mechanisms must be in place to regulate immune responsiveness to the 100 tons of food which is handled by the gastrointestinal tract over an average lifetime. Furthermore, the gut is colonized by over 500 species of bacteria numbering 10' ‘-10”/g in the colon. Thus, these control mechanisms must be capable of distinguishing non-pathogenic adherent bacteria from invasive pathogens, which would cause significant damage to the host. In fact, the intestinal flora contributes to defense of the host by competing with newly ingested potentially pathogenic micro-organisms.
Bacteria present in the human gastrointestinal tract can promote inflammation. Aberrant immune responses to the indigenous microflora have been implicated in certain disease states, such as inflammatory bowel disease. Antigens associated with the normal flora usually lead to immunological tolerance and failure to achieve this tolerance is a major mechanism of mucosal inflammation (6). Evidence for this breakdown in tolerance includes an increase in antibody levels directed against the gut flora in patients with inflammatory bowel syndrome (IBD).
The present invention is directed towards a Bzfidobacterium strain which has been shown to have immunomodulatory effects, by modulating cytokine levels or by antagonizing and excluding pro-inflammatory micro-organisms from the gastrointestinal tract.
Statements of Invention According to the invention there is provided Bzfidobacerium strain AHI206 (NClMB4l382) or mutants or variants thereof.
The mutant may be a genetically modified mutant. The variant may be a naturally occurring variant of Bzfidobaclerium.
The strain may be a probiotic. It may be in the form of a biologically pure culture.
The invention also provides an isolated strain of Bzfidobacterium NCIMB 41382.
In one embodiment of the invention Bifidobacterium strains are in the form of viable cells.
Alternatively Bzfidobacterium strains are in the form of non-viable cells.
In one embodiment of the invention the Bifidobacterium strains are isolated from infant faeces, the Bzfidobactermm strains being significantly immunomodulatory following oral consumption in humans.
The invention also provides a formulation which comprises the Bzfidobacterium strain of the invention.
In one embodiment of the invention the formulation includes another probiotic material.
In one embodiment of the invention the formulation includes a prebiotic material.
Preferably the formulation includes an ingestible carrier. The ingestible carrier may be a pharmaceutically acceptable carrier such as a capsule, tablet or powder. Preferably the ingestable carrier is a food product such as acidified milk, yoghurt, frozen yoghurt, milk powder, milk concentrate, cheese spreads, dressings or beverages.
In one embodiment of the invention the formulation of the invention further comprises a protein and/or peptide, in particular proteins and/or peptides that are rich in glutamine/glutamate, a lipid, a carbohydrate, a vitamin, mineral and/or trace element.
In one embodiment of the invention the Bzfidobacterium strain is present in the formulation at more than 106 Cfil per gram of delivery system. Preferably the formulation includes any one or more of an adjuvant, a bacterial component, a drug entity or a biological compound.
In one embodiment of the invention the formulation is for immunisation and vaccination protocols.
The invention filI'tl'l6I' provides a Bifidobacterium strain or a formulation of the invention for use as foodstuffs, as a medicament, for use in the prophylaxis and/or treatment of undesirable inflammatory activity, for use in the prophylaxis and/or treatment of undesirable respiratory inflammatory activity such as asthma, for use in the prophylaxis and/or treatment of undesirable gastrointestinal inflammatory activity such as inflammatory bowel disease e.g. Crohns disease or ulcerative colitis, irritable bowel syndrome, pouchitis, or post infection colitis, for use in the prophylaxis and/or treatment of gastrointestinal cancer(s), for use in the prophylaxis and/or treatment of systemic disease such as rheumatoid arthritis, for use in the prophylaxis and/or treatment of autoimmune disorders due to undesirable inflammatory activity, for use in the prophylaxis and/or treatment of cancer due to undesirable inflammatory activity, for use in the prophylaxis of cancer, for use in the prophylaxis and/or treatment of diarrhoeal disease due to undesirable inflammatory activity, such as Clastridium dzfiicile associated diarrhoea, Rotavirus associated diarrhoea or post infective diarrhoea, for use in the prophylaxis and/or treatment of diarrhoeal disease due to an infections agent, such as E. coli.
The invention also provides a Bzfidobacterium strain or a formulation of the invention for use in the preparation of an anti-inflammatory biotherapeutic agent for the prophylaxis and/or treatment of undesirable inflammatory activity or for use in the preparation of anti-inflammatory biotherapeutic agents for the prophylaxis and/or treatment of undesirable inflammatory activity.
In one embodiment of the invention the strain of the invention act by antagonising and excluding proinflammatory micro-organisms from the gastrointestinal tract.
The invention also provides a Bifidobacterium strain or formulation of the invention for use in the preparation of anti-inflammatory biotherapeutic agents for reducing the levels of pro- inflammatory cytokines.
The invention further provides a Bzfidobacterium strain for use in the preparation of anti- inflammatory biotherapeutic agents for modifying the levels of IL-1 0.
The invention may also provide for the use of a Bifidobacterium strain as an anti-infective probiotic due to their ability to antagonise the growth of pathogenic species.
The invention may also provide for the use of a Bzfidobacterium strain in the preparation of a medicament for treating asthma and/or allergy. The medicament may be in a form suitable for inhalation.
The invention may further provide for the use of a Bifidobacterium strain in the preparation of anti-inflammatory biotherapeutic agents for reducing levels of IgE.
We have found that particular strains of Bifidobacrerium elicit immunomodulatory effects in vitro.
The invention may therefore have potential therapeutic value in the prophylaxis or treatment of dysregulated immune responses, such as undesirable inflammatory reactions for example asthma and/or allergy.
Bzfidobacterium are commensal microorganisms. They have been isolated from the microbial flora within the human gastrointestinal tract. The immune system within the gastrointestinal tract cannot have a pronounced reaction to members of this flora, as the resulting inflammatory activity would also destroy host cells and tissue function. Therefore, some mechanism(s) exist whereby the immune system can recognize commensal non-pathogenic members of the gastrointestinal flora as being different to pathogenic organisms. This ensures that damage to host tissues is restricted and a defensive barrier is still maintained.
A deposit of Bzfidobacterium Zongum strain AHl206 was made at the NCIMB on March 15, 2006 and accorded the accession number NCIMB 41382.
The Bzfidobacterium longum may be a genetically modified mutant or it may be a naturally occurring variant thereof.
Preferably the Bzfidobacterium Zongum is in the form of viable cells.
Alternatively the Bzfldobacterium Zongum may be in the form of non-viable cells.
It will be appreciated that the specific Bzfidobacterium strain of the invention may be administered to animals (including humans) in an orally ingestible form in a conventional preparation such as capsules, microcapsules, tablets, granules, powder, troches, pills, suppositories, suspensions and syrups. Suitable formulations may be prepared by methods commonly employed using conventional organic and inorganic additives. The amount of active ingredient in the medical composition may be at a level that will exercise the desired therapeutic effect.
The formulation may also include a bacterial component, a drug entity or a biological compound. in addition a vaccine comprising the strains of the invention may be prepared using any suitable known method and may include a pharmaceutically acceptable carrier or adjuvant.
Throughout the specification the terms mutant, variant and genetically modified mutant include a strain of Bifidobacteria whose genetic and/or phenotypic properties are altered compared to the parent strain. Naturally occurring variant of Bifidobacterium Zongum includes the spontaneous alterations of targeted properties selectively isolated. Deliberate alteration of parent strain properties is accomplished by conventional (in vitro) genetic manipulation technologies, such as gene disruption, conjugative transfer, etc. Genetic modification includes introduction of exogenous and/or endogenous DNA sequences into the genome of a Bifidobacteria strain, for example by insertion into the genome of the bacterial strain by vectors, including plasmid DNA, or bacteriophages.
Natural or induced mutations include at least single base alterations such as deletion, insertion, transversion or other DNA modifications which may result in alteration of the amino acid sequence encoded by the DNA sequence.
The tenns mutant, variant and genetically modified mutant also include a strain of Bifidobacteria that has undergone genetic alterations that accumulate in a genome at a rate which is consistent in nature for all micro-organisms and/or genetic alterations which occur through spontaneous mutation and/or acquisition of genes and/or loss of genes which is not achieved by deliberate (in vitro) manipulation of the genome but is achieved through the natural selection of variants and/or mutants that provide a selective advantage to support the survival of the bacterium when exposed to environmental pressures such as antibiotics. A mutant can be created by the deliberate (in vitro) insertion of specific genes into the genome which do not fundamentally alter the biochemical functionality of the organism but whose products can be used for identification or selection of the bacterium, for example antibiotic resistance.
A person skilled in the art would appreciate that mutant or variant strains of Bifidobacteria can be identified by DNA sequence homology analysis with the parent strain. Strains of Bifidobacteria having a close sequence identity with the parent strain are considered to be mutant or variant strains. A Bifidobacteria strain with a sequence identity (homology) of 96% or more, such as 97% or more, or 98% or more, or 99% or more with the parent DNA sequence may be considered to be a mutant or variant. Sequence homology may be determined using on-line homology “BLAST” http://www.ncbi.nlm.nih,gov/BLAST/. algorithm program, publicly available at Mutants of the parent strain also include derived Bifidobacteria strains having at least 85% sequence homology, such as at least 90% sequence homology, or at least 95% sequence homology to the 16s — 23s intergenic spacer polynucleotide sequence of the parent strain. These mutants may fixrther comprise DNA mutations in other DNA sequences in the bacterial genome.
Brief Description of the Drawings Fig. l is is a BOX PCR (bioanalyzer) barcode profile for B. longum AHl206. Base pair sizes were determined using the Agilent 2100 software; Fig. 2 is a graph illustrating the faecal recovery of B. longum AI-H206 over an 8 day feeding period and demonstrates that AH1206 can survive the murine gastrointestinal tract; Fig. 3 is a bar graph showing the effect of B. longum AH1206 on ll.-10 cytokine production by human PBMCS. Results are expressed as mean +/- SE (n=6); Fig. 4 is a bar graph showing the effect of B. longum AI-U206 feeding on eosinophil recruitment to the lungs of sensitized mice. (A) total number of cells present in bronchoalveolar lavage (BAL) were reduced in AH1206 fed mice; (B) Differential cell counts on BAL revealed that the reduction in cell numbers was primarily in the eosinophil population. (Cell number is expressed on the y—axis (X104); *p<0.05 versus placebo); Fig. 5 A and B are graphs showing the effect of probiotic bacterial strain AHl206 (A) and placebo (B) on total cell numbers in bronchoalveolar lavage fluid following ovalbumin (OVA) challenge in sensitised animals (n=I0/group, * = p<0.05 compared to OVA challenge alone); Fig. 6 A and B are graphs showing the effect of probiotic bacterial strain AH1206 (A) and placebo (B) treatment on airway responsiveness to methacholine, as assessed by changes in enhanced pause (Penh) in ovalbumin (OVA) — sensitised mice 24 hours after intranasal challenge with OVA or saline. Each data paint represents the mean i SEM (n=l0/groups * p = <0.05 compared to OVA alone); Fig. 7 is a graph showing the TNF cytokine level in brochoalveolar lavage (BAL) fluid from ovalbumin (OVA) — sensitised mice. Each column represents the mean :l: SEM (n=l0, * p<0.05 compared to OVA challenged, MRS broth treated control); ‘ Fig. .8A and B are graphs showing the effect of oral treatment with probiotic strain AH1206 an TNF (A) and IFN7 (B) cytokine production from activated splenocytes isolated from OVA ~ sensitised mice (CD3/CD28 stimulated splenocytes). Each column represents the mean dc SEM (n=l0, * p=<0.05 compared to OVA challenge, MRS broth treated control); Fig. 9 is a graph showing that the levels of OVA — specific IgE in serum isolated from mice fed AHl206 probiotic bacteria was significantly lower than the non — probiotic fed controls (**p = <0.0l); Fig. 10 is a graph illustrating the effect of oral treatment of probiotic strain AHl206 on TNFO. production from activated splenocytes isolated from OVA-sensitised mice (CD3/CD28 stimulated splenocytes). The mean is illustrated for each group (* p = <0.05, ** p =<0.0l compared to OVA and CT challenge, MRS broth treated control); Fig. 11 is a graph illustrating that CD4 + CD25+ cells from AHl206 fed animals substantially reduced proliferation (n=10 for all groups except the control, in which n=20); Fig. 12 A and B are graphs showing the percentage of cells in the CD4 + population that are also CD25+, as assessed by flow cytometry (n=11 for the unfed group, n=20 for placebo group, and n=l0 for the AHl206 fed group; and Figure 13. The percentage of CD4/CD25 + cells expressing the transcription factor Foxp3 is significantly upregulated in germ free mice consuming AHl206 (n=8 or 9 per group). *p<0.05 vs placebo.
Fig. 14 is a graph illustrating the stability of probiotic strain AH1206 over 3 months.
Detailed Description We have found that Bzfldobacterium longum strain AHl206 is not only acid and bile tolerant and transits the gastrointestinal tracts but also, surprisingly has immunomodulatory effects, by modulating cytokine levels or by antagonising and excluding pro-inflammatory or immunomodulatory micro-organisms from the gastrointestinal tract. Indeed, consumption of B.
Zongum AHl2i)6 significantly reduces recruitment of disease causing cells to the lungs of a murine asthma model.
The general use of probiotic bacteria is in the form of viable cells. However, it can also be extended to non-viable cells such as killed cultures or compositions containing beneficial factors expressed by probiotic bacteria. This could include thermally killed micro-organisms or micro- organisms killed by exposure to altered pH or subjection to pressure. With non-viable cells product preparation is simpler, cells may be incorporated easily into phannaceuticals and storage requirements are much less limited than viable cells. Lactobacillus casei YIT 9018 offers an example of the effective use of heat killed cells as a method for the treatment and/or prevention of tumour growth as described in US Patent No. US4347240.
It is unknown whether intact bacteria are required to exert an immunomodulatory effect or if individual active components of the invention can be utilized alone. Proinflammatory components of certain bacterial strains have been identified. The proinflammatory effects of gram-negative bacteria are medicated by lipopolysaccharide (LPS). LPS alone induces a proinflammatory network, partially due to LPS binding to the CD14 receptor on monocytes. It is assumed that components of probiotic bacteria possess immunomodulatory activity, due to the effects of the whole cell. Upon isolation of these components, pharmaceutical grade manipulation is anticipated.
IL-10 is produced by T cells, B cells, monocytes and macrophages. This cytokine augments the proliferation and differentiation of B cells into antibody secreting cells. IL-10 exhibits mostly anti-inflammatory activities. It up-regulates IL-IRA expression by monocytes and suppresses the majority of monocyte inflammatory activities. IL-10 inhibits monocyte production of cytokines, reactive oxygen and nitrogen intermediates, MHC class II expression, parasite killing and IL-10 production via a feed back mechanism (7). This cytokine has also been shown to block monocyte production of intestinal collagenase and type IV collagenase by interfering with a PGE2—cAMP dependant pathway and therefore may be an important regulator of the connective tissue destruction seen in chronic inflammatory diseases.
The host response to infection is characterised by innate and acquired cellular and humoral immune reactions, designed to limit spread of the offending organism and to restore organ homeostasis. However, to limit the aggressiveness of collateral damage to host tissues, 21 range of regulatory constraints may be activated. Regulatory T cells (Tregs) serve one such mechanism. These are derived from the thymus but may also be induced in peripheral organs, including the gut mucosa. Deliberate administration of Treg cells suppresses inflammatory disease in a wide range of murine models including experimental autoimmune encephalomyelitis, inflammatory bowel disease, bacterial-induced colitis, collagen-induced arthritis, type 1 diabetes, airway osinophilic inflammation, graft-vs-host disease and organ transplantation. The forkhead transcription factor Foxp3 (forkhead box P3) is selectively expressed in Treg cells, is required for Treg development and function, and is sufficient to induce a Treg phenotype in conventional CD4 cells (19). Mutations in Foxp3 cause severe, multi-organ autoimmunity in both human and mouse. We have described a Bifldobacterium strain that generates CD25 positive/Foxp3 positive T regulatory cells in vivo.
The invention will be more clearly understood from the following examples.
Demonstration of Example 1: Characterisation of bacteria isolated from infant faeces. probiotic traits.
Isolation of Probiotic Bacteria Fresh faeces was obtained from a 2 day old male breast fed infant and serially dilutions were plated on TPY (trypticase, peptone and yeast extract) and MRS (deMann, Rogosa and Sharpe) media supplemented with 0.05% cysteine and mupirocin. Plates were incubated in anaerobic jars (BBL, Oxoid) using CO2 generating kits (Anaerocult A, Merck) for 2-5 days at 37°C. Gram positive, catalase negative rod-shaped or bifurcated/pleomorphic bacteria isolates were streaked for purity on to complex non-selective media (MRS and TPY). Isolates were routinely cultivated in MRS or TPY medium unless otherwise stated at 37°C under anaerobic conditions.
Presumptive Bifidobacterium were stocked in 40% glycerol and stored at -20°C and -80°C.
Following isolation of a pure bifidobacteria strain, assigned the designation Al-H206, microbiological characteristics were assessed and are summarized in Table l below. AH]206 is a gram positive, catalase negative pleomorphic shaped bacterium which is FructosePhoshate Phosphoketolase positive confirming its identity as a bifidobacterium. Using minimal media in which a single carbon source was inserted, AH1206 was able to grow on all carbon sources tested (Glucose, Lactose, Ribose, Arabinose, Galactose, Raffinose, Fructose, Malt Extract, Mannose, Maltose, Sucrose).
Table 1 Physiochemical characteristics of B. Iongum AH1206 Strain Characteristics B.longum AH1206 Gram Stain + Catalase - Motility - F6PPK* + Milk coagulation + °C anaerobic culture 45°C aerobic culture CHO Fermentation: Glucose Lactose Ribose Arabinose Galactose Raflinose Fructose Malt Extract Mannose Maltose Sucrose +++++++++++ * signifies FructosePhoshate Phosphoketolase Assay Species identification s Intergenic spacer (IGS) sequencing was performed to identify the species of bifidobacteria isolated. Briefly, DNA was isolated from AH1206 using 100 pl of Extraction Solution and 25 pt] of Tissue Preparation solution (Sigma, XNAT2 Kit). The samples were incubated for 5 minutes at 95°C and then 100 pl of Neutralization Solultion (XNAT2 kit) was added. Genomic DNA solution was quantified using a Nanodrop (RTM) spectrophotometer and stored at 4°C. PCR was performed using the IGS primers, IGS L: 5’—GCTGGATCACCTCCT"TTC-3’ (SEQ ID NO. 3) which is based on SEQ ID NO. 1 and IGS R: 5’-CTGGTGCCAAGGCATCCA-3’ (SEQ ID NO. 4) which is based on SEQ ID NO. 2. The cycling conditions were 94°C for 3 min (1 cycle), °C for 30 sec, 53°C for 30 sec, 72°C for 30 sec (28 cycles). The PCR reaction contained 4 ul (50ng) of DNA, PCR mix (XNAT2 kit), 0.4 pM IGS L and R primer (MWG Biotech, Germany).
The PCR reactions were performed on an Eppendorf thermocycler. The PCR products (10 ul) were ran alongside a molecular weight marker (100 bp Ladder, Roche) on a 2% agarose EtBr stained gel in TAE, to determine the IGS profile. PCR products of Bifidobacterium (single band) were purified using the Promega Wizard (RTM) PCR purification kit. The purified PCR products were sequenced using the primer sequences (above) for the intergenic spacer region.
Sequence data was then searched against the NCBI nucleotide database to determine the identity of the strain by nucleotide homology. The resultant DNA sequence data was subjected to the NCBI BLAST (http://www.ncbi.nlm.nih.gov/BLAST/). The nearest match to the sequence was identified and standard nucleotide-to-nucleotide homology search engine then the sequences were aligned for comparison using DNASTAR MegAlign software. The sequences obtained can be viewed in the sequence listing in which SEQ ID NO. l is the IGS forward sequence and SEQ ID NO. 2 is the [GS reverse sequence. Searching the NCIMB database revealed that AH1206 has a unique IGS sequence with its closest sequence homology to a Bifidobacterium Zongum.
In order to develop a barcode PCR profile for AHLZ06, PCR was performed using BOX primers (8). The cycling conditions were 94°C for 7 min (1 cycle); 94°C for 1 minute, 65°C for 8 minutes, (30cycles) and 65°C for 16 minutes. The PCR reaction contained 50ng of DNA, PCR mix (XNAT2 kit) and 0.3 uM BOXAIR primer (5’-CTACGGCAAGGCGACGCTGACG-3’) (SEQ ID NO. 5) (MWG Biotech, Germany).
Eppendorf thermocycler.
The PCR reactions were performed on an The PCR products (1 ul) were ran alongside a molecular weight marker (DNA 7500 ladder, Agilent, Germany) using the DNA 7500 LabChip® on the Agilent 2100 Bioanalyzer (RTM) (Agilent, Germany). determined using the Agilent Bioanalyzer (RTM) software where peak number (PCR products) The barcode (PCR product profile) was and size were identified (Fig. 1).
Antibiotic sensitivity profiles Antibiotic sensitivity profiles of the B. longum strain was determined using the ‘disc susceptibility’ assay. Cultures were grown up in the appropriate broth medium for 24-48h spread-plated (l00ul) onto agar media and discs containing known concentrations of the antibiotics were placed onto the agar. Strains were examined for antibiotic sensitivity after 1-2 days incubation at 37°C under anaerobic conditions. Strains were considered sensitive if zones of inhibition of 1mm or greater were seen. The minimum inhibitory concentration (MIC) for each antibiotic was independently assessed. The MIC for clindamycin, vancomycin and metronidazole were 0.32, 0.75 and 0.38 respectively.
Intestinal transit To determine whether Bzfidobacterium langum could survive at low pH values equivalent to those found in the stomach, bacterial cells were harvested from fresh overnight cultures, washed twice in phosphate buffer (pH 6.5) and resuspended in TPY broth adjusted to pH 2.5 (with 1M HCI). Cells were incubated at 37°C and survival measured at intervals of 5, 30, 60 and 120 minutes using the plate count method. AH1206 survived well for 5 minutes at pH 2.5 while no viable cells were recovered after 30 minutes.
Upon exiting the stomach, putative probiotics are exposed to bile salts in the small intestine. In order to determine the ability of B. longum to survive exposure to bile, cultures were streaked on TPY agar plates supplemented with 0.3% (W/v), 0.5%, 1%, 2%, 5%, 7.5% or 10% porcine bile.
B. longum AH1206 growth was observed on plates containing up to 1% bile.
In a murine model, the ability of B. longum AH1206 to transit the gastrointestinal tract was assessed. Mice consumed 1x109 AH1206 daily and faecal pellets were examined for the presence of the fed micro-organism. Detection of AH1206 was facilitated by isolating a spontaneous rifampicin resistanct variant of the bifidobacteria — incorporation of rifampicin in the TPY plates used to assess transit ensured that only the fed rifampicin resistant bifidobacteria was cultured. Faecal samples were collected daily and B. longum transit through the gastrointestinal tract was confirmed (Fig. 2).
Anti-microbial activi The indicator pathogenic micro-organisms used in this study were propagated in the following medium under the following growth conditions: Salmonella typhimurium (37°C, aerobic) in Tryptone Soya broth/agar supplemented with 0.6% yeast extract (TSAYE, Oxoid), Campylobacterjejuni (37°C, anaerobic) and E. coli OlS7:H7 (37°C, anaerobic) on Blood agar medium, Clostridium difiicile (37°C, anaerobic) in reinforced Clostridial medium (RCM, Oxoid). All strains were inoculated into fresh growth medium and grown overnight before being used in experiments.
Antimicrobial activity was detected using the deferred method (9). Briefly, B. longum AH1206 was incubated for 36-48 h. Ten-fold serial dilutions were spread-plated (l00ul) onto TPY agar medium. After overnight incubation, plates with distinct colonies were overlayed with the indicator bacterium. The indicator lawn was prepared by inoculating a molten overlay with 2% (V/v) of an overnight indicator culture which was poured over the surface of the inoculated TPY plates. The plates were re-incubated overnight under conditions suitable for growth of the indicator bacterium. Indicator cultures with inhibition zones greater than 1 mm in radius were considered sensitive to the test bacterium. B. Iongum AH1206 inhibited the growth of all pathogenic organisms tested, with zones of clearing measuring 14, >80, 13.33 and 17 mm for Salmonella typhimurium, Campylobacter jejuni, E. coli Ol57:H7 and Clostridium dzflicile respectively.
Example 2: Cytokine production by PBMCS in response to B. longum.
Peripheral blood mononuclear cells (PBMCS) were isolated from healthy donors by density gradient centrifugation. PBMCS were stimulated with the probiotic bacterial strain for a 72 hour period at 37°C. At this time culture supernatants were collected, centrifuged, aliquoted and stored at -70°C until being assessed for IL-10 levels using cytometric bead arrays (BD BioSciences). AH1206 induced significant secretion of the anti-inflammatory cytokine IL-10 by human PBMCS (Fig. 3) suggesting this strain may be useful as a anti-inflammatory agent in vivo.
Example 3: B. Longum AH1206 attenuates respiratory disease in a murine model of asthma.
This study utilized a Balb/c ovalbumin (OVA) sensitized mouse model of allergic airway inflammation. Mice were sensitized by i.p. injection of OVA and disease was initiated by intranasal challenge with OVA. Twenty-four hours afier the last challenge (day 15), mice were subjected to measurements of airway responsiveness followed by BAL procedure. OVA- sensitized, saline-challenged mice served as controls. Commencing on day l (i.e at time of first OVA sensitization), animals received B. longum AH1206 via a gavaging needle for 14 consecutive days. Animals gavaged with MRS broth served as controls.
Airway inflammation was assessed by inflammatory cell counts in brochoalveolar lavage (BAL) fluid. Cells were removed from BAL fluid by centrifugation and cells were resuspended in phosphate-buffered saline (1 ml). BAL cells were stained with trypan blue, and viable cells were counted using a hemocytometer. Smears of BAL cells were prepared with a Cytospin (Thermo Shandon, Pittsburgh, PA) and stained with HEMA 3 reagent (Biochemical Sciences, Swedesboro, NJ) for differential cell counts, were a total of 200 cells were counted for each lavage. Consumption of B. longum AH1206 significantly reduced the total BAL compared to placebo with the majority of this difference being seen in the eosinophil population (Figure 4).
This study was repeated to further investigate whether the probiotic bacteria strain Bifidobacterium longum AH1206, suppresses allergic responses in an OVA sensitized mouse model of allergic airway inflammation. Briefly, adult male BALB/c mice were sensitized by i.p. injection of OVA day 0 and day 6. On days 12 and l4, mice were challenged intranasally with OVA. Twenty-four hours after the last challenge (day 15), mice were subjected to measurements of airway responsiveness followed by BAL procedure. OVA/alum—sensitized, saline-challenged mice served as controls. Animals received probiotic or placebo throughout the trial. Airway inflammation (cytokine and cell counts) was assessed by inflammatory cell counts in bronchoalveolar lavage (BAL) fluid. Airway responsiveness was also measured using the Buxco whole-body plethysmograph. Splenocytes were also isolated from OVA sensitized mice and were incubated in the presence of anti-CD3 and anti-CD28 antibodies afier which cytokine levels were measured in the supematants by flow cytometry.
B. longum AH1206 treatment resulted in a significant reduction in cells recovered from BAL fluid following OVA challenge, when compared to broth fed animals (Fig. 5). Airway responsiveness was measured and challenge of sensitized mice with OVA resulted in an enhancement of AHR to methacholine when compared with saline-challenged mice. However no modulation of this enhanced airway responsiveness to methacholine, as assessed by changes in enhanced pause was seen (Fig. 6).
BAL cytokine levels were measured by cytometric bead array no significant differences were noted for IL-10, IFN-7, IL-6 and CCL2 levels. AH1206 significantly reduced TNF-on levels (compared to OVA control (Fig.7).
Cytokine levels in splenocyte supernatants were quantified by cytomeric bead array following in vitro OVA or anti-CD3 and anti-CD28 stimulation. Increased IL-10 release from OVA stimulated splenocytes, associated with in vivo OVA sensitization, was not observed in AH1206 fed mice. There was no significant difference in IL-6, TNF and MCP-1 (CCL2) levels. IL-10 release from CD3/CD28 splenocytes was not increased in AH1206 fed animals. However, secretion of the pro-inflammatory cytokines TNF-ct and IFN-7 were significantly reduced in the spienocyte culture supematants of AHl206-fed animals (Fig. 8). No significant changes were noted for the other cytokines measured.
Example 4: OVA feeding model The aim of this study was to investigate whether the probiotic bacteria, Bzfidobacterium longum AHl206 suppresses allergic responses in an ovalbumin (OVA)-induced allergy mouse model.
BALB/c mice were divided into groups (8/group) and fed Placebo, Bzfidabacterium longum AHl206 and Distilled H20 for four weeks.
Ovalbumin and Cholera Toxin in 300uls PBS excluding one of the dH2O groups which were All mice were oraily gavaged weekly with orally gavaged with 300uls PBS only as a control. After four weeks of treatment, a blood sample from each mouse was collected via facial vein puncture and a subsequent ELISA performed to measure OVA-specific IgE levels. The spleens and mesenteric lymph node cells were isolated and stimulated in vitro with LPS and antiCD3/CD28 and the immunodominant OVA peptide. Th1 and Th2 cytokines were measured by cytomeric CBA.
There was significantly less OVA-specific IgE induced in the probiotic fed group compared to the placebo and positive control groups (Fig. 9). The negative control group and the AHI206 fed groups were not different suggesting that AI-11206 feeding completely inhibited the induction of an OVA-Specific IgE response. Statistics were done using the unpaired T test.
Splenocytes were isolated from probiotic, placebo and dH2O fed BALB/c mice and either left unstimulated or stimulated with LPS, antiCD3/CD28 and the immunodominant OVA peptide and then analyzed for cytokine production of TNF-ct, IL-2, IFN-y, IL-4 and IL-5 by Thl/Th2 cytometric bead array. Cytokine results are summarized in Table 2.
Table 2 Cytokine summary Unstimulated Splenocytes Strain TNF-alpha IL-2 IFN-gamma IL-4 IL-5 AH1206 LPS-Stimulated Splenocytes Strain TNF-alpha IL-2 IFN-gamma IL-4 IL-5 AH1206 t t rt NC rt NC antiCD3/CD28-stimulated splenocytes Strain TNF-alpha IL-2 IFN-gamma IL-4 IL-5 AH1206 I rtNC ‘I’ rtNC ‘I‘nNc ‘I’nNc PC lrtPC lnpc lnpc In 1nPC RT = Relative to NC= Negative control (water fed, PBS challenged) PC= Positive control (water fed, OVA and CT challenge In un-stimulated splenocytes, no alterations were observed compared to control animals. TNF-or and IFN-ry release from LPS stimulated splenocytes was significantly greater for AH1206 fed animals compared to the negative controls but these levels were consistent with those observed with the OVA sensitized and cholera toxin administered positive controls. CD3/CD28 stimulation revealed profound alterations in lymphocyte signaling in the probiotic fed group.
AH1206 fed animals secreted significantly less TNF-or compared to the positive controls but levels were higher compared to negative controls (Fig. I0). AHl206 fed animals had significantly lower levels of IFN-7, IL-2, IL-4 and IL~5 compared to the non-probiotic fed positive controls.
’ Example 5: Treg effector model This study investigated the effect of probiotic consumption on regulatory T cell number and activity in healthy mice. BALB/c mice (10/group) were fed Bzfidobacterium longum AHl206 or placebo for three weeks. Following probiotic/placebo consumption, CD4+CD25+ T-regulatory cells were isolated and their in vitro suppressive activity was detennined by measuring proliferation of anti-CD3/CD28 stimulated CFSE-labelled CD4+ responder T cells using flow cytometry. CD4+ responder T cells were co-incubated with CD4+CD25- T cells as a control.
The percentage of CD4+CD25+ cells (Regulatory T cells) in murine splenocytes that are also FoxP3 positive was determined in the spleens of probiotic or placebo-fed mice.
The % of CD4+ cells that proliferated when co-incubated with CD4+CD25+ cells from the probiotic/placebo fed mice was compared to the % of CD4+ cells that proliferated when co- incubated with CD4+CD25- cells from the same trial mouse. In each case, T cell proliferation was less in cultures containing CD4+CD25+ cells compared in cultures containing CD4 cells alone and depleted of the CD25+ cells (Fig. l 1).
The % of cells in the CD4+ population that were also CD25+ was determined (Fig. 12). The Bifidobacterium longum AHl206 fed group had significantly more CD4+ T cells that were CD25+ (i.e. T-Regulatory cells) than their placebo-fed counterparts (p= 0.008l). This suggests that the % of T—Regulatory cells within the CD4+ population was increased significantly by feeding with AH1206.
The number of CD4+CD25+F0xP3+ cells in the whole splenocyte populations of probiotic or placebo-fed mice was also determined. The number of CD4+CD25+ T-Regulatory cells expressing FoxP3 was unchanged in the spleens of probiotic fed mice relative to placebo or unfed mice.
Example 6: Germ free model Germ free mice were purchased at 6 weeks of age and maintained in the germ-free unit at the biologicalservices unit in UCC. Animals consumed the probiotic strain Bifidobacterium longum AHl206 for '14 days or remained germ free. Induction of T regulatory cells was assessed by flow cytometry.
The numbers of CD4+CD25+Foxp3+ cells in the spleen of AHl206 fed germ-free animals was significantly increased following feeding (Fig. I3). Total CD3/CD4 or CD3/CD8 counts remained unaltered.
Example 7: Stability results The stability of probiotic strain-AHl206 was assessed over 3 months at 30°C (Fig. I3).
These results indicate that Lactobacillus rahmosus GG was a poor performer over the test period with a 2 log drop over the 3 month period whereas AH 1206 was quite stable with no viability loss recorded over the period.
Immunomodulation The human immune system plays a significant role in the aetiology and pathology of a vast range of human diseases. Hyper and hypo-immune responsiveness results in, or is a component of, the majority of disease states. One family of biological entities, termed cytokines, are particularly important to the control of immune processes. Pertubances of these delicate cytokine networks are being increasingly associated with many diseases. These diseases include but are not limited to inflammatory disorders, immunodeficiency, inflammatory bowel disease, irritable bowel syndrome, cancer (particularly those of the gastrointestinal and immune systems), diarrhoea] disease, antibiotic associated diarrhoea, paediatric diarrhoea, appendicitis, autoimmune disorders, multiple sclerosis, AIzheimer’s disease, rheumatoid arthritis, coeliac disease, diabetes mellitus, organ transplantation, bacterial infections, viral infections, fungal infections, periodontal disease, urogenital disease, sexually transmitted disease, HIV infection, HIV replication, HIV associated diarrhoea, surgical associated trauma, surgical-induced metastatic disease, sepsis, weight loss, anorexia, fever control, cachexia, wound healing, ulcers, gut barrier function, allergy, asthma, respiratory disorders, circulatory disorders, coronary heart disease, anaemia, disorders of the blood coagulation system, renal disease, disorders of the central nervous system, hepatic disease, ischaemia, nutritional disorders, osteoporosis, endocrine disorders, epidermal disorders, psoriasis and acne vulgaris. The effects on cytokine production are specific forthe probiotic strain—examined. Thus specific probiotic strains may be selected for normalising an exclusive cytokine imbalance particular for a specific disease type.
Customisation of disease specific therapies can be accomplished using either a single strain of AHl206 or mutants or variants thereof or a selection of these strains.
Immune Education The enteric flora is important to the development and proper function of the intestinal immune system. In the absence of an enteric flora, the intestinal immune system is underdeveloped, as demonstrated in germ free animal models, and certain functional parameters are diminished, such as macrophage phagocytic ability and immunogiobulin production (10). The importance of the gut flora in stimulating non-damaging immune responses is becoming more evident. The increase in incidence and severity of allergies in the western world has been linked with an increase in hygiene and sanitation, concomitant with a decrease in the number and range of infectious challenges encountered by the host. This lack of immune stimulation may allow the host to react to non-pathogenic, but antigenic, agents resulting in allergy or autoimmunity.
Deliberate consumption of a series of non-pathogenic immunomodulatory bacteria would provide the host with the necessary and appropriate educational stimuli for proper development and control of immune fimction.
Inflammation Inflammation is the term used to describe the local accumulation of fluid, plasma proteins and white blood cells at a site that has sustained physical damage, infection or where there is an ongoing immune response. Control of the inflammatory response is exerted on a number of levels (1 1). prostaglandins, reactive intermediates and leukotrienes. Cytokines are low molecular weight The controlling factors include cytokincs, hormones (e.g. hydrocortisone), biologically active proteins that are involved in the generation and control of immunological and inflammatory responses, while also regulating development, tissue repair and haematopoiesis.
They provide a means of communication between leukocytes themselves and also with other cell types. Most cytokines are pleiotrophic and express multiple biologically overlapping activities.
Cytokine cascades and networks control the inflammatory response rather than the action of a particular cytokine on a particular cell type (12). Waning of the inflammatory response results in lower _concentrations of the appropriate activating signals and other inflammatory mediators leading to the cessation of the inflammatory response. TNFOL is a pivotal proinflammatory cytokine as it initiates a cascade of cytokines and biological effects resulting in the inflammatory state. Therefore, agents which inhibit TNFOL are currently being used for the treatment of inflammatory diseases, e.g. infliximab.
Pro-inflammatory cytokines are thought to play a major role in the pathogenesis of many inflammatory diseases, including inflammatory bowel disease (IBD). Current therapieswfor treating IBD are aimed at reducing the levels of these pro-inflammatory cytokines, including IL- 8 and TNFOL. Such therapies may also play a significant role in the treatment of systemic inflammatory diseases such as rheumatoid arthritis.
The strains of the present invention may have potential application in the treatment of a range of inflammatory diseases, particularly if used in combination with other anti-inflammatory therapies, such as non-steroid anti-inflammatory drugs (NSAIDS) or Infliximab.
Cytokines and Cancer The production of multifunctional cytokines across a wide spectrum of tumour types suggests that significant inflammatory responses are ongoing in patients with cancer. It is currently unclear what protective effect this response has against the growth and development of tumour cells in viva. However, these inflammatory responses could adversely affect the tumour-bearing host. Complex cytokine interactions are involved in the regulation of cytokine production and cell proliferation within tumour and normal tissues (13, 14). It has long been recognized that weight loss (cachexia) is the single most common cause of death in patients with cancer and initial malnutrition indicates a poor prognosis. For a tumour to grow and spread it must induce the formation of new blood vessels and degrade the extracellular matrix. The inflammatory response may have significant roles to play in the above mechanisms, thus contributing to the decline of the host and progression of the tumour. Due to the anti-inflammatory properties of Bzfidobacterium longum irzfantis these bacterial strains they may reduce the rate of malignant cell transformation. Furthermore, intestinal bacteria can produce, from dietary compounds, substances with genotoxic, carcinogenic and tumour-promoting activity and gut bacteria can activate pro-carcinogens to DNA reactive agents (15). In general, species of Bzfidobacterium have low activities of xenobiotic metabolizing enzymes compared to other populations within The introduction of probiotic organisms is accomplished by the ingestion of the micro-organism the gut such as bacteroides, eubacteria and clostridia. Therefore, increasing the number of Bifidobacterium bacteria in the gut could beneficially modify the levels of these enzymes.
Vaccine/Drug Deliveg The majority of pathogenic organisms gain entry via mucosal surfaces. Efficient vaccination of these sites protects against invasion by a particular infectious agent. Oral vaccination strategies have concentrated, to date, on the use of attenuated live pathogenic organisms or purified encapsulated antigens (16). Probiotic bacteria, engineered to produce antigens from an infectious agent, in vivo, may provide an attractive alternative as these bacteria are considered to be safe for human consumption (GRAS status).
Murine studies have demonstrated that consumption of probiotic bacteria expressing foreign antigens can elicit protective immune responses. The gene encoding tetanus toxin fragment C (TTFC) was expressed in Lactococcus lactis and mice were immunized via the oral route. This system was able to induce antibody titers significantly high enough to protect the mice from lethal toxin challenge. ln addition to antigen presentation, live bacterial vectors can produce bioactive compounds, such as immunostimulatory cytokines, in vivo. L. Zaclis secreting bioactive human IL-2 or IL-6 and TTFC induced 10-15 fold higher serum lgG titres in mice immunized intranasally (17). However, with this particular bacterial strain, the total IgA level was not increased by coexpression with these cytokines. Other bacterial strains, such as Streptococcus gordonii, are also being examined for their usefulness as mucosal vaccines.
Recombinant S. gordonii colonizing the murine oral and vaginal cavities induced both mucosal and systemic antibody responses to antigens expressed by this bacterial (18). Thus oral immunization using probiotic bacteria as vectors would not only protect the host from infection, but may replace the immunological stimuli that the pathogen would normally elicit thus contributing to the immunological education of the host.
Prebiotics in a suitable carrier. It would be advantageous to provide a medium that would promote the growth of these probiotic strains in the large bowel. The addition of one or more oligosaccharides, polysaccharides, or other prebiotics enhances the growth of lactic acid bacteria in the gastrointestinal tract. Prebiotics refers to any non-viable food component that is specifically fermented in the colon by indigenous bacteria thought to be of positive value, e.g. bifidobacteria, lactobacilli. Types of prebiotics may include those that contain fructose, xylose, soya, galactose, glucose and mannose. The combined administration of a probiotic strain with one or more prebiotic compounds may enhance the growth of the administered probiotic in vivo resulting in a more pronounced health benefit, and is termed synbiotic.
Other active ingredients It will be appreciated that the probiotic strains may be administered prophylactically or as a method of treatment either on its own or with other probiotic and/or prebiotic materials as described above. In addition, the bacteria may be used as part of a prophylactic or treatment regime using other active materials such as those used for treating inflammation or other disorders especially those with an immunological involvement. Such combinations may be administered in a single formulation or as separate formulations administered at the same or different times and using the same or different routes of administration.
The invention is not limited to the embodiment herein before described which may be varied in detail.
References.
McCracken V.J. and Gaskins H.R. Probiotics and the immune system. In: Probiotics a critical review, Tannock, GW (ed), Horizon Scientific Press, UK. 1999, p.85-113.
Savage D.C. Interaction between the host and its microbes. In: Microbial Ecology of the Gut, Clark and Bauchop (eds), Academic Press, London. 1977, p.277-310.
Kagnoff M,F. Immunology of the intestinal tract. Gastroenterol. 80. ; 105 (5); 1275- Lamm M.E. Interaction of antigens and antibodies at mucosal surfaces. Ann. Rev.
Microbiol. 1997; 51: 3 I 1-40.
Raychaudhuri S., Rock KL. Fully mobilizing host defense: building better vaccines. Nat biotechnol, I998; 16: 1025-31.
Stallmach A., Strober W, MacDonald TT, Lochs H, Zeitz M. Induction and modulation of gastrointestinal inflammation. Immunol. Today, 1998; I9 (10): 438-41. de Waal Malefyt R, Haanen J, Spits H, Roncarolo MG, te Velde A, Figdor C, Johnson K, Kastelein R, Yssel H, de Vries JE. Interleukin 10 (IL-10) and viral IL-10 strongly reduce antigen-specific human T cell proliferation by diminishing the antigen-presenting capacity of monocytes via downregulation of class 11 major histocompatibility complex expression. J Exp Med 1991 Oct 1;174(4):915-24.
Masco L, Huys G, Gevers D, Verbrugghen L, Swings J. Identification of Bifidobacterium species using rep-PCR fingerprinting. Syst Appl Microbiol. 2003 Nov;26(4):557-63.
PMID: 14666984.
Tagg, JR, Dajani, AS, Wannamaker, LW. Bacteriocins of Gram positive bacteria.
Bacterial Rev. 1976; 40: 722-756.
Crabbe P.A., H. Bazin, H. Eyssen, and J.F. Heremans. The nomial microbial flora as a major stimulus for proliferation of plasma cells synthesizing IgA in the gut. The germ free intestinal tract. Into. Arch. Allergy Appl Immunol, 1968; 34: 362-75.
Henderson B., Poole, S and Wilson M. 1998. health and disease. Portland Press, 79-130.
In “Bacteria-Cytokine interactions in Arai KI, Lee F, Miyajima A, Miyatake S, Arai N, Yokota T. Cytokines: coordinators of immune and inflammatory responses. Annu Rev Biochem 1990;59:783-836.
McGee DW, Bamberg T, Vitkus SJ, McGhee JR. A synergistic relationship between TNF-alpha, IL-1 beta, and TGF-beta I on IL-6 secretion by the IEC-6 intestinal epithelial cell line. Immunology 1995 Sep;86(l):6-I 1.
Wu S, Meeker WA, Wiener JR, Berchuck A, Bast RC Jr, Boyer CM. Transfection of ovarian cancer cells with tumour necrosis factor alpha (TNF-alpha) antisense mRNA abolishes the proliferative response to interleukin-I (IL-I) but not TNF-alpha. Gynecol Oncol 1994 Apr; 53(1):59-63.
Rowland I.R. Toxicology of the colon: role of the intestinal microflora. In: Gibson G.R. (ed). Human colonic bacteria: role in nutrition, physiology and pathology, I995, pp I55- I74. Boca Raton CRC Press.
Walker, R.I. New strategies for using mucosal vaccination to achieve more effective immunization. Vaccine, 1994; I2: 387-400.
Steidler L., K. Robinson, L. Chamberlain, K.M Scholfield, E. Remaut, R.W.F. Le Page and J.M. Wells. Mucosal delivery of murine interleukin-2 (IL-2) and IL-6 by recombinant strains of Lactococcus Iactis coexpressing antigen and cytokine. Infect.
Immun., 1998; 66:3183~9.
I Medaglini 13., G. Pozzi, T.P. King and V.A. Fischetti. Mucosal and systemic immune responses to a recombinant protein expressed on the surface of the oral commensal bacterium Streptococcus gordonii afier oral colonization. Proc. Natl. Acad. Sci. USA, ;92:6868-72 McCracken V.J. and Gaskins H.R, ‘Probiotics a critical review’, Horizon Scientific Press, UK 1999, p.278.
. Marson, A., Kretschmer, K., Frampton, G.M., Jacobsen, E.S., Polansky, J.K., Maclsaac, K.D., Levine, S.S., Fraenkel, E., von Boehmer, H and Young, R.A. Foxp3 occupancy and regulation of key target genes during T-cell stimulation. Letters to Nature, 2007.
Claims (1)
1. Claims An isolated strain of Bzfidobacterium deposited at NCIMB with accession number 41382. A Brfidobacrerium strain as claimed in claim 1 in the form of viable cells. A Bafldobacterium strain as claimed in claim 1 in the form of non-viable cells. A formulation which comprises a Bzfidobacterium strain as claimed in any of claims I to A formulation as claimed in claim 4 which further comprises another probiotic material. A formulation as claimed in any of claims 4 or 5 which further comprises a prebiotic material. A formulation as claimed in any of claims 4 to 6 further comprising an ingestable carrier. A formulation as claimed in claim 7 wherein the ingestable carrier is a pharmaceutically acceptable carrier such as a capsule, tablet or powder. A formulation as claimed in claim 7 wherein the ingestable carrier is a food product such as acidified milk, yoghurt, frozen yoghurt, milk powder, milk concentrate, cheese spreads, dressings or beverages. A formulation as claimed in any of claims 4 to 9 which fiirther comprises a protein and/or peptide, in particular proteins and/or peptides that are rich in glutamine/glutamate, a lipid, a carbohydrate, a vitamin, mineral and/or trace element. A formulation as claimed in claims 4 to 10 wherein the Bzfidobacterium strain is present in an amount of more than 106 cfu per gram of the formulation. A formulation as claimed in claims 4 to 1 1 which further comprises an adjuvant. A formulation as claimed in claims 4 to 12 which further comprises a bacterial component. A formulation as claimed in claims 4 to 13 which further comprises a drug entity. A formulation as claimed in claims 4 to 14 which further comprises a biological compound. A foodstuff comprising a Bifidobacterium strain as claimed in any of claims 1 to 3 or a formulation as claimed in any of claims 4 to 15. A Bifidobacterium strain as claimed in any of claims 1 to 3 or a formulation as claimed in any of claims 4 to 15 for use as a medicament. A Bzfidobacterium strain as claimed in any of claims 1 to 3 or a formulation as claimed in any of claims 4 to 15 for use in the prophylaxis and/or treatment of undesirable inflammatory activity. A Bzfidobacterium strain as claimed in any of claims 1 to 3 or a formulation as claimed in any of claims 4 to 15 for use in the prophylaxis and/or treatment of undesirable gastrointestinal inflammatory activity such as inflammatory bowel disease eg. Crohns disease or ulcerative colitis, irritable bowel syndrome; pouchitis; or post infection colitis. A Bgfidobacterium strain as claimed in any of claims 1 to 3 or a formulation as claimed in any of claims 4 to 15 for use in the prophylaxis and/or treatment of autoimmune disorders due to undesirable inflammatory activity. A Bifidobacterium strain as claimed in any of claims 1 to 3 or a formulation as claimed in any of claims 4 to 15 for use in the prophylaxis and/or treatment of diarrhoeal disease due to undesirable inflammatory activity, such as Clostridium dzflicile associated diarrhoea, Rotavirus associated diarrhoea or post infective diarrhoea or diarrhoeal disease due to an infectious agent, such as E.colz'. An anti-inflammatory biotherapeutic agent comprising a Bzfidobacterium strain as claimed in any of claims 1 to 3 or a formulation as claimed in any of claims 4 to 15 for use in the prophylaxis and/or treatment of undesirable inflammatory activity. A Bifidobacterium strain as claimed in any of claims 1 to 3 for use in the prophylaxis andz’or treatment of allergy, asthma or respiratory disorders. A Bzfidobacterium strain as claimed in any of claims 1 to 3 for use in the prophylaxis andIor treatment of allergic airway inflammation. A Bifidobacterium strain as claimed in any of claims 1 to 3 or a formulation as claimed in any of claims 4 to 15 for use in the prophylaxis and/or treatment of asthma and/or allergy. Use as claimed in claim 25 wherein the strain or formulation is in a form suitable for inhalation. An anti-inflammatory biotherapeutic agent comprising a Bzfidobacterium strain as claimed in any of claims 1 to 3 or a formulation as claimed in any of claims 4 to l5 for use in reducing the levels of pro inflammatory cytokines. An anti-inflammatory biotherapeutic agent comprising a Bzfidobacterium strain as claimed in any of claims 1 to 3 or a formulation as claimed in any of claims 4 to l5 for use in reducing levels of IgE.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
IE2008/0245A IE86045B1 (en) | 2008-03-28 | Probiotic bifidobacteria strains |
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
IEIRELAND28/03/20072007/0218 | |||
IE20070218 | 2007-03-28 | ||
IE2008/0245A IE86045B1 (en) | 2008-03-28 | Probiotic bifidobacteria strains |
Publications (2)
Publication Number | Publication Date |
---|---|
IE20080245A1 IE20080245A1 (en) | 2008-12-24 |
IE86045B1 true IE86045B1 (en) | 2012-08-15 |
Family
ID=
Similar Documents
Publication | Publication Date | Title |
---|---|---|
AU2008231466B2 (en) | Probiotic Bifidobacterium strains | |
US8709398B2 (en) | Probiotic Bifidobacterium strains | |
US11225641B2 (en) | Probiotic Bifidobacterium strain | |
US20030092163A1 (en) | Probiotic bifidobacterium strains | |
US20120207713A1 (en) | Probiotic bifidobacterium strains | |
IE86045B1 (en) | Probiotic bifidobacteria strains | |
IE20080245A1 (en) | Probiotic Bifidobacteria strains | |
IE86044B1 (en) | Probiotic bifidobacteria strains | |
IE20080244A1 (en) | Probiotic Bifidobacteria strains | |
ZA200400555B (en) | Probiotic bifidobacterium strains. |