Influence of exposure to microbes and polluents on the

development ofatopy and asthma

Prof. dr. K.Desager

Department of Pediatrics

University of Antwerp

Dr C. Vael

Department of Microbiology

University of Antwerp

Hygiene hypothesis

Rising prevalence of atopic disorders in infants and children

‘Westernized life style’ Modified pattern of microbial

exposure in young children

Asthma phenotypes

Martinez 2002

Asthma Predictive Index< 3 yr and frequent episodes of wheeze

2 minor criteria:

allergic rhinitis

Wheeze without cold

eosinophilia

75% : active asthma at school age

1 major criterium

Parental asthma

atopic dermatitisofor

enand

Castro-Rodriguez AJRCCM 2000;162: 1403 -1406

Aim

to evaluate the bacterial flora of infants in relation to environmental pressure and to atopy

Materials and methods

Prospective cohort study: started 10/2002

• recruitment of 159 healthy neonates• cord blood polluents: Cd, Pb, DDE, PCB, dioxine• fecal sampling: 3w, 6m,12m n= 431• questionnaires: prenatal, 3w, every 6 mths till 3yr• clinical, eNO, SPT: 3 yr

Characteristic Number %

Metropolitan 102/158 65

Sex (male) 73/138 53

Mother dd astma ever 12/147 8

Father dd asthma ever 8/138 6

Skin Prick Test positive 15/116 13

Positive mAPI 18/111 16

0

5

10

15

20

25

30

wheeze

Regional differences

Characteristic metropolitan rural p

mAPI 29% 24% 0.67

SPT 18% 4% 0.045*

parental asthma 19% 4% 0.01*

antibiotics yr1 48% 55% 0.48

passive smoke 12% 21% 0.22

Outcome SPT OR 95% Confidence intervals

Parental asthma 4,1* 1,1 15,7

Passive smoke 1,9 0,3 11,6

Region (metropolitan vs rural) 4,0 0,8 20,1

eNO

• In 121 children eNO attempted.– technical difficulties: 14– refusal: 11 children– leakage or irregular breathing: 43 – acceptable measurements: 38 (min 2 and max 5

attempts)

• SPT: 15 positive SPT for at least one allergen– grass pollen: 6– house dust mite: 10– egg: 3– cow’s milk: 2

eNO - mAPI

• children with positive mAPI were differentiated from those with negative mAPI at eNO of 3.4 ppb

• sensitivity: 71% • specificity: 71%

mAPI

negative positive

eNO(ppb)

< 3.4 17 4

> 3.4 7 10

(p < 0.05 )

eNO - SPT

• cut-off value reflecting the best combination of sensitivity and specificity occurred at 4.9 ppb

• sensitivity: 80%• specificity: 88%

SPT

negative positive

eNO(ppb)

<4.9 29 1

>4.9 4 4

(p < 0.05 )

Wheeze 2 yrFamily history asthma or allergy

+

Birth weight +

Birth height +

Sex +

Pb cord blood +

DDE cord blood +

Antibiotics +

Effect Point Estimate

95% WaldConfidence Limits

Birth height 1,603 1,196 2,149

DDE (fat, LOG) 1,930 1,078 3,454

Number of days antibiotics 1,093 1,038 1,150

polluents

Sunyer Env Health Persp 2005; 113: 1787

DDE - wheeze

DDE - asthma

• causal pathway DDE and asthma?• Immune system: associated with changes in

– immune cells (Vine 2001)– immunoglobulins (Cooper et al. 2004; Vine 2001)– cytokines (Bilrha 2003; Daniel 2002)

• hormone-like activity of DDE: – interfere with mast cells in airways that express

estrogen/progesterone receptors, favoring the role of DDE in TH2 immune differentiation

– Direct effect on the airway through altering b2-adrenergic responsiveness and increasing the production of prostaglandins

conclusions

• Region: No difference mApi, less sensitisation in rural region due to less parental asthma

• eNo relationship with mAPI– Difficult to perform in 3 yr old– in agreement with data in older children, adults: atopy,

family history

• Prenatal exposure DDE related to wheeze 2 yrs– In agreement Sunyer

Factors modifying infant gut flora

1. Breastfeeding: because fecal pH? (3w)

Lactobacilli/Streptococci

Bifidobacteria

Bacteroides

2. Cesarian delivery: delayed colonization?

Bifidobacterium(1w)

Bacteroides (6m!)

Clostridium perfringens (1m)

(Harmsen H., J.Ped. Gastr.Nutr.,2000)

(Gronlund M., J.Ped.Gastr.Nutr.,1999)

Infant GI flora in allergy/atopy

3. Allergy/Atopy:

Lactobacillus (24m)

Bifidobacterium (1w,3m,12m,24m)

Bacteroides (12m,24m)

Enterococcus (1w,1m)

Clostridia (3m)

S. aureus (6m)

Yeasts (3m)

Early (<3m): Clostridium, Yeasts, Enterococcus, Bifidobacterium

Late (>3m): Lactobacillus,Bifidobacterium,Bacteroides,S. aureus

other species

(Bjorksten B.,Cl.Exp.All.,1999)

(Bjorksten B.,JACI,2001)

(Kalliomaki, JACI,2001)

(Ouwehand A.,JACI,2001)

Results: Breast feeding at age 3 weeks

Log CFU/g

0 1 2

enkel BV BV en FV FV

0,00

2,00

4,00

6,00

8,00

10,00 Bifidobacteria

Enterococcus

Clostridium

(p<0.01 Kruskal-Wallis)

Feeding Number(%)

BF 88 (57)

BF+FF 21 (14)

FF 45 (29)

BF: breast feeding

FF: formula feeding

BF BF+FF FF

Results: Breast feeding at age 6 months

,00 1,00 2,00

BV BV+FV FV

0,00

2,00

4,00

6,00

8,00

10,00 Stafylococcus

Bacteroides

LactobacillusFeeding Number (%)

BF 14 (10)

BF+FF 19 (13)

FF 109 (77)

Log CFU/g

(p<0.05 , * P = 0.08, Kruskal-Wallis)

*

BF: breast feedingFF: formula feeding

BF BF+FF FF

Results: wheezing at age 1 year

0 1

neen piepen 12m ja

0,00

2,00

4,00

6,00

8,00

10,00 Bacteroides 3w

Bacteroides 6m

Tot.Anaeroben 3w

Gr Neg aeroob 12m

(p < 0.05 Mann-Whitney U )

Log CFU/gWheezing

Nr of children (%)

Yes No

33 (21) 126

non-wheezers at 1 year wheezers

Tot. Anaerobes

3w

MCC

1 yr

Bacteroides

6m

Bacteroides

3w

,00 1,00

mAPI

0,00

2,00

4,00

6,00

8,00

10,00logBBE

logCAA

mAPI at age 3 years

Log CFU/g

(p < 0.05 Mann-Whitney U )

mAPI

Nr of children (%)

Yes No

33 (27) 88

Bacteroides 3w

Tot. Anaerobes 3w

Neg. Pos.

Bacteroides culture 3 wks - mAPI

mAPI

Bacteroides colonisation

Neg Pos

Neg 59 7

Pos 34 11

Specif.63%

Sens.

61%

pos. mAPI prevalence = 16%

(p < 0.05 Fisher exact)

Toll-like receptors and intestinal microflora

Gr. pos bact. (Staf., PG) TLR 2 Th2Asthma

Bacteroides Lipoprotein *

Gr. neg. bact. (E. coli, LPS) TLR 4 Th1

(Agrawal S, J immunol., 2003)

* (Netea M. et al., Eur. J. Imm., 2004)

http://www.jci.org/content/vol112/issue5/images/large/JCI0319545.f1.jpeg

Copyright ©2002 BMJ Publishing Group Ltd.

Kirjavainen, P V et al. Gut 2002;51:51-55

Figure 1 Scattergram showing faecal concentrations of bacteroides and Escherichia coli versus serum total IgE concentration in each individual. Regression lines with 95% confidence intervals

(broken lines); Spearman's rho and p values are given for both slopes. HSG, highly sensitised group; SG, sensitised group.

2) 716 high school children:

If atopic disease

significantly higher Bacteroides vulgatus IgG titers

(Fukuda S et al., J.Adolesc. Health, 2004)

1)

Conclusions:

• Breastfeeding during the first year of life has an influence on the infant intestinal microflora.

• Changes in the intestinal flora in the first month of life are associated with wheezing at the age of 1 year and mAPI at the age of 3 years.

• Early postnatal intestinal colonization with Bacteroides fragilis is associated with a positive mAPI. Children with a positive mAPI have a 4 -10x higher risk of having subsequent asthma (age 6-13y).

Funded by the Flemish government within the project :

“Health and Environment, subdivision Asthma”.

Weisglas-Kuperus Tox lett 2004

Corrected breastfeeding, parity, education, smoking, fam history atopy, daycare

PCB’s: immunomodulating?

DDE - infections

• decreased response to viruses, bacteria?

• inconsistent results– moderate increase of acute infections during

first year of life (Dallaire et al. 2004)– not in 343 German school children (Karmaus

et al. 2003) – not in 207 Dutch infants (Weisglas-Kuperus et

al. 1995)

DDE – immunity, asthma

• changes in T-cell– mediated immune cytokines related with allergy (IL-4) (Bilrha 2003; Daniel 2002)

• similar effects: hexachlorobenzene (HCB) (Michielsen 1999) and polychlorinated biphenyls (PCBs) (Van Den Heuvel 2002)

• cross-sectional study school children Germany DDE related with increases in total IgE and asthma (Karmaus 2001, 2003)

• increase of asthma prevalence, mortality in adults was found among an older cohort of DDT sprayers (Beard 2003)

• prevalence of wheeze increased with a variety of pesticides among current applicators (Hoppin 2002)