Lactobacillus casei Shirota

21/October/2010Probiotics and Immunity:Benefits and Application ofLactobacillusMasanobu NannoYakult Central Institutefor Microbiological Research, JapanIV Simpósio Internacional ILSI Brasil Sobre Alimentos FuncionaisMaksoud Plaza Hotel São Paulo, Brasil

1. Lactobacillus as ProbioticsBy J.O.

Intestinal microbiota1. More than 10 11 per gram feces(More than 10 14 per person)2. More than 300 species per person

Intestinal microorganismsProduceusefulsubstancesProducenoxioussubstancesInhibitcolonization ofpathogensBifidobacteriumLactobacillusInvade thehostMaintainintegrity ofepithelial layerEnterobacteriaceaeBacteroidesDisrupt integrityof epitheliallayerEnhanceimmuneresponsesBeneficialeffectsMRSAC. perfringensIntestinal MicrobiotaInduceinflammatoryresponsesHarmfulinfluences

Intestinal microbiota in the hostIntestinal microbiota• Enhance digestion and absorption offoods.• Synthesize and supply vitamin K.• Produce short chain fatty acids.• Aid maturation of intestinal epithelialcells.• Compete pathogenic microorganisms.• Augment intestinal immune system.

Intestinal microbiota and diseaseUnbalanceddietProbioticsDiarrheaSeverestressAgingAntibioticsNormalizationDisturbanceof intestinalmicrobiotaAllergyIrritablebowel syndromeInflammatorybowel diseaseInfectionCancer

Lactobacillus casei Shirota (LcS)Lactobacillus casei Shirota• Survives in the intestine and is recovered alivefrom the feces.• Normalizes the intestinal microbiota andimproves bowel movement.• Decreases the production of noxious substancesand allows their clearance.

%%Effect of L. casei Shirota1008060on bowel movementOccurrenceof constipation1008060Occurrenceof hard stools404020200Before L. casei Placebo0Before L. caseiPlaceboAfterAfterKoebnick C et al., Can J Gastroenterol, 17:655-659 (2003)

Mutagen activity(No. of mutant colonies)Effect of L. casei Shirotaon detoxification of mutagens3 weeks’ ingestion of LcS6 healthy subjectsDetection of mutagens in urine4003002001000A B C D E FSubjectBefore AfterHayatsu H and Hayatsu T, Cancer Lett, 73:173-179 (1993)

p-cresol in urine (%)Effect of L. casei Shirotaon transformation of amino acidstyrosineOH2L. casei PlaceboH 2 Np-cresolCOOHOH1CH 3Promotion ofcarcinogenesis0BeforeAfterBeforeAfterV De Preter et al., Br J Nutr, 92:439-446 (2004)

Definition of ProbioticsLive microorganisms which, whenadministered in adequate amounts,confer a health benefit on the host(FAO/WHO, 2001)Probiotics1. can survive in the intestine.2. can normalize intestinal microbiota.3. can give benefits to the host.

2. Beneficial effects ofL. casei Shirota-prevention of carcinogenesis-By J.O.

Annual mortality trendfor leading causes of death in Japan(Rate per 100,000)250200TuberculosisCerebrovascular diseases150100Malignant neoplasmsHeart diseases50Diseases of liverPneumonia01930 1940 1950 1960 1970 1980 1990 2000Derived from CANCER STATISTICS IN JAPAN-2003

Incidence of cancer (%)Incidence of cancer (%)Effect of L. casei Shirotaon chemically induced cancerC57BL/6 miceBeige mice505040controlL. casei Shirota40controlL. casei Shirota30P=0.0543020100201001 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10Weeks after 3-methylcholanthrene injectionTakagi A et al., Carcinogenesis, 22:599-605 (2001)

Effect of L. casei Shirota on therecurrence of superficial bladder cancerSubjects: patients with superficial bladder cancer (n=78)Samples: placebo or LcS (3 x 10 10 CFU/day)74.3%LcS62.1%PlaceboPrevention of recurrenceAso Y et al., Eur Urol, 27:104-109 (1995)

Recurrence-free rate (%)Effect of L. casei Shirota on therecurrence of superficial bladder cancerSubjects: patients with superficial bladder cancer (n=202)Samples: epirubicin or epirubicin+LcS (3 x 10 10 CFU/day)10080epirubicin+LcS74.6%6040Prevention of recurrenceepirubicin59.9%2000 12 24 36 48 60 72 84Time after TUR-BT (Months)Naito S et al., J Urol, 179:485-490 (2007)

Effect of fermented milk productson the incidence of bladder cancerCase: diagnosed as primary bladder cancer (n=180)Control: no bladder cancer (n=442)0 times1-2 times/month1-2 times/week3-4 times/weekControlCase7 times/week0 10 20 30 40 50 60Relative distribution (%)Ohashi Y et al., Urol Int, 68:273-280 (2002)

Cancer incidence (%)No. of cancers/mouseEffect of L. casei Shirotaon colitis-associated cancerDSS (9 times at intervals of 1 wk)Schedule1 wkAdministration of LcS or saline (5 days/wk)80706050403020100Saline LcS210SalineMatsumoto S et al., Immunology, 128:e170-e180 (2009)LcS

Relative riskEffect of L. casei Shirotaon the development of colorectal tumorSubjects: patients with colorectal tumors (n=398)Samples: none or LcS (3 x 10 10 CFU/day)1.0Prevention of developmentNormal0.50.800.6502 years 4 yearsPolypIshikawa H et al., Int J Cancer, 116:762-767 (2005)

Beneficial effects of L. casei Shirotaconfirmed in animals and humansCancerInfectionAllergyAutoimmunityAnimal Expt.Yes3MC-inducedCACYesBacteriaVirusYesOVA-inducedYesCIA, SLE,Diabetes, EAEClinical TrialYesBladder cancerColon cancerYesRTIYesRhinitisOn planning

3. Immunomodulationby L. casei ShirotaBy J.O.

Intestinal Microbiota and CancerHarmful・Carcinogens・InflammationIncidenceIntestinalmicrobiotaWesternizationof dietProbioticsCancerBeneficial・Short chain fattyacids・Detoxification・Excretion・ImmunomodulationPrevention

Incidence rateNK activity and cancer incidence0.10Men0.05Women0.080.06lowhigh0.040.03lowmedium0.040.020.02medium0.01high0.000.000 2 4 6 8 10 12 0 2 4 6 8 10 12Time after measurement (years)Imai K et al., Lancet, 356:1795-1799 (2000)

Importance of NK activityin the defense against infection andcarcinogenesis• Virus infection is repeated in the patientcongenitally lacking NK cells.(Biron CA et al., 1989)• Lethal infection is more common in elderly havinglower NK activity.(Ogata K et al., 2001)• Incidence of lymphoma is higher in Chediak-Higashisyndrome, recessive mutation causing lack of NKactivity.(Roder JC et al., 1980)• NK activity is deteriorated in cancer patients.(Hersey P et al., 1979; Sibbitt WL Jr et al., 1984;Mickel RA et al., 1988)

Factors that affecting NK activity• Bad factors (Deteriorations):agingsevere mental stressbad life style (smoking, rare exercise,excessive alcohol-drinking, etc.)• Good factors (Improvement):moderate mental stressgood life style (adequate exercise, wellbalanceddiet, sufficient sleeping, etc.)

Change of NK activity(%)Effect of L. casei Shirotaon NK activity in healthy adults (1)Subjects: 20~40 years old; male n=4, female n=5Samples: fermented milk containing LcS (4 x 10 10CFU/80 ml) or placebo16128PlaceboLcS-milk***40Ingestion0 1 3 6 12(Week)Nagao F et al., Biosci Biotech Biochem, 64:2706-2708 (2000)

NK activity (%)Nk activity (%)Effect of L. casei Shirotaon NK activity in healthy adults (2)Subjects: 69~97 years old; male n=3, female n=7Samples: fermented milk containing LcS (4 x 10 10CFU/80 ml) or placebo50Before50After4040*30203020*100PlaceboLcS-milk20 10E/T ratioTakeda K et al., Clin Exp Immunol, 146:109-115 (2006)100PlaceboLcS-milk20 10E/T ratio

Disability ScoreDisturbance ScoreNK cell activity (%)Effect of L. casei Shirotaon NK activity in HAM patientsSubjects: 34~62 years old; HAM patients; male n=3, female n=7HAM=HTLV-1 associated myelopathySamples: fermented milk containing LcS (8 x 10 10 CFU/160 ml)76Motor disturbanceP=0.16Urinary disturbance76P

Induction of Lactobacillito induce IL-10/IL-12 productionL.casei 1L.casei 2L.rhamnosusL.zeaeIL-12 (ng/ml)IL-10 (ng/ml)126IL-10Conc. (mg/ml)0.111010009045Conc. (mg/ml)0.1110100IL-120L.fermentumL.gasseriL.johnsoniiL.acidophilusL.delbrueckiiL.helveticusL.plantarumShida K et al., J Dairy Sci, 89:3306-3317 (2006)

FreshCulturedFreshCulturedFreshCulturedNK cell activity(L.U./10 6 cells)Enhancement of NK activitywith L. casei Shirota in vitro6050**150*40100302010**500FreshNone LcS aCD3Cultured0Total CD56 + CD56 -LcS:Heat-killed L. casei Shirota was added at 1 mg/ml.aCD3:Anti-CD3 mAb was immobilized on the well.Takeda K et al., Clin Exp Immunol, 146:109-115 (2006)

IFN-g (ng/ml)IL-6 (ng/ml)NK cell activity(L.U./10 6 cells)IL-12 is mediatedin LcS-induced enhancementof NK activity in vitro54321*432140302010*0None Cont IgGaIL-120None Cont IgGaIL-120None Cont IgGaIL-12control IgG:MOPC21 (mouse IgG1)aIL-12: clone 8.6 (anti-human IL-12p40/p70; mouse IgG1)Takeda K et al., Clin Exp Immunol, 146:109-115 (2006)

Lytic Unit/10 6 cellsMonocyte is criticalin the enhancement of NK activity600500400300LcS activates CD14 + cellsto produce IL-12 andenhance NK activity.2001000rIL-12 (4 ng/ml)LcSLcSLcSLcSCD14 +CD14 +CD14 +CD14 -CD14 -CD14 -CD14 -Takeda K et al., Clin Exp Immunol, 146:109-115 (2006)

No. of tumorsColitis-associated cancer isIL-6-mediatedIntestinal bacteriaScheduleDSS (9 times at intervals of 1 wk)Intestinalepithelialcells1 wkInjection of sgp130Fc (4 times)TACEsIL-6RaIL-6/sIL-6Racomplex2.52.01.51.0Effect of sgp130Fc***IL-6Macrophage or dendritic cellsin lamina propria0.50.0Control sgp130Fc(500mg)sgp130Fc(50mg)Matsumoto S et al., J Immunol, 184:1543-1551 (2010)

PSPG-I, but not PSPG-II, inhibitsdysregulated IL-6 productionPSPG-IIL-6 inhibition in vitroPSPSPG-IIPGM G M G MM G M G MM G M G MM N-acetylmuramic acidG N-acetylglucosamineMatsumoto S et al., Immunology, 128:e170-e180 (2009)

Involvement of LcS PSPG-Iin anti-tumor activityTumor numberIL-6CytokinesSOCS3SalineSalineLcS DPSPG-ILcS DPSPG-ILcSP

LcS enhances NK activity anddown-regulates IL-6 transsignaling,resulting in suppression of cancer.So skillfully!By J.O.L. caseiShirotaIL-12Th1 cellIFN-gIL-12killingMΦ/DCNK cellCancer cellIL-6carcinogenesisIEC

Future questions• How does ingested LcS activate thelowered immune system in vivo?• Can LcS normalize the dysregulatedimmune system in allergy orautoimmune diseases?• Is LcS safe in any kind of subjectsincluding immunodeficient andautoimmune disease patients likehealthy subjects?By J.O.

AcknowledgmentsJuntendo UniversityFumiko Nagao, Kazuyoshi Takeda,Ko OkumuraThank you foryour attention!Kurume UniversityKei-ichi Mitsuyama, Michio SataYakult Central InstituteKan Shida, Junko Shibata,Tomomi Suzuki, Rumi Kaji,Masato Nagaoka, Koichi Watanabe,Satoshi Matsumoto,Mayuko Yamamoto