the reason why exposure to common environmental antigens induces allergic responses in some people and not others remains undetermined .
atopy and asthma have a complex genetic background , and multiple genes can contribute to their development through main effect , gene - gene , and gene - environmental interactions . allergen - specific cd4 + helper t - cell ( th ) generation is the initial event leading to the development of allergic disease .
th2 subtypes are pivotal to the inflammatory cascade through the production of il-4 , il-5 , il-13 , and il-9 .
th1 cells ( secreting mainly il-12 and ifn- ) may contribute to the chronicity and effector phase of these diseases .
several genetic polymorphisms of these genes have been studied in relation to allergic diseases and asthma [ 13 ] . however , other important molecules have been associated with phenotypes of asthma , including tumor necrosis factor- ( tnf- ) . this proinflammatory cytokine has been found in increased concentrations in asthmatic airways and the inhalation of tnf- has been shown to cause airway hyperresponsiveness and increased sputum neutrophil counts in healthy volunteers .
tnf- is a member of the tnf gene superfamily located within the human major histocompatibility complex on chromosome 6p , linked to atopic asthma in several studies [ 5 , 6 ] .
several polymorphisms have been reported in the promoter region of the tnfa gene , with tnf-308g > a being the polymorphism most widely studied in relation to this disease .
in addition to the inflammatory events , regulatory t - cell dysfunction is associated with development of complex genetic conditions such as atopy and asthma .
peripheral t - cell tolerance is characterized by functional inactivation of the cells in contact with the antigen , which in turn eliminates both proliferate response and cytokine secretion [ 8 , 9 ] . in humans ,
several t - cell subtypes with an immune - suppressive function , generically named regulatory t cells ( treg ) , have been extensively studied [ 10 , 11 ] .
the main role of all these cell subsets is to maintain the integrity of the body by avoiding excessive immune responses that may result in harmful immune pathology , as well as preserve a state of tolerance to innocuous substances . il-10 and
tgf- secreted by these tregs play an important role in the immune regulatory response [ 1315 ] .
several common polymorphisms have been identified in the promoter region of both cytokines , including 1082g > a and 592c > a for il10 and 509c > t for tgfb genes , some of these polymorphisms being associated with allergic diseases [ 1619 ] .
olive tree pollen is one of the most important causes of respiratory allergy in the mediterranean area .
olea europaea pollen induces mainly nasal and conjunctive symptoms , although it may also induce asthma exacerbations in areas with high levels of o. europaea pollen in the atmosphere . in jan , a region in southern spain
, there is a very high level of pollen ( 500 to 1000 grains / m during pollen season , with peaks of more than 5000 grains / m ) and a high prevalence of asthma . to date , at least 20 proteins with allergenic activity have been described in olive pollen . among them , ole e 1 is the most frequent sensitizing allergen .
besides ole e 1 , twelve additional allergens have also been isolated and purified from olea europaea pollen extract , some of which are major allergens in areas with high levels of pollen exposure , such as ole e 2 and ole e 10 [ 21 , 22 ] .
previously our group described specific genetic and environmental factors associated with olive pollen allergy [ 2327 ] , as also a strong association between ole e 10 and ole e 2 specific sensitizations and bronchial asthma clinical phenotype , being these sensitizations restricted by different hla class ii antigens .
more recently we have described how , during the pollen season , olive pollen allergic patients showed a statistically significant decrease of tgf- ( regulatory cytokine ) .
this result was consistent with a significant decrease in relative foxp3 mrna expression ( marker of regulatory t - cell cytokines ) and with the lower number of regulatory t cells , indicating a lack of regulatory mechanisms in olive pollen allergic subjects during the pollen season .
the subjects were selected from a region in southern spain with particularly high pollen counts during the pollen season and a high prevalence of asthma .
taking into account all of these previous results , we analyzed the role of 6 polymorphisms of genes previously associated with allergy and asthma , in a population of olive pollen allergic patients , with an extremely high incidence of asthma : tnfa ( g-308a , c-857 t , and c-1031 t ) as proinflammatory gene and il10 ( c-571-a and a-117 g ) and tgfb ( c-509 t ) as genes related to regulatory response .
one hundred forty - six unrelated patients were recruited at the allergy service of the  complejo hospitalario de jan .
 all patients fulfilled the following criteria : seasonal rhinitis and/or asthma from april to june , a positive skin prick test for o. europaea pollen extract ( alk abell , madrid , spain ) , and no previous o. europaea immunotherapy .
fifty healthy subjects from the same geographic area were recruited as a control group . clinical assessment performed in these patients
patients recorded symptom scores , drug requirements , and peak expiratory flow rates ( pefr ) every day from april to june ( pollen season ) .
individual nasal and eye symptoms ( sneezing , blockage , running , redness , and itching ) and chest symptoms ( breathlessness , wheezing , and tightness ) were recorded on a scale of 03 ( 0 = no symptoms ; 1 = mild symptoms ; 2 = moderate symptoms ; 3 = severe symptoms ) .
an asthma day fulfilled at least one of the following criteria : asthma symptoms score 2 , morning pef 20% lower than the mean morning pef of the last 7 days before pollination , and daytime use of salbutamol twice or more compared with the mean use of the last 7 days before pollination .
olive tree pollen and olive pollen allergens were isolated as previously was described [ 29 , 30 ] .
total serum ige levels and specific o. europaea extract ige antibodies were determined by pharmacia systems ( ige enzyme immunoassay and uni - cap , uppsala , sweden ) .
allergen - specific ige antibody measurements ( ole e 1 , ole e 2 , and ole e 10 ) of allergic sera were performed by enzyme - linked immunoassay ( elisa ) .
all 146 patients and 50 controls were tested for olive pollen allergy by a skin prick test with o. europaea whole extract and olive pollen purified allergens , following the recommendations of the european academy of allergy and clinical immunology ( eaaci ) .
the patients were also tested with a panel of common aeroallergens , including dermatophagoides pteronyssinus , blattella germanica , dog and cat dander , alternaria alternata , aspergillus fumigatus , cladosporium herbarum , lolium perenne , cynodon dactylon , cupressus sempervirens , platanus acerifolia , artemisia vulgaris , chenopodium album , salsola kali , and parietaria judaica ( abell , madrid , spain ) .
tnf- and il-10 sera levels were analyzed by flow cytometry , using the bd cytometric bead array ( cba ) , human th1/th2 cytokine kit ( becton dickinson , bd , san diego , ca ) .
flow cytometric analysis was performed using a facscalibur flow cytometer ( bd immunocytometry systems , san jose , ca ) .
tgf- sera levels were analyzed by a commercial elisa ( bd , san diego , ca , usa ) .
genomic dna from all individuals was genotyped for 6 polymorphisms : tnfa ( g-308a , c-857 t , and c-1031 t ) , il10 ( c-571-a and a-1117 g ) , and tgfb ( c-509 t ) .
polymorphisms were analyzed according to the applied biosystems allelic discrimination assay - by - design protocol ( ab-7500 real time pcr system ) .
single nucleotide polymorphisms ( snps ) in research and their identifier names , locations , alternative names , and dbsnp identification are shown in table 1 .
comparisons of phenotypic ( olive pollen sensitized versus controls , asthmatic versus rhinitis ) or genotypic frequencies between groups were measured by fisher 's exact test or by chi - square test .
cochran and mantel - haenszel and 2-factor anova tests were used for the statistical comparison of clinical parameters ( qualitative and quantitative , resp . ) between sensitized and nonsensitized groups .
multiple regression analysis was used to examine quantitative traits ( total ige , specific ige to o. europaea whole extract , specific olive pollen allergens ige , and soluble cytokines serum levels ) .
the control group included 33 females ( 66% ) and 17 males ( 34% ) , with a mean age of 37.6 years ( range 2358 years ) .
these individuals were selected from the same geographical area and were free from any allergic symptoms .
all the allergic subjects ( n = 146 ) showed significant ige antibody levels against pollen crude extract , but different frequencies of sensitization were observed for the purified o. europaea allergens ( table 2 ) .
ole e 1 , ole e 2 , and ole e 10 are major allergens in our population ( > 50% of recognition frequency ) .
genotype and allelic frequency distribution in the patient and control groups and the genotype and allelic distribution according to asthma or rhinitis in our patient population are shown in table 3 .
the only statistically significant polymorphism associated with o. europaea allergy was related to the tgfb - c509-t polymorphism .
we found a statistically significant increase in the phenotypic percentage of the ct heterozygous genotype in patients compared with controls ( 56.8% versus 40% , p = 0.03 , odds ratio , or , 1.97 ; 95% confidence interval , ci , 1.023.8 ) ( table 3 ) .
the sera of ct heterozygous patients showed the lowest levels of soluble tgf- ( 10849.71  12613 
pg / ml ) homozygous patients , but the differences were not statistically significant ( data not shown ) . when we analyzed the genotype distribution of the 6 polymorphisms according to the clinical phenotype of asthma or rhinitis , no relevant associations were found ( table 3 ) . however , the analysis according to specific ole e 2 and ole e 10 sensitizations showed several relevant results which are summarized in table 4 .
firstly , the study of 2 polymorphisms of il10 ( c-571-a and a-1117 g ) in patients with ole e 2 sensitization showed a statistically significant decrease of cc homozygous il10 - 571c > a genotype compared with ole e 2 nonsensitized patients ( 50% versus 70.5% , p = 0.029 , or , 0.41 ; 95% confidence interval , ci , 0.190.82 ) . this protection was higher when patients with asthma and ole e 2 sensitization were compared with ole e 2 nonsensitized patients with asthma ( 49.4% versus 78.6% , p = 0.008 , or , 0.26 ; 95% ci , 0.090.72 ) .
this genotype showed intermediate levels of il-10 cytokine ( 7.77  5.4  pg / ml ) compared with the other 2 genotypes ( aa homozygous : 5.9  6.1  pg / ml , ca heterozygous : 8.76  5.3  pg / ml ) . 

ca heterozygous il10 - 571c > a genotype was increased in patients with asthma and ole e 2 sensitization compared with ole e 2 nonsensitized patients with asthma ( 45.7% versus 21.4% , p = 0.02 , or , 3.08 ; 95% ci , 1.138.4 ) .
secondly ( table 4 ) , we have found a statistically significant increase of ga heterozygous tnfa-308g > a genotype in ole e 10 sensitized patients compared with olive pollen allergic patients without sensitization to ole e 10 allergen ( 25.3% versus 11.9% , p = 0.04 , or , 2.5 ; 95% ci , 1.026.1 ) .
this polymorphism showed intermediate levels of tnf- cytokine ( 7.77  5.4  pg / ml ) compared with the other 2 genotypes ( gg homozygous : 7.01  4.8  pg / ml , aa homozygous : 9.3  6.1  pg / ml ) .
sensitization to specific allergens is a complex response controlled by both genetic and environmental factors .
preliminary studies reported that the allergograms of olive - allergic patients living in areas with high level of pollen production ( such as some regions as andalusia in spain ) are notably different from those obtained in areas of low - level exposure ( e.g. , madrid in the central region of the country ) . besides , the level of each allergenic protein in the pollen grain could be a second parameter to be taken into consideration .
in fact , a great variability in the allergen composition of o. europaea pollen has been described .
the reasons behind this variability are unknown , but they could be due to the existence of several genetic strains , climatic conditions , and specific culture techniques .
previous works showed how , in areas with high levels of antigenic load , olive pollen allergic patients were sensitized to allergens that are  minor allergens  ( frequency of recognition lower than 50% ) in areas with low antigenic load [ 21 , 22 ] .
this sensitization ( mainly related to ole e 2 and ole e 10 ) was associated with a worse clinical prognosis , with a very high presence of asthma and more days of symptomatology .
this population was analyzed for inflammatory cytokine polymorphisms ( il-13 , il4ra , il5 , and2adr genes ) finding some relevant associations , mainly related to risk factor for olive pollen allergic sensitization .
very recently we demonstrated how several immuneregulatory elements are decreased ( tgf- and foxp3 ) in olive pollen allergic patients exposed to extremely high olive pollen antigenic load . .
with these previous results , the aim of this work was to determine whether some of the genes previously associated with asthma and immune regulation are important in these specific sensitizations .
to do so , we decided to analyze the relationship between 6 genetic polymorphisms of tnfa ( g-308a , c-857 t , and c-1031 t ) as a proinflammatory gene and il-10 ( c-571-a and a-117 g ) and tgfb ( c-509 t ) as being related to regulatory response and specific sensitization to o. europaea pollen in a well - characterized spanish population of patients with olive pollen allergy and a high prevalence of asthma ( 74.7% ) . in spite of the small size of our control population
firstly , we have shown that the ct genotype of tgfb c-509 t was the only polymorphism ( of the 6 analyzed ) associated with the genetic regulation of o. europaea allergy ( table 3 ) .
tgf- is a multifactorial cytokine that plays key roles in normal cellular processes and diseases , such as t - cell activation and proliferation , and may be essential in modulation of allergic airway inflammation and airway remodeling .
the tgf- gene is located on chromosome 19q and there are a number of polymorphisms within this gene that are believed to have a role in tgf- expression .
tgfb ( c-509 t ) is presented within a proximal negative regulatory region , and the t allele has been associated with higher tgf-1 plasma levels . due to the inconclusive results on the association of tgf- polymorphisms and asthma , 3 years ago , a meta - analysis   was reported studying the role of tgf- and asthma , based on 16-case control sets .
the authors concluded that the c-509 t polymorphism ( carriers of the t allele ) could be a risk factor for asthma , mainly in the asian population and adults .
our results are in agreement with the association of tt homozygous and the highest levels of soluble tgf- , but this genotype was infrequent in our population . however , the heterozygous ct genotype , which showed the lowest levels of tgf- , was a risk factor for olive pollen allergy in our population .
these data support our previous results in which we reported a tgf- decrease in the sera of olive pollen allergic patients , in an independent cohort of subjects , selected from the same area .
all of these results reinforce our previous idea that olive pollen allergic patients have a decreased tgf- dependent regulatory response , thereby pointing to the importance of evaluating , when feasible , the combination of levels and cytokine polymorphisms . according to ole e 2 and ole e 10 specific sensitizations
firstly , the cc homozygous il10 - 571c > a genotype could be a protective factor for ole e 2 sensitization and mainly for patients with asthma and ole e 2 sensitization compared with asthmatic - patients nonsensitized to ole e 2 .
this polymorphism showed intermediate levels of il-10 cytokine ( 7.77  5.4  pg / ml ) compared with the other 2 polymorphisms ( aa homozygous : 5.9  6.1  pg / ml , ca heterozygous : 8.76  5.3  pg / ml ) .
in contrast , the ca heterozygous il10 - 571c > a genotype was increased in patients with asthma and ole e 2 sensitization compared with ole e 2 nonsensitized patients with asthma ( 45.7% versus 21.4% , p = 0.02 , or , 3.08 ; 95% ci , 1.138.4 ) .
a recent meta - analysis suggested that il-10 promoter polymorphisms were associated with asthma risk .
this work revealed significant associations between 1082a / g and 592a / c polymorphisms and asthma .
however , there was no significant association between 819t / c polymorphism and asthma risk .
other studies showed that a polymorphism at the 592 position of il-10 is associated with its regulation of expression and recently the association of this polymorphism and immune - related diseases has been studied including type 2 diabetes with and without nephropathy , multiple sclerosis , and asthma . secondly
, we have found a statistically significant increase of the ga heterozygous tnfa-308g > a genotype in ole e 10 sensitized patients compared with olive pollen allergic patients without sensitization to the ole e 10 allergen ( 25.3% versus 11.9% , p = 0.04 , or , 2.5 ; 95% , ci , 1.026.1 ) ( table 4 ) .
this genotype showed intermediate levels of the tnf- cytokine ( 7.77  5.4  pg / ml ) compared with the other 2 genotypes ( gg homozygous : 7.01  4.8  pg / ml , aa homozygous : 9.3  6.1  pg / ml ) .
the 308g / a polymorphism in the tnf- gene has been extensively investigated for its association with asthma ; however , the results of different studies have been inconsistent .
two years ago a meta - analysis was carried out to analyze the association between the 308g / a polymorphism of the tnf- gene and asthma risk .
the results suggested that the tnf-   308g / a polymorphism contributes to susceptibility to asthma and , specifically , significantly elevated risks of asthma were associated with a allele carriers in the atopic population but not in the nonatopic population .
more recently , it has been described that the genetic polymorphisms of tgf-1 and tnf- are associated with asthma .
this same study also described tgf-1 as being a modulator of atopy , and both tgf-1 and tnf- were found to be elements that modulate clinical severity and airway obstruction in an additive manner .
overall , our results suggest a role of tgf-1 in olive pollen sensitization and tnf- and il-10 genotypes in the asthma induced by specific olive pollen allergens .
combined analysis of multiple genetic polymorphisms with different allergic phenotypes could be a useful tool for identifying profiles of risk or prevention of these disorders .
 sensitization to specific olive pollen - allergens ( ole e 2 and 10 ) has been correlated with a clinical pattern of asthma . 
 this study analyzes the association between several polymorphims of tnfa ( g-308a , c-857 t , and c-1031 t ) , il10 ( c-571a and a-1117 g ) , and tgfb ( c-509-t ) and these sensitizations . 
 these polymorphisms were genotyped by allelic discrimination , in olive pollen - allergic patients ( phenotyped for specific ole e 2 and 10 sensitizations ) and healthy controls . 
 levels of serum - soluble cytokines were correlated with specific genotypes and clinical phenotypes . 
 the results showed that heterozygous tgfb c-509 t genotype , besides having the lowest sera tgf- levels , was significantly increased in olive pollen - allergic patients compared with controls . according specific sensitizations , cc genotype of il10 c-571a could be a protective factor for ole e 2 sensitization and mainly for asthmatic ole e 2 sensitized patients compared with asthmatic non - ole e 2 sensitized patients ( or : 0.26 , p = 0.008 ) . 
 in contrast , heterozygous ca genotype was increased in ole e 2 asthmatic subjects compared to asthmatic non - ole e 2 sensitized patients . 
 lastly , heterozygous tnfa g-308a genotype was associated with ole e 10 sensitization ( or : 2.5 , p = 0.04 ) . in conclusion , 
 these results suggest a role of tgf-1 in olive - pollen sensitization and tnf- and il-10 genotypes in the asthma induced by specific olive - pollen allergens . 
1. Introduction
2. Material and Methods
3. Results
4. Discussion