Project leader:ProfessorTARI HAAHTELA,
Helsinki University Central Hospital, Skin and Allergy Hospital. P.O.Box 160, FIN-00029 HUS, Finland, +358 9 471 86302, tari.haahtela@hus.fi

Researchers:
Part I/Epidemiology
Von Hertzen L.,   HUCS/ Skin and Allergy Hospital 
Mäkelä M.,   HUCS/ Skin and Allergy Hospital
Jousilahti P. National Public Health Institute /Helsinki
Kosunen T.,Haartman Institute, Helsinki University
Laatikainen T., National Public Health Institute /Helsinki
Petäys T.,  HUCS/ Skin and Allergy Hospital
Vartiainen E.,National Public Health Institute /Helsinki

Key words: allergy, asthma, atopy, infections, Finland, Russia

Results

Epidemiology
We found earlier the prevalence of atopy, determined by skin prick tests (SPTs) and specific IgE measurements, and atopic diseases among adults were significantly lower in Russian Karelia as compared with adults in Finnish Karelia.(1)

This setting is of special interest as the East-West -gradient between these areas is considered as one of the widest in the world. Living conditions in Russian Karelia still today resembles those in Finland circa 50 years ago. However, due to the geographical proximity of the areas, the geo-climatic and vegetative conditions are similar. This setting offers a unique opportunity to examine the impact of living conditions and lifestyle on the occurrence of chronic diseases.

In the present study among school children and their mothers in the same areas (2), we found that disparities in occurrence of atopy (determined by SPTs) between children in Finland and Russia were even greater than those between mothers. Overall, the risk of atopy (any positive prick result of the 14 allergens tested) was four-fold higher among children in Finland as compare with their counterparts in Russia, whereas for mothers the risk in Finland was 2.4-fold. Several-fold differences were also found in the occurrence of atopic diseases both among children and their mothers. The factors associated with reduced risk of atopy were similar in both countries, and included parental farming and having a pet in early life, both of these factors are associated with increased exposure to saprophytes in soil and vegetation.  No signs of westernisation in Russian Karelia, using atopy as a proxy, were discernible in this study.

Microbiology and animal and in vitro models
We then asked which factors in the environment could be responsible for this substantial difference in atopy prevalence between the areas, and extended the studies to animal and and in vitro models. We had collected home dust samples from homes of the Finnish and Russian participants as well as drinking water samples from schools of the children.

Drinking water
Very few studies have examined the impact of the source and quality of drinking water on the occurrence of atopy, and no data from Europe are available. Our analyses revealed that in Russian Karelia, on average 9-fold higher number of microbial cells/ml, viable and non-viable, could be detected in drinking water in Russian Karelia, where surface water from Lake Ladoga and the associated rivers is used as drinking water, and mostly without any chemical treatment. On the Finnish side of the border, by contrast, treated ground water is used. Regression analysis showed that high microbial cell content in drinking water was strongly associated with reduced risk of atopy.

Farm barn dust vs. urban home dust
The analyses of home dust samples from Finnish and Russian Karelia are currently in progress. We have tested the hypothesis that a higher microbial diversity of farm dust may be more immunostimulatory than urban home dust. We have characterized  the microbial diversity of the dusts by several methods in parallel and administered  the dust suspensions  intranasally to Balb/c mice to see whether these dusts are able to induce any inflammatory responses in vivo.

1) Characterization of the dusts; microbiological analyses
Barn dust, as compared with urban home dust, had 5-fold higher bacterial content using  culture independent methods (PCR tests or fatty acid analyses), whereas using conventional culture methods, the difference was 40-fold.. Sequence analyses revealed that the bacterial diversity in barn dust was dramatically higher than that in home dust. In the latter, circa 90% of all the bacteria were members of the genus Bacillus (phylum Firmicutes), whereas in barn dust, bacteria of the phylum Actinobacteria predominated. All major genera of the dust bacteria belonged to genera that are void of endotoxin. Indeed, on the basis of fatty acid analyses, only 10% of the bacteria in barn dust showed to be gram-negative and the percentage was even smaller in urban home dust. The Limulus amoebocyte lysate (LAL) assay could reveal no major difference in endotoxin content between barn and home dust.

2) Immunological activity
We examined the immunological activity of barn and home dust by administrating dusts suspended in PBS intranasally to Balb/c mice  twice in a week for 3 weeks. We found that
barn dust elicited a vigorous inflammatory response as shown by elevated levels of neutrophils and lymphocytes, TNF-a-mRNA and –protein and chemokines MIP-1a and MIP-2, as well as increased expression of particularly TLR2 and TLR9, when compared with home dust or PBS (control). Interestingly, administration of home dust could increase only the levels of eosinophils.

3) Identification of the characteristic bacteria of dusts
As endotoxin levels were very low and virtually similar in both types of dusts, endotoxin could not be the compound responsible for the vigorous immune responses elicited by barn dust. We then isolated and identified the characteristic bacteria of the predominant phyla in both dusts, and found that Streptomyces spp., Macrococcus spp. and Sphingomonas spp. were typical of barn dust, whereas Bacillus (B. pumilus) was by far the most common (90%) in home dust. None of these bacteria possess endotoxin.

4) Immunostimulatory capacity of the signature bacteria in dusts
We then stimulated murine macrophages with these signature bacteria of both dusts, one by one, to see which of  these showed the highest immunostimulatory capacity. It was found that all the signature bacteria in barn dust,  Streptomyces sp. (as a representative of Actinobacteria), as well as Macrococcus and Sphingomonas could induce strong immune responses in a dose-dependent manner, shown by elevated  TNF-a-mRNA and –protein and  MIP-1a and MIP-2 levels in vitro. By contrast, the predominant bacterium in home dust, B. pumilus, elicited only minimal, if any, immune responses.

In sum, the above cited and further studies suggest that the diversity of bacteria, particularly those in the phylum Actinobacteria was overwhelming in barn dust as compared with home dust. Gram-negative bacteria represent only a minimal part of all bacteria in barn and home dust. Substantial immunological responses shown in mice after administration of barn dust could hardly be  related to endotoxin  but rather to structures of particularly Actinobacteria or endotoxin-free gramnegatives (Sphingomonas). These structures will be further clarified. The impact of endotoxin as an environmental immunomodulator may have been overestimated in the literature. Interestingly, the diversity of the cell wall components is largest among Actinobacteria of all members of the domain Bacteria. 
 
Selected publications:

von Hertzen L, Haahtela T. Immunization and atopy: possible implications of ethnicity Review. J Allergy Clin Immunol  2004;113:401-6.

von Hertzen L, Haahtela T. Asthma and atopy - the price of affluence? Allergy 2004;59:124-37.

Klemola T, Masyuk V, von Hertzen L, Haahtela T. Occurrence of atopy among Russian and Finnish school children. Allergy  2004;59:465-6.

von Hertzen L, Haahtela T. Signs of reversing trends in the prevalence of asthma. Allergy 2005;60:283-92.

Moverare R, Petäys T, Vartiainen E, Haahtela T. IgE reactivity pattern to timothy and birch pollen allergens in Finnish and Russian Karelia.  Int Arch Allergy Immunol 2005;136:33-38

von Hertzen L, Mäkelä MJ, Petäys T, et al. Growing disparities in atopy between the Finns and the Russians – a comparison of two generations. J Allergy Clin Immunol 2006;117:155-57.

von Hertzen L, Haahtela T.  Disconnection of man and the soil –reasons for the asthma and atopy epidemic? J Allergy Clin Immunol 2006;117:334-44.

Leino M, Alenius H, Fyhrquist-Vanni N, Wolff H, Reijula K, Hintikka EL, Salkinoja-Salonen M, Haahtela T, Mäkelä MJ. Intranasal Exposure to Stachybotrys chartarum Enhances Airway Inflammation in Allergic Mice. American Journal of Respiratory and Critical Care Medicine 2006; 173:512-18. 

von Hertzen LC, Laatikainen T, Mäkelä MJ,  Jousilahti P, Kosunen TU, Petäys T, Pussinen PJ, Haahtela T, Vartiainen E. Infectious burden as a determinant of atopy – a comparison between adults in Finnish  and Russian Karelia. Int Arch Allergy Asthma (in press)

Malmberg LP, Petäys T, Haahtela T, Laatikainen T, Jousilahti P, Vartiainen E, Mäkelä MJ. Exhaled  nitric oxide in healthy nonatopic school-aged children: determinants and height-adjusted reference values. Pediatr Pulm 2006, in press 

Reference: Vartiainen E, Petäys T, Haahtela T, et al. Allergic diseases, skin prick test responses, and IgE levels in North Karelia, adn the Republic of Karelia, Russia. J Allergy Clin Immunol 2002:109:643-48.

An abstract of the research plan (January 2003)