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Thiruttuu Voluntur THIRUTTUUUS009987313B2 VOLUNTUR (12 ) United States Patent ( 10 ) Patent No. : US 9 , 987 ,313 B2 Lorca (45 ) Date of Patent: * Jun . 5 , 2018 ( 54 ) LACTOBACILLUS SUPPLEMENT FOR (52 ) U .S . CI. ALLEVIATING TYPE 1 DIABETES CPC . .. .. A61K 35 / 747 (2013 . 01 ) ; A23C 9 / 1234 ( 2013 .01 ) ; A23C 19 /0323 ( 2013 .01 ) ; (71 ) Applicant: University of Florida Research (Continued ) Foundation , Incorporated , Gainesville , (58 ) Field of Classification Search FL (US ) None See application file for complete search history . (72 ) Inventor: Graciela Liliana Lorca , Gainesville , FL (US ) (56 ) References Cited (73 ) Assignee : University of Florida Research U . S . PATENT DOCUMENTS Foundation , Incorporated , Gainesville , 5 ,556 ,785 A 9 /1996 Kishida FL (US ) 6 ,214 , 336 B1 4 / 2001 Bukowska et al . (Continued ) ( * ) Notice : Subject to any disclaimer, the term of this patent is extended or adjusted under 35 FOREIGN PATENT DOCUMENTS U . S . C . 154 ( b ) by 308 days. JP 4044975 B 2 / 1997 This patent is subject to a terminal dis WO WO 2004 /076615 A2 9 / 2004 claimer . WO WO 2008 / 141989 11/ 2008 (21 ) Appl. No. : 14/ 656 ,209 OTHER PUBLICATIONS Kin Kwan Lai, Graciela L . Lorca, and Claudio F . Gonzalez , (22 ) Filed : Mar. 12 , 2015 Biochemical Properties of Two Cinnamoyl Esterases Purified from a Lactobacillus johnsonii Strain Isolated from Stool Samples of (65 ) Prior Publication Data Diabetes -Resistant Rats , 2009 , Applied and Environmental Micro US 2015 /0182567 A1 Jul. 2 , 2015 biology , vol. 75 , No. 15, p . 5018 - 5024. * (Continued ) Related U . S . Application Data Primary Examiner — Renee Claytor Assistant Examiner — Trent R Clarke (63 ) Continuation of application No . 13 /144 ,028 , filed as ( 74 ) Attorney , Agent, or Firm — Timothy H . Van Dyke ; application No . PCT /US2010 / 024575 on Feb . 18 , Beusse, Wolter , Sanks & Maire PLLC 2010 , now Pat . No . 9 , 474 , 773 . (57 ) ABSTRACT ( Continued ) Isolated Lactobacillus strains are useful in preventing or (51 ) Int . Cl. delaying the development of Type 1 Diabetes (T1D ) . A A61K 35 / 74 ( 2015 .01 ) probiotic composition comprising the Lactobacillus strains A61K 35 /747 ( 2015 .01 ) and use of the composition in T1D prevention are provided . (Continued ) 4 Claims, 12 Drawing Sheets (a3uelopo www Relativguptevan . INFO . US 9 , 987, 313 B2 Page 2 Related U . S . Application Data Calcinaro, F . et al ., " Oral probiotic administration induces interleukin - 10 production and prevents spontaneous autoimmune diabetes in the non -obese diabetic mouse ,” Diabetologia , 2005 , (60 ) Provisional application No . 61/ 153 ,516 , filed on Feb . 48 : 1565 - 1575 . 18 , 2009, provisional application No . 61/ 297 , 480 , Like , A . A . et al. , “ Influence of environmental viral agents on filed on Jan . 22 , 2010 . frequency and tempo of diabetes mellitus in BB /Wor rats ," Diabe tes , 1991, 40 ( 2 ) : 259 - 262 , abstract . (51 ) Int. CI. Matsuzaki, Takeshi et al . , " Prevention of onset in an insulin A23C 9 / 123 dependent diabetes mellitus model , NOD mice , by oral feeding of ( 2006 .01 ) Lactobacillus casei ,” Acta Pathologica , Microbiologica et A23C 19 / 032 ( 2006 .01 ) Immunologica Scandinavica ( APMIS ) , 1997 , 105 :643 -649 . A23G 9 / 36 ( 2006 . 01 ) McInerney , M . F . et al. , “ Prevention of insulitis and diabetes onset by C12N 1 /20 ( 2006 .01 ) treatment with complete Freund ' s adjuvant in NOD mice, " Diabe A23L 33 / 135 ( 2016 .01 ) tes , 1991, 40 ( 6 ) : 715 -725 , abstract . A61K 35 /00 ( 2006 .01 ) Sadelain , M . W . et al. , “ Prevention of type I diabetes in NOD mice (52 ) U . S . CI. by adjuvant immunotherapy, " Diabetes , 1990 , 39 ( 5 ) : 583 - 589 , abstract . CPC . .. A23G 9 /363 ( 2013 .01 ) ; A23L 33 / 135 Sadelain , Michael W . J . et al. , “ Prevention of Diabetes in the BB Rat (2016 .08 ) ; C12N 1 / 20 ( 2013 .01 ) ; A23V by Early Immunotherapy Using Freund ' s Adjuvant, ” Journal of 2002 /00 ( 2013 .01 ) ; A23Y 2220 /43 ( 2013 .01 ) ; Autoimmunity , 1990 , 3 :671 -680 . A23Y 2220 / 71 (2013 .01 ) ; A61K 2035 / 115 Salminen , Seppo et al. , “ Intestinal colonistation , microbiota and ( 2013 . 01 ) future probiotics? , ” Asia Pacific Journal of Clinical Nutrition , 2006 , 15 ( 4 ) :558 - 562 . ( 56 ) References Cited Satoh , Jo et al. , “ Treatment With Streptococcal Preparation ( OK 432 ) Suppresses Anti - Islet Autoimmunity and Prevents Diabetes in U . S . PATENT DOCUMENTS BB Rats, ” Diabetes , Sep . 1988 , 37 : 1188 - 1194 . Schwartz , R . F . et al. , “ Comment on : Brugman S . et al . ( 2006 ) 6 , 808 , 703 B2 10 / 2004 Park et al . Antibiotic treatment partially protects against type 1 diabetes in the 7 , 001, 756 B1 2 / 2006 Hsu et al. 7 , 189 ,390 B2 3 / 2007 Zink et al. Bio - Breeding diabetes -prone rat . Is the gut flora involved in the 7 , 390 , 519 B26 / 2008 Collins et al. development of type 1 diabetes ? Diabetologia , 49: .2105 - 2108 ," 2003 / 0235559 Al 12 / 2003 Sobol et al . Diabetologia , 2007 , 50 :220 -221 . 2005 /0100531 Al 5 / 2005 Bienenstock Shori, Amal B ., “ Antioxidant activity and viability of lactic acid 2006 / 0270020 AL 11/ 2006 Boileau et al . bacteria in soybean -yogurt made from cow and camel milk , " 2008 /0193485 AL 8 /2008 Gorbach et al. Journal of Taibah University for Science , 2013 , 7 : 202 - 208 . Vaarala , Outi et al ., “ The “ Perfect Storm ” for Type 1 Diabetes : The Complex Interplay Between Intestinal Microbiota , Gut Permeabil OTHER PUBLICATIONS ity , and Mucosal Immunity , " 2008 , Diabetes , 57 ( 10 ) :2555 -2562 . Wicker , Linda S . et al. , “ Genetic Control of Diabetes and Insulitis Kenneth Lau , Patrick Benitez , Alexandria Ardissone , Tenisha Wil in the Nonobese Diabetic (NOD ) Mouse , ” The Journal of Experi son , Erin Collins, Graciela Lorca, Nan Li, Dhyana Sankar , Clive mental Medicine , 1987 , 165 : 1639 - 1654 . Wasserfall, Josef Neu , Mark Atkinson , Desmond Shatz , Eric Yadav , Hariom et al. , “ Antidiabetic effect of probiotic dahi con Triplett , and Joseph Larkin , Inhibition of Type 1 Diabetes Corre taining Lactobacillus acidophilus and Lactobacillus casei in high lated to a Lactobacillus johnsonii N6 . 2 -Mediated Th17 Bias , 2011 , fructose fed rats, ” Nutrition , 2007 , 23 :62 -68 . J Immunol , vol . 186 , pp . 3538 - 3546 . * Yamano , Toshihiko et al. , “ Effects of the probiotic strain Lactobacil Brugman , S . et al . , “ Antibiotic treatment partially protects against lus johnsonii strain Lal on autonomic nerves and blood glucose in type 1 diabetes in the Bio - Breeding diabetes -prone rat. Is the gut rats ,” Life Sciences, 2006 , 79 : 1963 - 1967 . flora involved in the development of type 1 diabetes ? " , Diabetologia , 2006 , 49: 2105 - 2108 . * cited by examiner U . S . Patent Jun . 5 , 2018 Sheet 1 of 12 US 9 ,987 ,313 B2 [bpl 10382 7000 3000N 0097 0001 002– 008_2 081 Ladder?bp) NANI NAOOSE Ladder L 6 y=ExtractedBands DRBB- WWW 5 BB-DR 4 FIG.1 BB-DR 3 BB-DP 2 XXXXXXXXXXXXXXXX -DPBB 1 XXXXXXXX DPBB- *- LadderLadder www WWW. www | | | | | IT [bo]tbp 08801 0002 0002OOST 0001 084 500 300 01011 U . S . Patent Jun . 5 , 2018 Sheet 2 of 12 US 9 , 987, 313 B2 FIG . LA2A - 1 PCA - Qualitative - A . : . : . : . : . : . : . : . : . : . - - ' . - : 66%.explained16variationP3 1. * . .: Pevariation explained 17 .10 % . 0 :40 0: 20 002 : : -0 . 20 . P2 variation explained 16 .89 % 0. 40 FIG . 2A - 2 PCA - Quantitative - A 0 .50 0 . 40 0 . 30 13%.8explainedVariationP3 0 :20 0 : 10 0 .00 - 0 . 10 -020 op P1variation explained 32 ,7 % 0. 00 0. 00 0. 10 0. 20 0. 30 0. 40 0. 50 0. 30 p20. 20 variation explained 14. 9 % BioBreeding Diabetes prone BioBreeding Diabetes resistant U . S . Patent Jun . 5 , 2018 Sheet 3 of 12 US 9 ,987 , 313 B2 FIG . 2B - 1 PCA - Qualitative - B i 0 . 20 : . : : 0 , 10 88%explained6.variationP3 0 :00 - 0 . 10 ' + + . 1 - 0 . 20 . 9 *. :. *> : ••••••• . " ** * * * . ' ' - 0 . 30 0 . : . : . * - * . ' . * " . * . ' ' * . * * * . - 0 , 40 erede erweitern P1variation explained 9,88 % 0. 10 0. 00 0. 10 0. 20 P2variation explained 8 .09 % : -0 . 30 FIG . 2B - 2 PCA Quantitative - B 00%,explained8variationP3 P1variation Oexplained ooooo 20 .56 % 0.05 0. 10 0. 15 020 010 005 000 0. 15 P2 variation explained 9. 87 % BioBreeding Diabetes prone BioBreeding Diabetes resistant U . S . Patent Jun . 5 , 2018 Sheet 4 of 12 US 9 ,987 , 313 B2 Richness . IIIIIIII . 08OL mm.BB-DP(d) mwynBB-DR(0) 09 . Days BB-DR(HO) do-98 * OS0t0€OZOL FIG.3 index diversity weaver Shannon U . S . Patent Jun . 5 , 2018 Sheet 5 of 12 US 9 ,987 , 313 B2 FIG . 4A BE - OP BB- R N345???????ds}{??gJ????nu????? Lactobacillus spp . Bifidobacterium spp. FIG . 4B BB - DP MW BB -OR Logofthenumbercellsper5ngDNA . Bifidobacterium spp . Lactobacillus spp , U . S . Patent Jun . 5 , 2018 Sheet 6 of 12 US 9 ,987 ,313 B2 Lachnospiracea Clostridiaceae Ruminococcaceae Flavobacteriaceaetheoptions Ruminococcaceae FIG.5 Lactobacillaceae Prevotellacea Porphyromonadaceae Bifidobacteriaceae U . S . Patent thanJun5 . 52014 , 2018 Sheet 7 of 12 US 9 ,987 ,313 B2 FIG . 6 ? withDPratsofBB-Percentage bacterialtranslocation $ Control L . johnsonii L . johnsonii L . reuteri L . reuteri 106lu 108108 10109 108 FIG . 7 Onset of T1D Post -weaning ( W ) Pre -weaning (PW ) T ATUIT . IX . VIVITEITER . N . IT . REVIEVIVIRIS IIIIIIIIIIIIIIIIII - . I Y ani YY. Days 2 22 110 141 U . S . Patent Jun. 5, , 2018 201 Sheet 8 of 12 US 9 ,987 ,313 B2 FIG . 8A $ $ PercentEuglycemic $ W X X 5 10 15 * * * 20 Age (Weeks ) FIG . 8B $ $ PercentEuglycemic $ 5 10 15 20 Age (weeks ) U . S . Patent Jun . 5 , 2018 Sheet 9 of 12 US 9 ,987 ,313
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