×÷Õߣºmalak shaheen1, sherin el sayed2, ahmed abdel karim3, alfrid edward1 and magid ibrahim1    

¡¾ÕªÒª¡¿  objective: the aim of this study was to assess school indoor exposure to microbial products and prevalence of asthma and allergies in rural and urban children. methods: this study was carried on a rural and an urban school. environmental endotoxin level was measured in multiple samples of the ambient indoor air dust collected on special aseptic filter papers from the two schools. for two hundred children history taking, clinical examination, allergen skin prick test and basic pulmonary function test were preformed. results: environmental endotoxin levels showed significantly higher mean values (p<0.01) in rural school (3 eu/mg) as compared to the urban school (0.1 eu/mg) with (or=5.163; 95% ci: 0.95ª²28). history of allergic symptoms was significantly more in urban than rural students (p=0.01). mean values of pulmonary function parameters were significantly lower values in urban students compared to rural students. skin prick test results showed significant reactions to all tested allergens in urban children compared to rural children (p<0.05). conclusion: there is an inverse association between environmental exposure to endotoxins and susceptibility for allergic manifestations in school children.

¡¾¹Ø¼ü´Ê¡¿  endotoxin; environmental; school; urban; rural and pediatric asthma

¡¡¡¡introduction

¡¡¡¡there has been a significant increase in the prevalence of allergic diseases over the past 2 to 3 decades[1]. to explain these observations, environmental factors associated with more industrialized and urban living have been studied intensively. however, there is little consistent evidence to suggest that obvious risk factors, such as increased exposure to indoor allergens, pollution, or changes in diet and breastfeeding, could account for the rise in atopic diseases[2].

¡¡¡¡interestingly, atopic conditions have become more prevalent at the same time that childhood infectious diseases have become less prevalent worldwide[3]. these findings suggested that in fact infections early in life might protect from developing asthma and allergies. this is the soª²called hygiene hypothesis proposed by strachan[4] in 1989.

¡¡¡¡however, not all study findings support the "hygiene hypothesis"[5]. infections of the respiratory tract have also been identified as risk factors for wheeze and asthma. this discrepancy may in part be attributable to the phenotype of wheeze under study, early transient wheeze being positively associated with infections of all kinds, whereas the atopic phenotype may be protected from increased infectious exposure[4]. furthermore, the type of infection may play a role[5].

¡¡¡¡recently, endotoxin ª² a lipopolysaccharide cellª²wall component of gramª²negative bacteria ª²level has been considered as a marker for environmental hygiene[6].

¡¡¡¡the aim of this study was to assess the relation between environmental exposure to microbial products (endotoxins) and the susceptibility for childhood asthma and allergies in rural and urban school children.

¡¡¡¡material and methods

¡¡¡¡study population

¡¡¡¡this crossª²sectional pilot study was conducted in urban and rural areas of nagaa hammadi governorate, upper egypt, on 205 school children with their ages ranging between 6ª²12 years, presented in 2 schools, from an urban school (alumnium city, nagaa hammadi) and from a rural school (ezbet elbossa ,nagaa hammadi). each child was subjected to the following before the start of the study: history (detailed history of asthma and atopic diseases was assessed with a questionnaire filled by the investigator) and full clinical examination (general examination and local examination of chest, heart and abdomen). the final analysis was restricted to 200 children with complete data. the two hundred school children were randomly selected, with a mean age of 9 ¡À 3 years, 100 children were from a rural school (near a farm) and 100 children were from an urban school. for the included children; skin prick test for different allergens and basic pulmonary function tests (fvc, fev1) were done.

¡¡¡¡approval to conduct the survey was obtained from the local ethics committee for human studies in ain shams university and from the principals of the schools attended by the children. written informed consent was obtained from the parents of all children.

¡¡¡¡dust sampling

¡¡¡¡multiple dust samples (twenty samples for each school including different floors) of the ambient air from children schools were collected on special aseptic filter papers provided by associated corporate consultants, inc. (acc). all field workers were centrally trained to ensure similarity of sampling technique and timings. the specimens were collected aseptically in non pyrogenic containers. the ph of the reaction was adjusted to 6.8 by hcl or naoh.

¡¡¡¡specimen preparation: (i) 0.2 ml of the test specimen was added to the "single test vial" using an automatic pipetter; (ii) lal reagent water negative control was included with each batch of specimens tested. it was used to dilute standard endotoxin and was treated as a negative control; (iii) the reaction tubes were placed in a 37¡ãc dry bath for 60 minutes.

¡¡¡¡measurements of endotoxin levels

¡¡¡¡endotoxin content was measured by a kinetic limulus assay "limulus amebocyte lysate, pyrotell". endotoxin results were expressed as endotoxin units per milligram of dust as described by hollander et al[7].

¡¡¡¡single test vial: for detection and qantitation of gram negative bacterial endotoxins (lipopolysaccharides). company: associated corporate consultants, inc. (acc). address: public and corporate sectors 411 stable drive | franklin, tn 37069, 615ª²599ª²4448 | fax 615ª²599ª²4449. net address: accusa.com.results and interpretations: (i) endotoxin was quantified by finding the endpoint in a series of specimen dilutions , ¦Ë is 0.1 eu/mg. (ii) the concentration of endotoxin in this specimen was calculated by multiplying the pyrotell sensitivity (¦Ë) by the reciprocal of the dilution at the endpoint (e) : conc. = ¦Ë ¡Á1/e.

¡¡¡¡questionnaire and interview

¡¡¡¡the symptoms of atopic diseases (allergic rhinitis, allergic conjunctivitis, food allergy, urticaria or eczema), asthma and potential explanatory and confounding factors were assessed by a questionnaire filled by the investigators from all the included children. this questionnaire included the questions of the international study of asthma and allergies in childhood (isaac study), in arabic formula[8]. rural children were all living near by a farm. in an interview with the parents as part of the home visit, we obtained details of the timing of the child's exposure to farm animals, stables and to farm milk. exposure to farming during the first year of life was defined in all the included rural children and was an exclusion criterion for the urban school children.

¡¡¡¡skin prick test

¡¡¡¡skin prick test for allergy against common inhaled allergens (dust, dust mite, cat hair, dog hair, cows' milk, sheep wool and pigeon feather) was done for all the included children. the test depends on the introduction of allergen extract into the dermis resulting in an igeª²mediated response, which is characterized by an immediate wheal and flare reaction.

¡¡¡¡skin prick test was performed on the volar or inner aspect of the forearms avoiding the flexures and the wrist areas. the procedure was first explained to the children.

¡¡¡¡the skin must be clean and free of active eczema. a grid is marked with a pen at 2 cm intervals and a drop of the relevant allergen placed on the arm at the end of each line. the pattern follows a corresponding list of allergens used for easy identification.

¡¡¡¡a lancet with 1 mm point was used to prick the skin through the drop. with the soª²called "prick through drop" method it was unnecessary to scratch or lift the skin (no blood should be drawn). the lancet was wiped with dry gauze between each prick, in order to prevent carryª²over of allergens.

¡¡¡¡º£ÄÏҽѧԺѧ±¨ vol.15 no.4 apr.2009the solutions were blotted off the test site. reactions occured within 10ª²15 minutes after which, the results were assessed. positive and negative controls were included in each series of tests. the negative control solution was the diluents used to preserve the allergen extract. the positive control solution was a 1 mg/ml histamine hydrochloride solution.a reaction of 3 mm greater than the negative control was regarded as positive. grading (table 1) was measured according to weissenbacher et al[9].

¡¡¡¡table 1 grading of skin sensitization

¡¡¡¡(weissenbacher et al. 2006)

¡¡¡¡+no wheal, 3 mm flare++2.3 mm wheal with flare+++3ª²5 mm wheal with flare++++>5 mm wheal, may have pseudopodia

¡¡¡¡pulmonary function tests

¡¡¡¡pulmonary function tests were performed using mir spirobank, eª²mail: [email protected] which is a multifunction pocket spirometer and can measure both flow and volumes by infrared mini flow sensors. all the data memorized can be downloaded to personal computer software, allow visualization, graphic representation, comparison and printing of the functional parameters and curves. measured parameters; fvc: forced vital capacity and fev1: volume expired in the 1st second of test.

¡¡¡¡statistical analysis

¡¡¡¡descriptive data are expressed as means ¡À sd. continuous data were compared by the student t test. categorical data were compared by 2 test analysis. endotoxin levels were log10ª²transformed. multivariate logisticª²regression analyses, in which the endotoxin level was treated as a continuous variable, were performed. analysis of associations was expressed as pvalue, or and 95% ci. statistical significance was established at p<0.05. the statistical analysis was performed using the statistical package for the social sciences (spss) for windows (version 11.5; spss inc., chicago, il, usa; 2006) with adjustment for age, sex, study area and number of older siblings (the basic model).

¡¡¡¡results

¡¡¡¡complete data were available for 200 children, 100 from farming families and 100 from non farming families. the mean (¡Àsd) age was 9 ¡À3 years. the adjusted odds ratios for of asthma symptoms (attacks of dyspnea and wheezing), hay fever symptoms (sneezing and running nose), allergic conjunctivitis and skin urticaria were significantly less in rural students compared to urban students (p<0.05). however, there was insignificant difference regarding food allergy symptoms and eczema (p>0.05). the relations between farming status and environmentalª²exposure variables and health outcomes are shown in table 2.

¡¡¡¡there was a highly significant difference between rural and urban students as regard the history of environmental exposure to microbial products (endotoxins) by living near farm with contact to farm animals (figure 1).

¡¡¡¡figure 1: mean of environmental endotoxin levels was significantly higher (p<0.01) in rural school (3 eu/mg) as compared to that of urban school (< 0.1 eu/mg) with (or=5.163; 95% ci: 0.95ª²28).

¡¡¡¡the results of multivariate logisticª²regression analyses estimating the effect of the environmental endotoxin level on the rates of symptoms and disease, showed a strong inverse association with symptoms of hay fever, repeated wheezing, dyspnea, skin allergy and allergic conjunctivitis. mean values fev1 and fvc percentage of predicted for age and sex showed significantly lower values in urban students compared with rural students (p<0.01 and p<0.05 respectively).

¡¡¡¡again, strong negative associations between the level of endotoxin exposure and all skin sensitisation outcomes were observed (table 3). table 2 environmentalª²exposure variables and health outcomes

¡¡¡¡variablerural school children

¡¡¡¡(n=100)urban school children

¡¡¡¡(n=100)p valueor (95% ci)environmental exposure endotoxin level*3 (+ 0.9) eu/mg 0.1 (+ 0.002) eu/mg<0.015.163 (0.95ª²28)health outcomes #3588<0.010.07 (0.03ª²0.16)sneezing running nose73850.0560.48 (0.22ª²1.02)attacks of dyspnea8330.010.18 (0.07ª²0.43)attacks of wheezing75920.040.26 (0.1ª²0.65)skin urticaria9300.030.23 (0.09ª²0.55)eczema5511 (0.24ª²4.14)food allergy10200.070.44 (0.18ª²1.07)itchy eyes10220.040.39 (0.16ª²0.94)pulmonary function test fvc105.8¡À 24.3292.72¡À 23.23< 0.05fev1110.9 ¡À 19.6992.32 ¡À 24.22< 0.01

¡¡¡¡* endotoxin level mean value (+ standard deviation) is measured as (eu/mg of dust)

¡¡¡¡# expressed as number of positive cases reported as history of repeated attacks in the previous year

¡¡¡¡¡ì expressed as mean + standard deviation of percentage of predicted for age and sex

¡¡¡¡or= adjusted odd ratio, (95% ci) = 95% confidence interval

¡¡¡¡fvc= forced vital capacity

¡¡¡¡fev1= forced expiratory volume in the first second

¡¡¡¡table 3 skin prick test results in studied children

¡¡¡¡grouptestª²ve+1+2¦Ö2por (95% ci)ruraldust908211.7<0.010.14(0.06ª²0.31)urban553312ruraldust mite702554.3< 0.010.43 (0.23ª²0.8)urban503812ruraldog hair982ª²8.3<0.050.06 (0.01ª²0.28)urban75205ruralcat hair982ª²16.3<0.010.03 (0.01ª²0.15)urban62308ruralpigeon 928ª²6.7<0.050.29 (0.11ª²0.73)urbanleather73225ruralwool9010ª²13.1<0.010.12 (0.05ª²0.27)urban523216ruralcow milk95ª²512.5<0.010.12 (0.04ª²0.35)urban70255

¡¡¡¡discussion

¡¡¡¡a remarkable increase in the prevalence of allergic disease appears to parallel the postª²industrial lifestyle changes in westernized societies[2]. insufficient microbial exposure during infancy and early childhood is one of the proposed explanations for the increase in prevalence of atopic sensitization and associated clinical phenotypes (asthma, allergic rhinitis, skin allergy)[9].

¡¡¡¡there is growing evidence that one of the inadvertent consequences of the fight against infectious diseases and our obsession with cleanliness might have been the interference with the immune system development[10]. the changing pattern of microbial exposure, with the decline in some infectious diseases, may lead to a slower maturation of the immune system, with a delayed development of the optimally balanced immune responses. this notion forms the immunological basis of the hygiene hypothesis. however, the hygiene hypothesis originated from epidemiological studies[11].

¡¡¡¡in the light of these facts, our findings may suggest that environmental exposure to microbial products, as measured by the environmental endotoxin levels, was accompanied with a significant reduction in the manifestations of hay fever, atopic skin sensitization, asthma, allergic conjunctivitis and comprised lung functions in childhood. this protective effect was observed in children from farming environments that might favourably influence the susceptibility for allergic diseases in those children.

¡¡¡¡the mechanisms by which endotoxin exposure may protect against the development of atopic immune responses and diseases are not fully understood and other environmental factors could be also accused. nonetheless, our findings may suggest that by the time a child reaches school age, high levels of environmental exposure to endotoxin have resulted in a marked suppression of the innate immune system, whereas lipopolysaccharide (component of endotoxin) stimulation triggers an innate immune response by activating mainly antigenª²presenting cells and also activates t cells, resulting in a somewhat different pattern of cytokine production.

¡¡¡¡reduced responsiveness to stimulation with lipopolysaccharide after previous stimulation with lipopolysaccharide is a phenomenon referred to in the literature as lipopolysaccharide tolerance[12]. our results could be explained by such a downª²regulation occurs in vivo as a consequence of longª²term exposure to environmental endotoxin. whether this downª²regulation is merely a biologic marker of the exposure or is causally related to the decreased rate of atopy cannot be determined on the basis of our data; it is an area in which further exploration is needed.

¡¡¡¡it has been suggested that the innate immune response has an instructive role in adaptive immunity. differential expression of lipopolysaccharide receptors in children from farming and nonfarming households has recently been reported[13], suggesting that the innate immune system responds to the high microbial burden of the farming environment.

¡¡¡¡although only current endotoxin exposure was measured, the levels are likely to reflect longª²term exposure. therefore, longª²term, highª²level environmental exposure may favour a state of tolerance, which may prevent the development of allergic immune responses.

¡¡¡¡we demonstrated that exposure during the first year of life to stables and other aspects of farm life that are likely to reflect exposure to microbial products has a strong protective effect against the occurrence of asthma and atopy (p<0.05) at school age (independently of the current level of endotoxin exposure). however, independent of and in addition to this effect, endotoxin exposure at school age was associated with a markedly decreased risk of atopic outcomes.

¡¡¡¡the protective effect of endotoxin exposure at school age was observed only for hay fever, allergic conjunctivitis and asthma, not for food allergy or eczema. variations in genetic background, different environmental factors, and the interplay among them are likely to account for the varying clinical presentations of wheeze and atopic diseases[14].

¡¡¡¡in studies of human exposure and in studies of animals[15, 16], endotoxin has been shown to induce airway hyperª²responsiveness in healthy, non atopic subjects but to decrease airway responsiveness in sensitized animals, supporting the notion that the effect is modified by atopy, as we observed.

¡¡¡¡environmental endotoxin levels are therefore likely to reflect longerª²term exposure to microbial compounds. however, the crossª²sectional design of our study limited our ability to determine precisely the duration of exposure represented by current endotoxin measurements, and prospective studies are clearly needed.

¡¡¡¡much is unknown about the changes in immune responsiveness following microbial stimulation and resulting in tolerance towards environmental allergens[3]. it seems, however, likely that complex mechanisms, where genetic and environmental factors interact to influence critical components in the recognition of bacterial products, affect the development of atopic disease in humans[14]. this dynamic interaction educates the immune system to establish a robust regulatory network, possibly the key to controlling allergic diseases[2].

¡¡¡¡evidence suggesting that farming lifestyle is protective against allergic disease is also consistent, and has been reproduced in a number of studies from different countries[14, 17ª²18]. though, many unsolved questions remain from the findings of crossª²sectional surveys in farmers and non farmers children. therefore, a consortium of investigators all with previous experience in conducting large, multicentre studies came together to develop and run the pasture birth cohort[3].

¡¡¡¡growing up on farms is associated with exposure to endotoxin, helminths, lactobacilli and saprophytic mycobacteria, whilst anthroposophic lifestyle is related to less interference with common childhood infections because of reduced antibiotic use and vaccination, and consumption of naturally fermented vegetables and biodynamic food[19ª²20].

¡¡¡¡therefore, rather than considering a single environmental exposure that may prevent allergic disease, all these indicate that a number of different environmental exposures which differ between modern and traditional lifestyles may act together in impacting on the development of allergic disease[21ª²22].

¡¡¡¡in this study, we did not assess other bacterial components, or cellª²wall components from atypical mycobacteria or gramª²positive bacteria such as lipoteichoic acid, which are known to affect immune responses in ways similar to that of endotoxin. the observed protective effect associated with endotoxin levels in school enviromental dust is therefore likely to reflect the effect of exposure to a much broader spectrum of microbial compounds than gramª²negative bacteria alone.

¡¡¡¡finally, the result of our study (however small sample size and being a cross sectional study) spotlights that environmental exposure to microbial products ª²as assessed by the measurement of school environment endotoxin levelsª² may be related to the development of tolerance toward ubiquitous allergens found in natural environments. this could attract wider epidemiological studies especially on children populations for understanding this unique hostª²parasite interaction and identifying the distinct parasite molecules that have immunomodulatory effects that will help to combat allergy without paying the price of becoming infected with noxious pathogens.

¡¡¡¡acknowledgment

¡¡¡¡we are indebted to the spirit of professor a. h. abdel karim ª²professor of environmental and public health, national research centreª² the great scientist and the kind father. his broad scientific mind and undefeated spirit has made impossible thoughts to come true and all difficulties to melt out. he passed away after completing this work. however, he will be always an inspiration for all of us. we also thank all the participating children, their families and the schools' supervisors for all their help and understanding.

¡¾²Î¿¼ÎÄÏס¿
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