Allergiprevention med probiotika Thomas Abrahamsson, Barnläkare Institutionen för klinisk och experimentell medicin, avd för Pediatrik, Linköpings universitet Tänk på uttalet på eczema=först stavelsen skall betonas. Good afternoon everybody. My name is…Om inte det är sagt. I would like to thank the organizers for giving the opportunity to presnt this study on primary prevention of allergic disease with probiotics

Hygien hypotesen Börjar först med lite bakgrund. The hygiene hypothesis says that the increase of allergic disease in affluent societies is due to an alteration of the microbial exposure (föränfring av exponeringen för mikrober) And there are several other studies indicating that microbial pressure is important. But to evaluate the relevance of this findings, intervention studies are needed. Probiotics may be useful for that. Sudo, Björksten Riedler, von Mutius, Braun-Fahrländer Krämer ” early entrance.. Alm Strachan Matricardi McKeever, Farooqi

108/dos L reuteri ATCC 55730 Spädbarns-kolik Gastroenterit Infektioner Savino, Pediatrics. 2007 Jan;119(1):e124-30. Gastroenterit 108/dos Infektioner Immunsvar i tunntarmsbiopsier Shornikova främst signifikant effekt vid log9, men Eom effekt vid log8 Weizman gav log7/g formula Valeur 4xlog8 (dock vuxna som har >4 ggr större Gi kanal Savino har nu även visat en effekt med log8 på kolik. The strain the dose we have used. Valeur: B lymphocytes in duodenum, CD4+ in mucosa in ileum biopsies. Shornikova, Pediatr Infect Dis J 1997;16:1103-07 Eom, Korean J Pediatr 2005;48:986-90 Weizman,Pediatrics 2005;115:5-9 Valeur, Appl Environ Microbiol 2003;70:1176-81

Studiedesign Allergisk sjukdom, Pricktest, Specifikt IgE n=117 n=115 Lactobacillus reuteri 108/d Placebo 12 mån 12 mån Så för att testa hygienhyptesen och utvärdera en ny strategi för allergiprevention har vi genomfört en DB randomiserad PC studie med just LBr. Familjer rekryterades på MVC under graviditeten….. Ange antal som rekryterades och som fullföljde. 2-6 veckor 2-6 veckor 2 år 2 år n=95 n=93 Allergisk sjukdom, Pricktest, Specifikt IgE

Sensibilisering och IgE-associerat eksem Definitioner: Sensibilisering = minst ett pos Prick Test* och/eller Specifikt IgE** IgE-associerat eksem = Eksem + Sensibilisering IgE-associerad sjukdom= Allergiskt sjukdom *** + Sensibilisering *SPT med äggvita, färsk komjölk och standardiserade katt, björk och timotej extrakt **Cirkulerande IgE antikroppar mot äggvita och komjölk samt en mix av födoallergen, fx5 ***Eksem, astma, allergisk rhinokonjunktivit, gastrointestinal allergi och allergisk urtikaria The cut off level was 0.35 kU/L

Kumulativ incidens vid 2 år Hur gick det? Nej, vi kunde se någon effekt på symptom av allergisk sjukdom. Tex var incidensen av eksem i Lbr gruppen… Eczema 36% vs 34% Scorad 12 m 12-9, 24m 11-8p Astma 7-11, ARC 1-4, Urticaria 3-1, GI 2-2 The numbers were ti low ti achieve significance. Ifall någon frågar: Any symptom: 46% vs 45 % (incl wheeze) Any parameter: 62% vs 57%

Prevalens vid 6, 12 and 24 månader eksem L reuteri eksem placebo 3 * IgE-associerat eksem placebo IgE-associerat eksem L reuteri % 2 1 Födelse 0-6 mån 6-12 mån 1 2 - 2 4 mån *p=0.02. X 2 test.

Kumulativ incidens vid 2 år p=0.04 p=0.07 And if we looked at senstized infants with any symptom of allergic disease, as we defined as it IgE ass disease, the cumulative indicidence was lower in the infants receiving L reuteri There was also a trend for lower SPT in the treated group, and X 2 test.

Naturalförlopp av spädbarnseksem Eksem vid 2 års ålder: Icke-sensibiliserat Sensibiliserat barn Eksem Eksem 14% 59% Novembre E, Allergy 2001;56:452-53 * Astma vid 10-12 år: So what? The obvious question is whether a lower incidence of IgE associated disease important although the symptoms of allergic disease were not affected? Well, Infant Eczema is conidered as a predictor of asthma and ARC in school age. But the increased risk is rather limitted to sensitized infants with eczema, and not non-sensitized infants with eczema, as you can se in these two publications. (It seems that this is true prefaerably for sensitized infants with eczema) Thus you might therefore speculate that the group with treated infants possibly run a reduced risk to develop asthma and ARC in school age, than the placebo group This would be in contrast to the Finnsih study with Lactobacillus GG by Kalliomäki 14% 60% Wütrich B, Allergy 2002;57:267-68 ** * Sensibilisering= SPT+ **Sensibilisering=SPT+ och/eller specific IgE

Tidigare preventionstudie Lactobacillus GG, 109/dos Placebo 6 mån 6 mån 2 år 2 år How is our result related to other intervention studies with probiotics. The first allergy prevention study by Kalliomäki i Finland, another lactobacilli strain LGG reduced eczema with 50%, but SPT and specific IgE was not affected. Eksem SPT+ RAST 46%* 14% 25% 23%* 18% 27% Kalliomäki, Lancet 2003

Tidigare preventionstudie Lactobacillus GG, 109/dos Placebo 6 mån 6 mån 2 år 2 år Ingen skillnad i SPT här heller 33% vs 31%. Effekten verkar inte vara IgE medierad. 7 år Eksem 46%* 23%* Astma ARC 17% 5% Kalliomäki, JACI 07 23% 10%

Allergi prevention studier Produkt Graviditet Odds ratio SPT or sens. Eksem IgE ass eksem Abrahamsson et al, JACI 2007;119:1174-80 L reuteri Ja Nej 1.13 (0.53-2.37) 0.53 (0.27-1.06) 0.35 * (0.14-0.88) 0.36 * (0.16-0.82) 1.37 (0.47-4.07) ? 0.74 * (0.55-0.98) 0.86 (sens.) (0.65-1.14) 0.66 * (0.46-0.95) 1.18 (0.62-2.26) 2.04 * (1.01-4.15) 2.18 (0.95-5.20) 188 fullföljde LGG Kalliomäki et al, Lancet, 2001: 357;1076-79 132 fullföljde Very recently,another 2 allergy prevention studies with probiotics have been reported. Another really large stusy has been performed in Finland with LGG in a mixture with another 3 bacterial strains. Still LGG reduced eczema, but the odds ratio was not as impressive as in the first study. Still there was no efect on sensitization. As You can see, the interventions studies with LGG has had effect mainly on eczema, but not sensitization. While our study with L reuteri had an effect on IgE ass eczema, just because the the effect on senitization in infants with eczema. A study by Taylor et al in Australia did not see any preventive effect by another strain. Endpoint was at 12 months. Instead sensitization was more common among treated infants. The main difference, besides that a different strain was used was that they did not give the study product to the mother during pregancy. Maybe the supplemention to the mother during pregnany is crucial? We have data that indicates that…. MIX + LGG 925 fullföljde Kukkonen et al, JACI 2007;119:192-198 L acidophilus Taylor et al, JACI 2007;119:184-191 178 fullföljde

Allergi prevention studier Produkt Graviditet Odds ratio SPT or sens. Eksem IgE ass eksem Abrahamsson et al, JACI 2007;119:1174-80 L reuteri Ja Nej 1.13 (0.53-2.37) 0.53 (0.27-1.06) 0.35 * (0.14-0.88) 0.36 * (0.16-0.82) 1.37 (0.47-4.07) ? 0.74 * (0.55-0.98) 0.86 (sens.) (0.65-1.14) 0.66 * (0.46-0.95) 1.18 (0.62-2.26) 2.04 * (1.01-4.15) 2.18 (0.95-5.20) 188 fullföljde LGG Kalliomäki et al, Lancet, 2001: 357;1076-79 132 fullföljde MIX + LGG 925 fullföljde Kukkonen et al, JACI 2007;119:192-198 L acidophilus Taylor et al, JACI 2007;119:184-191 178 fullföljde

Allergi prevention studier Produkt Graviditet Odds ratio SPT or sens. Eksem IgE ass eksem Abrahamsson et al, JACI 2007;119:1174-80 L reuteri Ja Nej 1.13 (0.53-2.37) 0.53 (0.27-1.06) 0.35 * (0.14-0.88) 0.36 * (0.16-0.82) 1.37 (0.47-4.07) ? 0.74 * (0.55-0.98) 0.86 (sens.) (0.65-1.14) 0.66 * (0.46-0.95) 1.18 (0.62-2.26) 2.04 * (1.01-4.15) 2.18 (0.95-5.20) 188 fullföljde LGG Kalliomäki et al, Lancet, 2001: 357;1076-79 132 fullföljde MIX + LGG 925 fullföljde Kukkonen et al, JACI 2007;119:192-198 L acidophilus Taylor et al, JACI 2007;119:184-191 178 fullföljde

** * * * Barn till MAMMOR med allergisk sjukdom Kumulativ incidens Because the mother received the study product in late pregnancy, we stratified the material according to maternal allergic disease. And yes, the effect was really pronounced when only infants to allergic mothers were included. As you can see, also the difference of sensitization became significant. The cumulative incidence differ significantly for…… 71 in the L reuteri an 72 in placebo group were included in this analyses. *p<0.05, ** p<0.01 X 2 test.

Barn till PAPPOR med allergisk sjukdom Kumulativ incidens P=0.09 Whereas the effect of treatment almost disappeared when including only infants to fathers with allergic disease. Still there was a trend for lower incidence of IgE ass disease, however. Om jag hinner: Det här är ju jätteintressant eftersom det skull kunna tyda på att man skall fokusera på den gravida mamman vid primär prevention av allergsik sjukdom hos barnen. Flera epidemiologiska stduier talar för det. Som många av er vet verkar det vara skyddande att växa upp på bondgård med kreatur och man har tänkts sig att det beror på ett högre mikrobiellt tryck. Nu har de nyligen visat att effekten blir betydligt större ifall den gravida mamman har jobbat med kreatur under graviditeten. Sedan verakr den gravida kvinnans kost spela roll för allergiutv och det kommer ju Catrin berätta för er alldelses strax. Thus, obviously this findings suggest that the supplementation to the mothers during late pregancy is of a crucial importance for the effect.

Arbete i stallet under graviditet minskar allergi hos barnen OR P-värde Prenatal exponering: 0.58 0.007 Aktuell exponering: 0.96 Nu har det kommit ytterligare en studie som fokuserat på exponering under graviditeten. Det visade sig att det ffa var om mammorna hade arbetat med djur under graviditeten som uppväxt på bonngård skyddade. Exponeras för mikrober under graviditeten. Samma mönster som i vår studie. Även i dessa studier i Tyskland sett en omvänd relation mellan endotoxin från bakterier och allergiutveckling. : Ege, J Allergy Clin Immunol. 2006;117(4):817-23. TA

L reuteri behandlade mammor IL-10 och TGF-b2 i bröstmjölk från L reuteri behandlade mammor Kolostrum median (25-75 percentilen) 1m mjölk median (25-75 percentilen) L. reuteri placebo p-värde IL-10 (pg/ml) 6.6 (4.5-19.0) 4.8 (1.15-12.7) 0.046 1.15 (1.15-5.5) (1.15-4.0) n.s. TGF-b1 (pg/ml) 570 (382-748) 653 (443-948) 321 (231-497) 310 (219-593) TGF-b2 (pg/ml) 674 (455-1153) 965 (557-1691) 0.02 399 (265-672) 378 (233-810) TNF (pg/ml) 11 (3.9-21) 10 (3.9-23) 4 (4-12) (4-10) sCD14 (pg/ml) 17 (13-19) 15 (12-20) 9 (7-10) 8 (6-10) sIgA (mg/ml) 2.8 (1.7-4.0) 2.9 (1.6-4.3) 1.2 (0.9-1.5) 1.1 (0.9-1.4) Na/K 0.8 (0.7-1.1) 0.9 (0.7-1.2) 0.7 (0.6-0.9) 0.6 (0.5-0.8) Frågan är då om Lbr påverkan mammans immunförsvar. Jo, vi har fynd som skulle kunna tala för det. Vi mätte cytokiner i bröstmjölk från mmorna och den antiinflammoriska cytokinen IL-10 var högre och TGF beta var lägre hos mammor som fått Lbr. Att TGF beta vara kägre var väl en överraskning och liten besvikelse först. TGF-Beta var ännu lägre hos de mammor som hade Lbr i feces. MFB

Icke-sensibiliserade barn har fått bröstmjölk med lägre nivåer TGF-b2 4000 6 m 12 m 24 m Kum 6-24 m TGF-b2 (pg/ml) p=0.10 p=0.18 p=0.01 p=0.04 2000 Sensibilisering + - + - + - + - Och vi kunde se att barn sp, fått BM med ……..Denna skillnad kvarstod trots att man justerade för att barnen fått Lbr eller inte. Man skulle ju annars kunna tänka sig att det påverkan på mamman och det låga TGF beta vara ett paralellefenomen och att det egentligen var att barnen i Lbr gruppen beh med Lbr som hade effekt på sensibilisering. Nu verkade beh i sig vara viktig. Det utesluter ju inte att beh av barnet också är viktigt. 17 74 26 66 25 66 32 50 n= MFB

* CRP (ng/ml) IgE-associerad allergisk sjukdom 0-24 months Navelsträng + 200 400 600 800 1000 2000 3000 4000 5000 6000 CRP (ng/ml) We have analyzed immunological parameters in blood. Low grade inflammation has been associated with lower risk of allergis disease in a bery recent publication from Finland and we could confirm this. Infants developing IgE associated allergic disease and eczema hade lower CRP the first yeAR OF LIFE.

L. reuteri i moderns avföring och colostrum perinatalt var relaterat till högre CRP hos deras barn CRP navelsträng CRP vid 24 mån median (ng/ml) p median (ng/ml) (interkvart. range) Ja 64 0.07 279 0.002 (24-68) (60-1301) Nej 30 70 (22-64) (62-172) 63 0.03 116 0.30 (27-66) (70-1121) 28 77 (3-64) (66-336) Moderns feces 1 v postpartum: Colostrum 1-3 dagar And infants to mothers with L reuteri in stool and colostrum had higher CRP in umbilical cord and at 24 months.. The duration of supple…. Wich also indicates that the treatment of the mother was important for the preventive effect. Durationen av L. reuteri intag av mödrarna under graviditeten korrelerade med CRP i navelstränen (rho=0.25, p=0.015)

KONKLUSIONER FRÅN VÅR STUDIE En preventiv effekt på eksem kunde inte bekräftas Behandlade barn hade en lägre incidens av IgE associerad allergisk sjukdom och lägre frekvens av IgE-associerade eksem vid 2 års ålder. Effekten av behandling var mer uttalad om modern hade allergisk sjukdom När jag läser upp den andra punkten kan jag kägga till att de därmed kan löpa en mindre risk att urveckla astma och allergsik rhinokonjunktivit Samt nämna att vi planerar en 7-årsuppföljning just pga detta När jag läser upp det utgå från abstract text: A preventive effect of probiotics on infant eczema was not confirmed. The treated infants, however, had less IgE-associated eczema at 2 years of age and a lower incidence of skin prick test reactivity herefore possibly run a reduced risk to develop later respiratory allergic disease.

7 år 7-årsuppföljning Astma, ARC, sensibilisering, NO Placebo Lactobacillus reuteri 108/d 12 mån 12 mån 2 år 2 år Ingen skillnad i SPT här heller 33% vs 31%. Effekten verkar inte vara IgE medierad. 7 år IgE assoc. sjd 35%* 20%* Astma, ARC, sensibilisering, NO

Meta-analyser: Review: Osborn DA, Sinn JK. Cochrane Database Syst Rev. 2007 Oct 17;(4):CD006475. “There is insufficient evidence to recommend the addition of probiotics to infant feeds for prevention of allergic disease “ Lee J, Seto D, Bielory L. J Allergy Clin Immunol. 2008 Jan;121(1):116-121. “Current evidence is more convincing for probiotics' efficacy in prevention than treatment of pediatric atopic dermatitis” Review: So what can we conclude from all this studies. Is there a place for probiotics in prevention of eczema? The last year thre have been at least two meta-analyses trying to answer this. The first, from cochrande, concluded…. The second one in JACI concluded. There have also been a lot of reviews. Presctott and Björksten have the sam conclusion as Osborn: that there isinsufficient evidence and that more studies are neeeded. They also pointed out robust meta-analysis are problematic to perform. And I agree with them Prescott SL, Björksten B. J Allergy Clin Immunol. 2007 Aug;120(2):255-62. “ …robust meta-analyses problematic to perform.”

Meta –analysis av Lee, 2008 I´ve chosen a picture from the meta-analysis by Lee to illustrate this. They have listed the 6 articles that were included in the analysis. However the last three ones are actuallöy from the same study, the study by Kalliomäki. It is the 2-yar follow up, the 4 year follow up and the artcle with breat milk analysis. This sudy was counted three times. If this is corrected 4 studies remain nad these are the same as I´ve already mention. And as I mention all these studies used different probiotics strains. This is problematic. Different bacterial strains have probably different effect,. They have different immunlogical proerties, different survival rate in the GI tract and different effect on the gut microbiota. It is like compareing apple and pears. To create reliable a meta-analyis for reccommendation of probiotics You need several studies with the same strain. And that is not possible Yet. You might easily be mislead by a meta-analysis. A i have choosen the meta-analysis ny Lee as an example. Firstly, we can notice that the Kalliomki stusy was countes tthree times. Two yars follow up, 4 year follow up and the article with breast milk analysis So the article included in this meta-analysis are the four ones I went through before. Ax I meantion all these have differnet strains differ i several aspects: immunological properties, survival in the GI tract, effect on the gut microflora etc. Therefore it is probelmetaic to include them in the same meta-analysis. Because one strain works it is not certian that all other do. To perform a relevant meta-analysis we need several studies with the same strain. Lee J, J Allergy Clin Immunol. 2008 Jan;121:116-121

Negativ studie med låg statistisk power Lactobacillus GG 5x109/d Placebo 6 månader 6 månader 2 år 4-6 veckor 4-6 veckor 2 år However, the fact is that very recently a new study with LGG was published. Kopp and collegues tried to mimic the first prevention study with LGG to see wether theu could confirm the effect ny this probiotics strain. Unfortuinaltely this Study had a too low statistcal power. Only 94 infants completed the study wich was tthe half compared to our study. So, Although the study did not confirm any effect of L GG, the authors have not proved that LGG doesn´t work since the power was all to low. 2. But, nevertheless, the incidence figures for eczema are very similiar. Could the differemt results be explained by different gut microbiota in Finland and Germany. Hardly, but You might not exclude that. 3. This study also points out the imortance that studies are reproduced. Unfortunately it was too small. 4. An interesting finding was that the number og infants with recurrent wheezing was higher in the LGG group. This is a similar finding as in the first study with LGG. The incidence of asthma tended to be higher in the treated group at 7 yeras of age. n=50 n=44 Eksem: Bronkit (>5): 28% 26%* 27% 9%* Kopp et al, Pediatrics. 2008 Apr;121(4):e850-6.

KONKLUSION Det finns otillräcklig evidens för att rekommendera probiotika för allergiprevention Ytterligare studier som bekräftar effekten av enskilda probiotiska stammar behövs So, the conclusion still must be that När jag läser upp det utgå från abstract text: A preventive effect of probiotics on infant eczema was not confirmed. The treated infants, however, had less IgE-associated eczema at 2 years of age and a lower incidence of skin prick test reactivity herefore possibly run a reduced risk to develop later respiratory allergic disease.

STORT TACK TILL Elisabeth Andersson Linnea Andersson Eivor Folkesson   STORT TACK TILL våra ovärderliga studiesköterskor och lab personal! Elisabeth Andersson Linnea Andersson Eivor Folkesson Lena Lindell Ann-Marie Fornander

Och mina medarbetare! Avd för Pediatrik, Linköpings universitet   Avd för Pediatrik, Linköpings universitet Maria Jenmalm Malin Fagerås-Böttcher Mats Fredrikson Karolinska institutet, Stockholm Bengt Björkstén Barnkliniken, Ryhov, Jönköping Göran Oldaeus Barnkliniken, Vrinnevisjukhuset, Norrköping Ted Jakobsson

Stöd från: BioGaia AB, Stockholm Ekhaga Stiftelsen   Stöd från: BioGaia AB, Stockholm Ekhaga Stiftelsen Hjärt- och lungfonden FORSS Astma- och allergiförbundet Vetenskapsrådet Universitet i Linköping. Thank you very much for your attention!