BACKGROUND
celiac disease  is an immune-mediated enteropathy triggered by the ingestion of gluten-containing grains  in genetically susceptible individuals  <cit> .
its estimated prevalence in western countries is near 1%  <cit> .
it is generally agreed that cd is a t-cell mediated disorder in which gliadin derived peptides activate lamina propria t lymphocytes which release proinflammatory cytokines  <cit> .
to date, several peptides including alpha- and gamma-gliadins, have been reported to activate cd4+ lymphocytes via their interaction with hla-dq <dig> and -dq <dig> heterodimer on antigen presenting cells   <cit> .
recently, scientific evidence showed microecological changes in the intestinal tract of celiac infants, suggesting a potential role of gut microbiota in cd.
alterations in the composition of faecal short-chain fatty acids in cd patients compared with those of healthy controls have been demonstrated  <cit> .
imbalance in the composition of duodenal microbiota or in faecal bacterial communities of children with cd has also been reported  <cit> .
rod-shaped bacteria have been observed in both gluten-free diet -treated and untreated pediatric patients'mucosa, along with a distinctive lectin pattern  <cit> .
the present study was carried out to add further information on the characterization of intestinal microbiota of cd patients, a variable that may represent a new piece of the intriguing puzzle of cd illness.
for this purpose we analyzed by ttge the composition of duodenal mucosa-associated microbiota in the same cohort of gfd untreated and treated cd children and in controls.
this prospective study was performed to compare the influence of the disease status on gut microbial composition and to study whether the microbial imbalance could be a peculiar characteristic of the disease.
RESULTS
agglomerative hierarchical classification
the ttge profiles of pcr amplicons obtained with universal primers were firstly analyzed by xlstat software.
the resulting dendrogram presented two major well-defined clusters .
the first cluster  groups 8/ <dig> of control patients, while the second one  groups 18/ <dig> of cd patients .
these results highlighted the presence of a dominant microbiota related to the celiac disease, irrespectively to the disease status.
the average number of bands in ttge profiles, calculated by digidoc-it software, was significantly higher  in celiac children  than in controls , indicating that duodenal mucosa of cd patients showed a higher diversity of associated bacterial population.
the average number of bands in ttge profiles was also significantly higher in active disease than inactive one .
moreover, interindividual analysis showed a mean dice similarity index of ttge profiles of  <dig> % ±  <dig> % within active disease group,  <dig> % ±  <dig> % within inactive disease group and  <dig> % ±  <dig> % within control group.
otherwise, mean dice similarity index between celiac individuals before and after gfd treatment was  <dig> % ±  <dig> %.
ecological features
shannon-wiener index  analysis was performed to determine a measure of estimated diversity within each biopsy sample by ttge profiles.
mean shannon-wiener index value differed significantly between active  and inactive  cd patients, a similar result was obtained between active cd patients and controls.
the shannon-wiener index among inactive cd patients and controls was not significantly different.
.
the variance values  relative to active group revealed a minor data dispersion than inactive and control ones, indicating a more similar microbial biodiversity between its members .
the carrying capacity of the duodenal system showed mean rr values of:  <dig>  ±  <dig> ,  <dig>  ±  <dig> ,  <dig>  ±  <dig>  for active, inactive and control group respectively.
the mean rr values were highly different among the three groups .
factor discriminating analysis
to improve the analysis of ttge profiles the more discriminating fda approach was performed.
the principal component analysis  transformed data showed a well-defined separation between controls, active and inactive celiac groups , with a confusion matrix of  <dig> % .
results from this analysis indicated that the ttge profiles were sufficient to predict the patient category  with 100% predictiveness, suggesting the importance of duodenal microbiota in this pathology.
partial least square discriminant analysis
pls-da was employed to investigate peculiar ttge bands having discriminatory power in separating ttge profiles in the three groups studied, utilizing the raw data .
the score plot confirmed a division between the patients' groups.
interestingly, in patients n.  <dig> and  <dig> the ttge profiles of inactive status resulted closer to those of control group.
on the basis of pls-da score plot, it could be seen that cd patients and controls were separated along principal component  <dig>  component, whilst active and inactive cd patients were separated along principal component  <dig>  component.
fig  <dig> shows hierarchical discriminatory importance of the ttge bands for pc <dig> component and pc <dig> component.
the variable importance  mainly reflected the correlation between the ttge bands and all the patients groups along a specific principal component axis .
the bands with vip larger than  <dig> were picked.
the ttge bands picked partitioning cd and non cd-diagnosed patients were:  <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>  the picked ttge bands separating active and inactive cd patients were:  <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>
statistical evaluation of ttge bands occurrence by pls-da
the selected ttge bands obtained by pls-da analysis were statistically evaluated for their occurrence as reported in table  <dig>  the ttge selected bands  dividing cd and controls resulted all statistically significant .
in the separation between active and inactive cd patients, bands resulted statistically significant were:  <dig>   <dig>   <dig>   <dig>   <dig> and  <dig>  moreover, some of selected ttge bands run parallel with e. coli, p. distasonis and b. vulgatus gel markers used.
the parallelism is reported in tab.
<dig>
§ bands were self numbered according to the order of appearance  on the ttge gel and are listed in descending order of importance  in the pls-da model.
between parentheses are reported the species used in the gel marker that run parallel to specific ttge bands.
mann-whitney u-test, α =  <dig>
wilcoxon signed rank test, α =  <dig>
age at 1st biopsy
gfd duration
none of the patients underwent previous endoscopic assessment
biopsies were taken in the second part of duodenum
species-specific pcr
results obtained revealed a highly significant difference  in the prevalence of b. vulgatus , and e. coli  in cd patients versus controls.
a significant difference  was found in the prevalence of b. vulgatus  and in the prevalence  of clostridium coccoides group  in active cd patients versus inactive cd one.
no significant difference was found in the prevalence of bifidobacterium spp.
between cd patients and controls  and between active and inactive cd .
discussion
this is the first longitudinal study on the duodenal mucosa-associated microbiota, carried out on the same cohort of cd pediatric patients , showing a distinctive 'microbial structure' in celiac pediatric patients.
the most important results of this study, obtained through multivariate statistical analysis of ttge profiles, were: i) a dominant duodenal microbiota that could be linked to the disease status , outlining differences in the microbiota composition before and after gfd treatment; ii) a significantly higher diversity in dominant microbiota in patients with active disease vs the same in remission state, as well as in patients with active disease vs controls, as revealed by shannon-wiener index.
this higher duodenal microbial diversity in cd patients could have a possible harmful impact on the duodenal homeostasis.
iii) a higher inter-individual similarity in cd patients than controls, indicating a more homogeneous structure among microbial communities of celiac patients.
analyzing ttge profiles, the lowest carrying capacity and the lowest median number of bands found in the duodenal system of the control group can be attributed to an environment particularly adverse or restricted to colonization.
the nature of duodenal habitat is radically changed in cd patients, where the carrying capacity and the median number of bands in ttge profiles are much higher than controls, consequently a thriving colonization could be due to a more habitable environment.
it could be speculated that in duodenum the microbial life could be largely inhibited by different factors such the rapid transit of food , pancreatic juices or the rapid mucosal turnover.
is therefore likely that a relative small number of definite microbial species or groups are highly adapted to this particular habitat, then the number of ttge bands found in our control duodenal samples was lower than others found in different intestinal tracts  <cit> .
the dice index analysis of ttge profiles revealed that mucosa-associated microbiota differed markedly from one patient to another in control group, whereas cd patients showed a high inter-individual similarity.
moreover, before and after gfd treatment, there's a loss of  <dig> % of inter-individual similarity.
specifically, the similarity is lost in a homogeneous way between all celiac individuals, as showed by the high similarity dice index within active and inactive groups.
we may speculate that the change in the mucosa lectin patterns both in active and remissive cd, as demonstrated by forsberg  <cit> , could create more selective microbial adhesive patterns in duodenal mucosa of these patients, promoting a more similar interindividual mucosal colonization.
ttge bands, having discriminatory power in separating the three patients'groups, have been selected.
some of these ttge bands run parallel with e. coli, p. distasonis and b. vulgatus gel markers used.
the genera bacteroides, as reported by previous works  <cit> , was significantly increased providing a strong correlation between this microbial group and cd  <cit> .
moreover a high prevalence of potentially pro-inflammatory gram negative bacteria was found in the celiac patients' duodenum  <cit> .
furthermore, the presence of bacteria such e. coli and bacteroides spp has been related by other authors  <cit>  with mucin degradation and an increase in small intestinal permeability.
although the technique we used does not allow a specific characterization of microbial species or groups of this particular intestinal habitat, it provides a picture of modifications encountered by dominant bacterial groups/species profile of a sample in relation to different factors .
the presence/absence of bacterial species/groups might act as 'key' or 'regulatory' species leading to a different relative abundance of the present species.
to assess this, we need to improve our data by direct sequencing of ttge bands.
ttge profiles of 18/ <dig> cd patients in remission, with a duodenal histology not fully normalized, clustered together and away from controls.
interestingly, ttge profiles of  <dig> cd patients  with a fully histological duodenal normalization at gfd, clustered close to controls as reported by the pls-da score plot.
this would indicate an association between inflammatory status of intestinal mucosa and the kind of colonizing microbiota.
partial recovery of microbiota composition in the  <dig> patients with full histological normalization seems to indicate that the mucosa inflammation status is not the only factor driving the kind of microbial composition, but certainly is an influencing factor.
CONCLUSIONS
in conclusion, our data show a potential role of the duodenal microbiota in the cd pathogenesis.
common ttge profiles in cd patients are probably due to a similar intestinal habitat creating selective pressures that shape a peculiar dominant microbiota.
in addition, the occurrence of distinctive ttge profiles in celiac patients before and after gfd treatment could open new therapeutic strategies aimed at restoring the intestinal ecosystem balance.
further studies will be necessary to develop such strategies, as well as a more accurate identification of bacterial species or groups having discriminatory properties in the cd spectrum.

 celiac disease is an immune-mediated enteropathy triggered by the ingestion of gluten-containing grains in genetically susceptible individuals. its estimated prevalence in western countries is near 1%. it is generally agreed that cd is a t-cell mediated disorder in which gliadin derived peptides activate lamina propria t lymphocytes which release proinflammatory cytokines. to date, several peptides including alpha- and gamma-gliadins, have been reported to activate cd4+ lymphocytes via their interaction with hla-dq and -dq heterodimer on antigen presenting cells. recently, scientific evidence showed microecological changes in the intestinal tract of celiac infants, suggesting a potential role of gut microbiota in cd. alterations in the composition of faecal short-chain fatty acids in cd patients compared with those of healthy controls have been demonstrated. imbalance in the composition of duodenal microbiota or in faecal bacterial communities of children with cd has also been reported. rod-shaped bacteria have been observed in both gluten-free diet -treated and untreated pediatric patients'mucosa, along with a distinctive lectin pattern. the present study was carried out to add further information on the characterization of intestinal microbiota of cd patients, a variable that may represent a new piece of the intriguing puzzle of cd illness. for this purpose we analyzed by ttge the composition of duodenal mucosa-associated microbiota in the same cohort of gfd untreated and treated cd children and in controls. this prospective study was performed to compare the influence of the disease status on gut microbial composition and to study whether the microbial imbalance could be a peculiar characteristic of the disease.  agglomerative hierarchical classification the ttge profiles of pcr amplicons obtained with universal primers were firstly analyzed by xlstat software. the resulting dendrogram presented two major well-defined clusters. the first cluster groups 8/ of control patients, while the second one groups 18/ of cd patients. these results highlighted the presence of a dominant microbiota related to the celiac disease, irrespectively to the disease status. the average number of bands in ttge profiles, calculated by digidoc-it software, was significantly higher in celiac children than in controls, indicating that duodenal mucosa of cd patients showed a higher diversity of associated bacterial population. the average number of bands in ttge profiles was also significantly higher in active disease than inactive one. moreover, interindividual analysis showed a mean dice similarity index of ttge profiles of % ± % within active disease group, % ± % within inactive disease group and % ± % within control group. otherwise, mean dice similarity index between celiac individuals before and after gfd treatment was % ± %. ecological features shannon-wiener index analysis was performed to determine a measure of estimated diversity within each biopsy sample by ttge profiles. mean shannon-wiener index value differed significantly between active and inactive cd patients, a similar result was obtained between active cd patients and controls. the shannon-wiener index among inactive cd patients and controls was not significantly different.. the variance values relative to active group revealed a minor data dispersion than inactive and control ones, indicating a more similar microbial biodiversity between its members. the carrying capacity of the duodenal system showed mean rr values of: ±, ±, ± for active, inactive and control group respectively. the mean rr values were highly different among the three groups. factor discriminating analysis to improve the analysis of ttge profiles the more discriminating fda approach was performed. the principal component analysis transformed data showed a well-defined separation between controls, active and inactive celiac groups, with a confusion matrix of %. results from this analysis indicated that the ttge profiles were sufficient to predict the patient category with 100% predictiveness, suggesting the importance of duodenal microbiota in this pathology. partial least square discriminant analysis pls-da was employed to investigate peculiar ttge bands having discriminatory power in separating ttge profiles in the three groups studied, utilizing the raw data. the score plot confirmed a division between the patients' groups. interestingly, in patients n. and the ttge profiles of inactive status resulted closer to those of control group. on the basis of pls-da score plot, it could be seen that cd patients and controls were separated along principal component component, whilst active and inactive cd patients were separated along principal component component. fig shows hierarchical discriminatory importance of the ttge bands for pc component and pc component. the variable importance mainly reflected the correlation between the ttge bands and all the patients groups along a specific principal component axis. the bands with vip larger than were picked. the ttge bands picked partitioning cd and non cd-diagnosed patients were: the picked ttge bands separating active and inactive cd patients were: statistical evaluation of ttge bands occurrence by pls-da the selected ttge bands obtained by pls-da analysis were statistically evaluated for their occurrence as reported in table the ttge selected bands dividing cd and controls resulted all statistically significant. in the separation between active and inactive cd patients, bands resulted statistically significant were: and moreover, some of selected ttge bands run parallel with e. coli, p. distasonis and b. vulgatus gel markers used. the parallelism is reported in tab. § bands were self numbered according to the order of appearance on the ttge gel and are listed in descending order of importance in the pls-da model. between parentheses are reported the species used in the gel marker that run parallel to specific ttge bands. mann-whitney u-test, α = wilcoxon signed rank test, α = age at 1st biopsy gfd duration none of the patients underwent previous endoscopic assessment biopsies were taken in the second part of duodenum species-specific pcr results obtained revealed a highly significant difference in the prevalence of b. vulgatus, and e. coli in cd patients versus controls. a significant difference was found in the prevalence of b. vulgatus and in the prevalence of clostridium coccoides group in active cd patients versus inactive cd one. no significant difference was found in the prevalence of bifidobacterium spp. between cd patients and controls and between active and inactive cd. discussion this is the first longitudinal study on the duodenal mucosa-associated microbiota, carried out on the same cohort of cd pediatric patients, showing a distinctive'microbial structure' in celiac pediatric patients. the most important results of this study, obtained through multivariate statistical analysis of ttge profiles, were: i) a dominant duodenal microbiota that could be linked to the disease status, outlining differences in the microbiota composition before and after gfd treatment; ii) a significantly higher diversity in dominant microbiota in patients with active disease vs the same in remission state, as well as in patients with active disease vs controls, as revealed by shannon-wiener index. this higher duodenal microbial diversity in cd patients could have a possible harmful impact on the duodenal homeostasis. iii) a higher inter-individual similarity in cd patients than controls, indicating a more homogeneous structure among microbial communities of celiac patients. analyzing ttge profiles, the lowest carrying capacity and the lowest median number of bands found in the duodenal system of the control group can be attributed to an environment particularly adverse or restricted to colonization. the nature of duodenal habitat is radically changed in cd patients, where the carrying capacity and the median number of bands in ttge profiles are much higher than controls, consequently a thriving colonization could be due to a more habitable environment. it could be speculated that in duodenum the microbial life could be largely inhibited by different factors such the rapid transit of food, pancreatic juices or the rapid mucosal turnover. is therefore likely that a relative small number of definite microbial species or groups are highly adapted to this particular habitat, then the number of ttge bands found in our control duodenal samples was lower than others found in different intestinal tracts. the dice index analysis of ttge profiles revealed that mucosa-associated microbiota differed markedly from one patient to another in control group, whereas cd patients showed a high inter-individual similarity. moreover, before and after gfd treatment, there's a loss of % of inter-individual similarity. specifically, the similarity is lost in a homogeneous way between all celiac individuals, as showed by the high similarity dice index within active and inactive groups. we may speculate that the change in the mucosa lectin patterns both in active and remissive cd, as demonstrated by forsberg, could create more selective microbial adhesive patterns in duodenal mucosa of these patients, promoting a more similar interindividual mucosal colonization. ttge bands, having discriminatory power in separating the three patients'groups, have been selected. some of these ttge bands run parallel with e. coli, p. distasonis and b. vulgatus gel markers used. the genera bacteroides, as reported by previous works, was significantly increased providing a strong correlation between this microbial group and cd. moreover a high prevalence of potentially pro-inflammatory gram negative bacteria was found in the celiac patients' duodenum. furthermore, the presence of bacteria such e. coli and bacteroides spp has been related by other authors with mucin degradation and an increase in small intestinal permeability. although the technique we used does not allow a specific characterization of microbial species or groups of this particular intestinal habitat, it provides a picture of modifications encountered by dominant bacterial groups/species profile of a sample in relation to different factors. the presence/absence of bacterial species/groups might act as'key' or'regulatory' species leading to a different relative abundance of the present species. to assess this, we need to improve our data by direct sequencing of ttge bands. ttge profiles of 18/ cd patients in remission, with a duodenal histology not fully normalized, clustered together and away from controls. interestingly, ttge profiles of cd patients with a fully histological duodenal normalization at gfd, clustered close to controls as reported by the pls-da score plot. this would indicate an association between inflammatory status of intestinal mucosa and the kind of colonizing microbiota. partial recovery of microbiota composition in the patients with full histological normalization seems to indicate that the mucosa inflammation status is not the only factor driving the kind of microbial composition, but certainly is an influencing factor.  in conclusion, our data show a potential role of the duodenal microbiota in the cd pathogenesis. common ttge profiles in cd patients are probably due to a similar intestinal habitat creating selective pressures that shape a peculiar dominant microbiota. in addition, the occurrence of distinctive ttge profiles in celiac patients before and after gfd treatment could open new therapeutic strategies aimed at restoring the intestinal ecosystem balance. further studies will be necessary to develop such strategies, as well as a more accurate identification of bacterial species or groups having discriminatory properties in the cd spectrum. 
recently, scientific evidence suggested a potential role of gut microbiota in cd. similarity index between celiac children before and after gfd treatment was % ± %. mean interindividual similarity index was % ± % for active disease, % ± % for remission state and % ± % for controls. overall, the results highlighted a peculiar microbial ttge profile and a significant higher biodiversity in cd pediatric patients' duodenal mucosa. the average number of bands in ttge profiles was significantly higher in active and inactive states than in controls. to have a snapshot of dominant duodenal microbiota we analyzed the mucosa-associated microbiota of children with cd, before and after a gluten-free diet regimen, and of controls. celiac disease is an autoimmune disorder of the small intestine in which dietary gluten ingestion leads to a chronic enteropathy. ttge profiles were analyzed by statistical multivariate analysis. bacteroides vulgatus and escherichia coli were detected more often in cd patients than in controls. differences in microbiota biodiversity were among active and remission state and amid active cd and controls.
 celiac disease is an autoimmune disorder of the small intestine in which dietary gluten ingestion leads to a chronic enteropathy. recently, scientific evidence suggested a potential role of gut microbiota in cd. to have a snapshot of dominant duodenal microbiota we analyzed the mucosa-associated microbiota of children with cd, before and after a gluten-free diet regimen, and of controls. total dna was extracted from duodenal biopsies and amplification products of 16s ribosomal dna were compared by temporal temperature gradient gel electrophoresis. ttge profiles were analyzed by statistical multivariate analysis.  the average number of bands in ttge profiles was significantly higher in active and inactive states than in controls. mean interindividual similarity index was % ± % for active disease, % ± % for remission state and % ± % for controls. similarity index between celiac children before and after gfd treatment was % ± %. differences in microbiota biodiversity were among active and remission state and amid active cd and controls. bacteroides vulgatus and escherichia coli were detected more often in cd patients than in controls.  overall, the results highlighted a peculiar microbial ttge profile and a significant higher biodiversity in cd pediatric patients' duodenal mucosa. the possible pathophysiological role of these microbial differences needs further characterization. 