allergic rhinitis ( ar ) is a common chronic illness affecting 10% to 20% of the children worldwide , and its prevalence has significantly increased among children over the last two decades .
ar is clinically defined as a symptomatic disorder of the nose induced by an ige - mediated inflammation after exposure to an allergen . in the immediate response to an antigen ( the early - phase allergic reaction ) ,
this causes the characteristic nasal symptoms which include sneezing , itching , rhinorrhoea , and nasal congestion .
a late - phase allergic reaction occurs approximately 4 to 12 hours after antigen exposure , with nasal congestion as the predominant symptom . during this phase , inflammatory cells such as eosinophils and t cells infiltrate the mucosa .
the th2-polarized immune response especially , with secretion of cytokines such as il-4 , il-5 and il-13 and a defect of th1 cells producing ifn - gamma , play a central role in the late phase allergic response [ 24 ] .
it has been observed that the immune response to allergens is the result of a fine balance between allergen - specific th2 cells and t regulatory cells ( tregs ) .
tregs include a broad spectrum of cd4 t - cell subpopulations such as natural thymic - derived treg cells ( ntreg ) characterized by their cd4cd25 phenotype expressing high levels of foxp3   and inducible tr1 cells ( itreg ) which suppress immune function by the secretion of il10 and tgf - beta [ 79 ] .
from the recent literature it becomes clear that it may be possible to interfere with the natural course of ar by specific immunotherapy ( sit ) [ 913 ] .
sit may induce changes that skew th2- to th1-type responses   and by means of tolerance induction .
one of the key mechanisms behind this tolerance induction includes induction of treg - type cytokines ( il-10 and tgf - beta ) [ 9 , 10 ] and increased frequencies of cd4cd25 cells .
there is an unexplained variability in the clinical course of allergic rhinitis  with persistence in some individuals and either progression or remission in others .
there is a lack of longitudinal investigations examining the natural history of ar . in the present study
, we investigate the natural course of ar regarding clinical manifestations of the disorder and immunological responses in a followup of 2 years in children with house dust mite allergy .
we evaluate clinical symptoms , in vivo skin responses due to allergen and in vitro allergen - specific effector t cells and treg cells during the natural course of the disease .
this study is a part of the randomized placebo - controlled trial isrctn91141483 which evaluates sublingual immunotherapy with low - dose house dust mite allergen ( 2.03  mcg / ml or 700  bu / ml der p1 ) in children with ar [ 15 , 16 ] .
the study was approved by the ethical review board of erasmus mc - university medical center rotterdam .   as described elsewhere ,   neither this low - dose form of slit was effective with regard to the reduction of allergy complaints , asthma complaints , intake of rescue medication and disease - specific quality of life , nor was there any difference in reported side effects ( both local and general ) between slit or placebo treatment , indicating that the dosis of the investigated product was a low to mediate reaction .
analysis of placebo ( n = 30 ) and verum ( n = 29 ) groups separately in children participating in the present study revealed neither differences between verum and placebo regarding clinical symptoms ( p value varied between 0.06 and 0.86 ) nor in vivo skin responses ( p value between 0.17 and 0.80 ) or in vitro measurements of pro - inflammatory markers ( p value between 0.16 and 0.85 ) or regulatory t cells ( p value between 0.10 and 0.89 ) .
therefore , we decided to merge the two groups and consider them as one group which received placebo during two years .
this merged group forms the basis for the present study . from the main trial ,
fifty - nine children were randomly selected and invited to participate in this elaborate study . as in the main study ,
children ( aged 618 years ) with ar and established hdm allergy were selected from the electronic medical records in general practice .
the inclusion criteria were presence of specific ige antibodies to hdm in serum ( 0.7 
ku / l ) , a history of allergic rhinitis during at least 1 year and a nasal symptom score of at least 4 out of 12 ( see below ) . before scoring symptoms ,
nasal corticosteroids were withheld for 4 weeks before the study period . during the total study period patients
were allowed to use rescue medication ( provided by us , i.e. , levocetirizine tablets , xylometazoline nasal spray , and levocabastine eyedrops ) or another allergy or asthma medication as long as they wrote it down on their diary cards ( see below ) .
the presence of asthma was assessed using the international study of asthma and allergies in childhood ( isaac ) core questionnaire .
all participants or their parents scored their nasal , eye , and asthma ( lung ) symptoms on diary cards at baseline ( 1 month in october or in november ) and after two years ( 3 months in september  december ) .
nasal symptoms ( sneezing , itching nose , watery running nose , and nasal blockage ) , eye symptoms ( itching , tearing , and redness ) , and asthma symptoms ( wheeze / breathless and dry cough during night ) were scored on a 03 scale ( 0 = none , 1 = mild , 2 = moderate , and 3 = severe ) . in total , a maximal daily cumulative nasal symptom score of 12 , eye symptom score of 9 , and lung symptom score of 6 could thus be obtained .  
a mean symptom score was determined by calculating the mean daily score over the entire diary period ( i.e. , one month at baseline and three months after 2 years ) .
only diaries with at least 50% of the filled - out pages were included in the analyses . in case patients used additional medication for their allergy or asthma , they were asked to document their use in the patient diary throughout the 2-year period .
allergy skin testing was performed at baseline and after 2 years by intracutaneous injection of 0.02  ml dermatophagoides pteronyssinus in the forearm ( concentration 30  sq 
u / ml , manufactured by alk - abell , nieuwegein , the netherlands ) .
we chose to perform an intracutaneous skin test rather than the usual skin prick test because intracutaneous injection of the allergen is the most feasible and convenient way to induce a late - phase response after the early - phase skin response .
as a positive control , histamine ( concentration of 0.01  mg / ml ) was injected , and the negative control was dilution buffer .
reactions were read after 15  min ( early response ) and after 6  h ( late response ) .
the area of the skin response in mm was measured by a specially developed scanning programme .
the early - phase response was expressed as a histamine equivalent intra - cutaneous index or heic index .
the late - phase response was expressed as the area of the skin response in mm .
serum ige antibodies to d. pteronyssinus were determined using the cap - rast system ( pharmacia , uppsala , sweden ) , according to the manufacturers instructions .
blood was drawn at baseline and after 2 years before skin testing . peripheral blood mononuclear cells ( pbmcs )
were isolated from heparinized blood by density centrifugation on ficoll - paque plus ( ge healthcare , uppsala , sweden ) .
cells were resuspended in complete medium ( rpmi + hepes + glutamax supplemented with gentamicin ( gibco , gibco brl , life technologies , rockville , md , usa ) and 5% heat - inactivated human serum ( sanquin , rotterdam , the netherlands ) and stimulated by culturing in the presence or absence of 2  ir / ml d. pteronyssinus ( stallergnes , france ) .
all cell cultures were performed in quadruplicate in a final volume of 200  ml with a cell concentration of 2  10  cells / ml in 96-well round - bottomed microtitre plates . to determine hdm - specific proliferative responses , cells were cultured for 5 days at 37c , 5% co2 , and 95% humidity . during the last 16  h , cells were pulsed with 0.5  ci / well h thymidine ( pharmacia , uk ) .
radioactivity was measured with a -plate reader , and the proliferative capacity was assessed by the stimulation index ( si ) , calculated as the ratio of mean h thymidine uptake in stimulated to that in non - stimulated cultures .
the si was considered positive when it exceeded 2.0 . to determine hdm - specific cytokine production , 1  ml of pbmcs with a concentration of 2  10 cells / ml was cultured in duplicate in a 24-well plate with or without 2  ir / ml d. pteronyssinus ( stallergnes france ) .
after 5 days of culture , supernatants were harvested and stored at 20c until testing .
il-5 , il-13 , ifn- , il10 , and tgf - beta cytokine production was measured following the manufacturer 's instructions ( ebioscience , san diego , ca , usa or r&d systems , abingdon , uk ) .
hdm - induced cytokine production was assessed by subtracting the cytokine concentration of nonstimulated from that of stimulated culture supernatants . for phenotypical analysis
, cells were washed with facs buffer ( 0.05% nan3 , 2% bsa in pbs ) and stained for 30  min on ice protected from light .
for the detection of cd4cd25 t cells the following anti - human antibodies were used : fitc - conjugated anti - hcd3 ( ucht1 , ebioscience ) , pe - conjugated anti - hcd25 ( m - a251 , bd ) apc - conjugated anti - hcd4 ( rpa - t4 , ebioscience ) .
aspecific binding was prevented by the use of 2% heat inactivated ( hi ) human ab serum during staining .
data acquisition was performed by flow cytometry ( facscalibur ; bd biosciences ) and data analysis was performed using flowjo software ( treestar , coata mesa , ca ) ) .
quantitative rt - pcr for hfoxp3 was performed on rna isolated from hdm - stimulated pbmc 's ( as described above ) .
total rna was isolated with rneasy mini kit ( qiagen ) and treated with dnasei , according to manufacturer 's protocol .
100  ng rna was used as a template for cdna synthesis , using superscript ii reverse transcriptase ( invitrogen ) and random hexamer primers .
quantitative pcr was performed with taqman universal pcr mastermix ( applied biosystems ) , preformulated primers ( hfoxp3 and housekeeping gene hhprt ) and probe mixes (  assay on demand  , applied biosystems ) .
pcr conditions were 2  min at 50c , 10  min at 95c , followed by 40 cycles of 15  s at 95c and 60c for 1  min using an abi prism 7300 ( applied biosystems ) .
pcr amplification of the housekeeping gene was performed during each run for each sample to allow normalization between samples .
values are presented as mean ( with standard deviation , sd ) , median ( with interquartile range , iqr ) , and median estimated difference between baseline ( year 0 ) and year 2 .
comparisons were performed by nonparametric analysis , using wilcoxon signed rank test for comparisons between baseline data and data after 2 years .
the spearman rank test ( rs ) was used to assess the correlations between year 0 and year 2 .
the baseline characteristics of the 59 children included in the study are described in an earlier study . of the 59 children , 42 ( 71.8% ) completed the 2-year study period .
mean and median values ( with sd or iqr ) of clinical symptoms , in vivo allergen - induced skin tests , and in vitro specific pro - inflammatory and regulatory responses are displayed in table 2 .
clinical symptoms of the nose , eye and lung all decreased significantly two years after baseline measurement .  
0.0001 ) for nose symptoms , 0.29 ( p = 0.02 ) for eye symptoms and 0.33 ( p < 0.0001 ) for lung symptoms . there was a significant positive correlation between values of year 0 and year 2 for all clinical symptoms ( rs between 0.56 and 0.61 ; p < 0.0001 ) .
reactions of in vivo hdm specific skin tests ( both early as well as late skin reactions ) did not significantly change when measured two years after the baseline measurement ( table 3 , p = 0.09 or p = 0.07 , resp . ,
table 3 ) . whereas there were no significant changes in time , skin responses showed a positive correlation when skin responses of year 0 and year 2 were compared ( rs = 0.44 ; p = 0.005 for the early skin response or rs = 0.5 ; p = 0.001 for the late skin response ) .
relevant proinflammatory variables for ar ( i.e. , hdm specific ige , t - cell proliferation , il-5 , il-13 , and ifn - gamma ) were comparable between year 0 and year 2 ( table 3 , p > 0.05 except for ifn - gamma which showed a significant decrease ) . whereas values did not significantly increase or decrease , there was a significant correlation between data from year 0 and year 2 for all these markers ( rs between 0.35 and 0.86 ) , except for il-5 ( rs = 0.27 ) .
the frequency of regulatory cd4cd25 cells had increased two years after baseline measurement . however ,
other markers characteristic for regulatory cells ( foxp3 , il-10 , tgf - beta ) did not increase in time ( in fact for il-10 a decrease could be detected , table 3 ) .
correlations between year 0 and year 2 for these variables were nonsignificant ( except for il-10 ) . 

figure 1 summarizes the above - described findings with a decrease of clinical symptoms after 2 years in combination with a positive correlation between data of year 0 and year 2 ( represented by nasal symptoms in figure 1(a ) ) , comparable skin - test values for year 0 and year 2 , with a positive correlation between the two years ( early skin test is presented in figure 1(b ) ) , no significant change in proinflammatory reactions ( in figure 1(c ) represented as spec ige ) with a high correlation between measurements during the two years , and an increase of cd4cd25 treg cells without a correlation between the two years of measurement ( figure 1(d ) ) . as can be seen in this figure ,
there is no difference in any of the values between patients receiving placebo treatment or verum - slit treatment ( which they received during a period of two years in the original trial ) .
results of the present study show clinical symptoms rated by allergic patients at baseline and two years later in combination with immunological parameters measured in vivo or in vitro . in a time period of two years , clinical symptoms of the nose , eye , and lung significantly decrease in children with house - dust - mite - induced ar .
although children rate their allergy complaints as being less than at the beginning of the study , this decrease is not reflected in a decrease of one of the immunological parameters .  
a possible explanation for the reduction of symptoms is that allergic rhinitis decreases in time .
whereas there are many studies determining symptoms during followup of slit treatment , there is little information on the possibility of spontaneous remission of allergic rhinitis [ 12 , 20 ] .
some studies have reported the relationship between age and improvement or decline of rhinitis over the years [ 2123 ] . during an 8-year follow - up of patients with allergic rhinitis ,  nihln et al .   found a 20% remission rate , which was higher in older patients .
described that remission with allergic rhinitis was more likely when the duration of the disease was shorter than 5 years .
another plausible explanation for the reduction of symptoms might be that , due to participation in a study , patients are more aware of their allergy and take more care of their complaints , which may result in a reduction of their symptoms . in this study
the effect of placebo treatment is substantial in patients participating in a study evaluating slit . whereas from this study
it becomes clear that complaints of patients with ar decrease over time , it is demonstrated in a birth cohort study that the prevalence of patients developing ar increases over the time . therefore , when children get older the change of developing ar is increasing , but for the patients with complaints of ar the change of having comparable complaints two years later is decreasing .
skin responses ( measured directly after intradermal allergen injection and a late response with induction of inflammatory cells ) remain constant over time . whereas these responses are comparable at baseline and after two years , these data show a positive correlation , which means that values which are high at baseline are also high after two years , and low values remain low during the two years .
this is underlined by a study of bodtger and linneberg   who describe that in adult patients with house dust mite allergy , only 5.5% of the patients show a remission of specific ige after eight years , whereas remission of symptoms occurred in 32.4% of the patients . also , a recent study of kong et al .  
ar is characterized by high levels of allergen - specific ige and secretion of the th2 cytokines il-4 , il-5 and il-13 [ 3 , 27 , 28 ] . in this study , we do not see a decrease of these inflammatory markers two years after the baseline measurement , whereas patients rate their symptoms as better than before .
in contrast to our findings , in patients who do not receive immunotherapy , allergen - specific t - cell responses can be reduced by means of specific immunotherapy ( sit ) .
the literature shows that sit is able to reduce allergic complaints in combination with a reduction of allergen - induced t - cell proliferation   and a shift in the th2/th1 cytokine balance , favouring type-1 t cells .
this shift could be a result of either decreased il-4 production after allergen stimulation   or enhanced ifn- expression .
some studies have found a reduction of allergen - specific ige in combination with an increase of specific igg levels due to sit [ 3032 ] . in the large randomized study ,
of which this present study is a component , we determined igg1 and igg4 levels in 163 children at year 0 and year 2 .
these results show that igg levels remain constant over the time with a very high correlation between year 0 and year 2 ( r is 0.74 for igg and 0.83 for igg4 ) , which emphasises that the dose of allergen administrated might have been insufficient to induce tolerance .
they describe a significant increase of il-10 , and tgf- in patients undergoing sit compared to untreated atopic patients ( or patients receiving placebo ) .
moreover , the number of cd4cd25 cells expressing the transcription factor foxp3 was increased after allergen stimulation in the immunotherapy group . in our study
, results related to regulatory t cells are conflicting . whereas we see an increase of cd4cd25 cells
, there is no correlation between data of year 0 and data of year 2 .
moreover , other markers regarding regulation of the immune response ( i.e. , foxp3 , il-10 and tgf - beta ) do not increase .
, it is unlikely that regulatory cells play a role in this present study ; self - rated allergy symptoms do decrease , but this is not the result of an increase of regulatory cells .
however , we have to keep in mind that the number of patients used to answer this question is small , and we should be conservative with respect to this statement .
a limitation of the present study is that only 42 out of 59 children ( 71% ) wanted to participate two years later .
some of these children were lost to followup ( a total of seven children ) , and others still participated in the main slit study but did not want to do additional skin testing or venapuncture ( a total of ten children ) .
however , the total number of 42 children is still an acceptable number to study possible immunological mechanisms in relation to symptoms in a period of two years . from this study
, it can be concluded that whereas , comparable to sit , allergic complaints decrease , the immunological changes of hdm - specific t - cell activity ( both effector cells and regulator cells ) which are observed after immunotherapy do not change .
this decrease in clinical symptoms might be explained by the placebo effect , since patients participated in a study and took ( placebo ) medication during a period of two years .
it may be concluded that sit plays an active role in tolerance induction in the patients immune system ; whereas during the two years of this study , allergic complaints reduce without any difference in immunological parameters as determined in this study .
 
 background . 
 allergic rhinitis is a disease with polarization towards th2 and a defect of regulatory t cells . 
 immunological changes have been reported after immunotherapy treatment . 
 however , there is not much known about the natural course of allergic rhinitis with respect to clinical manifestation and the relation with immunological responses . objective . to evaluate clinical symptoms of allergic rhinitis , in relation to in vivo allergen - specific skin responses and in vitro allergen - specific effector and regulatory t cells determined at baseline and after two years . 
 methods . from a large trial 
 , 59 children were randomly selected . 
 the following variables were compared : clinical symptoms , allergen skin tests , specific ige , t - cell proliferation , il-5 , il-13 , ifn - gamma , il-10 , tgf - beta , cd4+cd25hi cells , and foxp3 expression . 
 results . 
 allergic symptoms had decreased after two years . 
 whereas skin test reactions correlated between years 0 and 2 , there was no change in the size of the reaction . 
 also , proinflammatory reactions did not change after two years , with a positive correlation between years 0 and 2 . 
 no relevant changes were observed with respect to regulatory cells . 
 conclusion . whereas , comparable to immunotherapy , allergic complaints decrease , the immunological changes of specific t - cell activity ( both effector cells and regulator cells ) which are observed after immunotherapy , do not change . 
1. Introduction
2. Methods
3. Results
4. Discussion