BACKGROUND
celiac disease is a gluten-induced autoimmune disease of the small intestine characterized by small-bowel mucosal villous atrophy with crypt hyperplasia and a profound inflammation in the mucosa.
in addition to causing damage to the mucosa in genetically susceptible individuals, gluten also provokes the production of autoantibodies typically found in the sera of untreated celiac disease patients.
these autoantibodies recognize exclusively endomysial antigens now identified as transglutaminase  <dig> .
the autoantibodies are produced locally in the mucosa  <cit> , and besides being detectable in patient sera, they are also deposited extracellularly in vivo in the mucosa  <cit> .
furthermore, recent findings suggest that these tg2-targeted mucosal iga-autoantibody deposits are already present in the early phases of the disease process prior to manifest mucosal lesion  <cit>  and before autoantibodies appear in the serum  <cit> .
after adoption of a gluten-free diet, serum autoantibodies disappear and the small-bowel mucosa heals usually within one year  <cit> .
at the time the autoantibodies have disappeared from the circulation, there may still be residual autoantibody deposits present in the small-intestinal mucosa which will also in due course disappear on a strict gluten-free diet  <cit> .
for over twenty years the human small-intestinal organ culture method has been widely used in detecting the toxic effects of wheat gliadin in celiac disease in vitro  <cit> .
in earlier studies the toxicity of gliadin has commonly been demonstrated by an increased density of lamina proprial lymphocytes  <cit>  and reduced epithelial cell height   <cit>  in cultured biopsy samples from untreated and treated celiac disease patients.
however, when measuring endomysial autoantibody  secretion to culture supernatants from biopsies from treated celiac patients, the results have been contradictory.
picarelli and associates  <cit>  showed that in biopsies derived from treated celiac disease patients gliadin induces secretion of ema to culture supernatants.
in contrast, some studies report that the secretion of ema can only be achieved in half of  <cit>  or even no  <cit>  experiments carried out with treated celiac patient biopsies.
and even further, it has been suggested that ema secretion to the organ culture system is totally independent of gliadin challenge  <cit>  and histological damage  <cit> .
due to these discrepancies among previous studies concerning autoantibody secretion to the organ culture system, our aim was to establish in both short- and long-term treated celiac disease patients whether the antibody secretion to culture supernatants is dependent on the duration of patients' gluten-free diet .
furthermore, since in celiac disease the small-bowel mucosal extracellular tg2-specific iga deposits seem to disappear slowly after a gluten-free diet  <cit> , we hypothesized that the autoantibody secretion to supernatants is related to the existence of mucosal tg2-specific iga deposits in the cultured small-bowel biopsy samples.
RESULTS
celiac autoantibodies
in order to study the baseline serum autoantibody levels of study subjects, ema and tg <dig> antibody  titers were measured.
all five untreated celiac disease patients involved had positive ema  and tg2-ab titers  in serum.
in contrast, all  <dig> treated celiac disease patients and all six non-celiac controls had normal serum autoantibody levels.
furthermore, all celiac disease patients carried either the hla dq <dig> or the dq <dig> haplotype.
when the small-bowel biopsies from celiac disease patients were challenged in the organ culture system with peptic-tryptic digest of gliadin  for  <dig> hours, ema was secreted to the culture supernatants in all five untreated but in only  <dig> out of the  <dig> treated celiac cases .
moreover, no antibodies were found in supernatants from non-celiac controls .
when autoantibody secretion to culture supernatants was investigated in relation to the duration of the gluten-free diet, ema was detected in supernatants after pt-gliadin challenge in  <dig> out of  <dig> short-term  and in  <dig> out of  <dig> long-term  treated patient biopsy samples .
since the duration of the gluten-free diet was insufficient to explain ema secretion completely, we tested whether ema presence in the culture supernatants was dependent on pre-existing small-bowel mucosal iga-autoantibody deposits.
when the treated celiac patients were divided into two groups based on the presence or absence of mucosal iga autoantibody deposits , it was found that only biopsies derived from patients having positive autoantibody deposits were able to secrete ema to the culture supernatants .
in some treated celiac cases evincing mucosal iga deposits, ema was already secreted to the supernatant spontaneously without pt-gliadin stimulation, but both ema and tg <dig> antibody titers increased significantly after  <dig> hours' culture with pt-gliadin .
although the autoantibody titers increased in culture supernatants after pt-gliadin challenge, there was no significant change in the intensity of mucosal iga deposits in the cultured biopsy samples after the challenge .
nd = not determined
gfd = gluten-free diet
* p <  <dig> ; † p =  <dig>  compared to medium only  <dig> hours and pt-gliadin  <dig> hours.
gfd = gluten-free diet
* p >  <dig>  compared to medium only  <dig> hours and pt-gliadin  <dig> hours
determination of ech and the number of cd25+ lymphocytes
in further demonstrating the toxic effects of gliadin, ech and the number of lamina proprial cd25+ cells were calculated in cultured biopsy samples.
in summary, it was found that these parameters were also dependent on the presence of mucosal iga deposits.
the ech decreased and the density of cd25+ cells increased significantly only in the treated celiac disease patient biopsies with pre-existing iga deposits .
discussion
the results presented here show that gliadin toxicity, as measured by autoantibody secretion, decrease in ech and increase in the density of lamina proprial cd25+ cells, can be demonstrated in the treated celiac disease patient organ culture system only if small-intestinal mucosal tg2-specific iga autoantibody deposits are still present in the cultured biopsy samples.
patients with persisting mucosal autoantibody deposits, normal small-bowel mucosal villous structure and negative serum autoantibody levels are usually those who have been on a gluten-free diet for a short period of time.
our results explain the discrepancies reported in previous papers on ema secretion in biopsies derived from treated celiac disease patients.
vogelsang and colleagues  <cit>  reported that ema secretion after a 24-hour gliadin challenge was achieved in only  <dig> out of  <dig>  biopsy samples derived from celiac disease patients on a strict gluten-free diet with normal mucosal villous morphology.
in contrast, biopsies from 60% of patients with dietary lapses and 92% of untreated celiacs with ongoing mucosal inflammation responded to the challenge in respect of ema secretion.
similar results have been obtained by biagi and associates  <cit>  in a small-scale study involving only seven treated celiac disease patients.
ema secretion to the culture supernatant could not be stimulated after a 24-hour gliadin administration in any of the long-term  treated celiac patients with normal mucosal architecture.
samples responding to the gliadin insult were derived from patients still evincing mucosal damage despite a gluten-free diet.
in the above-mentioned studies the treated patients unable to react to gliadin as measured by ema secretion were well and long-term treated and may be hypothesized not to have small-bowel iga autoantibody deposits.
this absence of mucosal deposits could thus explain the discrepancy in results.
furthermore, it has been shown that  <dig> hours of gliadin treatment might not be a sufficient time to induce ema secretion, as shown by picarelli and colleagues  <cit>  who demonstrated that a 48-hour culture period in the presence of gliadin is needed to achieve ema secretion in all treated celiac disease patient samples, this also being in agreement with the results presented in the current study .
it remains to be elucidated how a gliadin challenge leads to ema secretion in the organ culture system.
basically there are two different possibilities.
firstly, ema could be actively secreted by plasma cells in the biopsy specimen  <cit> .
the inability of long-term treated patient biopsy samples to secrete autoantibodies to the supernatant could be explained by the overall absence of plasma cells and helper t cells as well as the failure of memory b cells to become activated within the timeframe of the organ culture  <cit> .
secondly, the secretion of ema to the culture supernatant might simply be due to detachment of the autoantibodies from the tissue deposits and their release into the supernatant.
however, we observed no decrease in the intensity of the mucosal autoantibody deposits after the organ culture.
we did nevertheless see changes in ech and the density of cd25+ cells, which speaks in favor of an active gliadin-induced process, this supporting the first proposition.
CONCLUSIONS
our results indicate that when studying the toxic effects of gliadin in an organ culture setting, biopsy samples from short-term treated celiac disease patients who are likely still to have mucosal tg2-specific iga deposits should be used.
altogether, the current study provides a platform for further validation of the organ culture method.

 celiac disease is a gluten-induced autoimmune disease of the small intestine characterized by small-bowel mucosal villous atrophy with crypt hyperplasia and a profound inflammation in the mucosa. in addition to causing damage to the mucosa in genetically susceptible individuals, gluten also provokes the production of autoantibodies typically found in the sera of untreated celiac disease patients. these autoantibodies recognize exclusively endomysial antigens now identified as transglutaminase. the autoantibodies are produced locally in the mucosa, and besides being detectable in patient sera, they are also deposited extracellularly in vivo in the mucosa. furthermore, recent findings suggest that these tg2-targeted mucosal iga-autoantibody deposits are already present in the early phases of the disease process prior to manifest mucosal lesion and before autoantibodies appear in the serum. after adoption of a gluten-free diet, serum autoantibodies disappear and the small-bowel mucosa heals usually within one year. at the time the autoantibodies have disappeared from the circulation, there may still be residual autoantibody deposits present in the small-intestinal mucosa which will also in due course disappear on a strict gluten-free diet. for over twenty years the human small-intestinal organ culture method has been widely used in detecting the toxic effects of wheat gliadin in celiac disease in vitro. in earlier studies the toxicity of gliadin has commonly been demonstrated by an increased density of lamina proprial lymphocytes and reduced epithelial cell height in cultured biopsy samples from untreated and treated celiac disease patients. however, when measuring endomysial autoantibody secretion to culture supernatants from biopsies from treated celiac patients, the results have been contradictory. picarelli and associates showed that in biopsies derived from treated celiac disease patients gliadin induces secretion of ema to culture supernatants. in contrast, some studies report that the secretion of ema can only be achieved in half of or even no experiments carried out with treated celiac patient biopsies. and even further, it has been suggested that ema secretion to the organ culture system is totally independent of gliadin challenge and histological damage. due to these discrepancies among previous studies concerning autoantibody secretion to the organ culture system, our aim was to establish in both short- and long-term treated celiac disease patients whether the antibody secretion to culture supernatants is dependent on the duration of patients' gluten-free diet. furthermore, since in celiac disease the small-bowel mucosal extracellular tg2-specific iga deposits seem to disappear slowly after a gluten-free diet, we hypothesized that the autoantibody secretion to supernatants is related to the existence of mucosal tg2-specific iga deposits in the cultured small-bowel biopsy samples.  celiac autoantibodies in order to study the baseline serum autoantibody levels of study subjects, ema and tg antibody titers were measured. all five untreated celiac disease patients involved had positive ema and tg2-ab titers in serum. in contrast, all treated celiac disease patients and all six non-celiac controls had normal serum autoantibody levels. furthermore, all celiac disease patients carried either the hla dq or the dq haplotype. when the small-bowel biopsies from celiac disease patients were challenged in the organ culture system with peptic-tryptic digest of gliadin for hours, ema was secreted to the culture supernatants in all five untreated but in only out of the treated celiac cases. moreover, no antibodies were found in supernatants from non-celiac controls. when autoantibody secretion to culture supernatants was investigated in relation to the duration of the gluten-free diet, ema was detected in supernatants after pt-gliadin challenge in out of short-term and in out of long-term treated patient biopsy samples. since the duration of the gluten-free diet was insufficient to explain ema secretion completely, we tested whether ema presence in the culture supernatants was dependent on pre-existing small-bowel mucosal iga-autoantibody deposits. when the treated celiac patients were divided into two groups based on the presence or absence of mucosal iga autoantibody deposits, it was found that only biopsies derived from patients having positive autoantibody deposits were able to secrete ema to the culture supernatants. in some treated celiac cases evincing mucosal iga deposits, ema was already secreted to the supernatant spontaneously without pt-gliadin stimulation, but both ema and tg antibody titers increased significantly after hours' culture with pt-gliadin. although the autoantibody titers increased in culture supernatants after pt-gliadin challenge, there was no significant change in the intensity of mucosal iga deposits in the cultured biopsy samples after the challenge. nd = not determined gfd = gluten-free diet * p <; † p = compared to medium only hours and pt-gliadin hours. gfd = gluten-free diet * p > compared to medium only hours and pt-gliadin hours determination of ech and the number of cd25+ lymphocytes in further demonstrating the toxic effects of gliadin, ech and the number of lamina proprial cd25+ cells were calculated in cultured biopsy samples. in summary, it was found that these parameters were also dependent on the presence of mucosal iga deposits. the ech decreased and the density of cd25+ cells increased significantly only in the treated celiac disease patient biopsies with pre-existing iga deposits. discussion the results presented here show that gliadin toxicity, as measured by autoantibody secretion, decrease in ech and increase in the density of lamina proprial cd25+ cells, can be demonstrated in the treated celiac disease patient organ culture system only if small-intestinal mucosal tg2-specific iga autoantibody deposits are still present in the cultured biopsy samples. patients with persisting mucosal autoantibody deposits, normal small-bowel mucosal villous structure and negative serum autoantibody levels are usually those who have been on a gluten-free diet for a short period of time. our results explain the discrepancies reported in previous papers on ema secretion in biopsies derived from treated celiac disease patients. vogelsang and colleagues reported that ema secretion after a 24-hour gliadin challenge was achieved in only out of biopsy samples derived from celiac disease patients on a strict gluten-free diet with normal mucosal villous morphology. in contrast, biopsies from 60% of patients with dietary lapses and 92% of untreated celiacs with ongoing mucosal inflammation responded to the challenge in respect of ema secretion. similar results have been obtained by biagi and associates in a small-scale study involving only seven treated celiac disease patients. ema secretion to the culture supernatant could not be stimulated after a 24-hour gliadin administration in any of the long-term treated celiac patients with normal mucosal architecture. samples responding to the gliadin insult were derived from patients still evincing mucosal damage despite a gluten-free diet. in the above-mentioned studies the treated patients unable to react to gliadin as measured by ema secretion were well and long-term treated and may be hypothesized not to have small-bowel iga autoantibody deposits. this absence of mucosal deposits could thus explain the discrepancy in results. furthermore, it has been shown that hours of gliadin treatment might not be a sufficient time to induce ema secretion, as shown by picarelli and colleagues who demonstrated that a 48-hour culture period in the presence of gliadin is needed to achieve ema secretion in all treated celiac disease patient samples, this also being in agreement with the results presented in the current study. it remains to be elucidated how a gliadin challenge leads to ema secretion in the organ culture system. basically there are two different possibilities. firstly, ema could be actively secreted by plasma cells in the biopsy specimen. the inability of long-term treated patient biopsy samples to secrete autoantibodies to the supernatant could be explained by the overall absence of plasma cells and helper t cells as well as the failure of memory b cells to become activated within the timeframe of the organ culture. secondly, the secretion of ema to the culture supernatant might simply be due to detachment of the autoantibodies from the tissue deposits and their release into the supernatant. however, we observed no decrease in the intensity of the mucosal autoantibody deposits after the organ culture. we did nevertheless see changes in ech and the density of cd25+ cells, which speaks in favor of an active gliadin-induced process, this supporting the first proposition.  our results indicate that when studying the toxic effects of gliadin in an organ culture setting, biopsy samples from short-term treated celiac disease patients who are likely still to have mucosal tg2-specific iga deposits should be used. altogether, the current study provides a platform for further validation of the organ culture method. 
in addition to providing an explanation for the discrepancies in previous publications, the present study also enables further validation of the organ culture method. since the mucosal autoantibodies disappear slowly after a gluten-free diet, our aim was to establish whether autoantibody secretion to organ culture supernatants in treated celiac disease patient biopsies is related to the duration of the diet and further to the pre-existence of mucosal tg2-specific iga deposits in the cultured biopsy samples. furthermore, in these biopsies autoantibody secretion could be stimulated fully only after a 48-hour gliadin challenge. however, these changes could be demonstrated only in biopsies from short-term treated celiac disease patients, where the small-intestinal mucosal tg2-specific iga autoantibody deposits were still present. the toxicity of gluten and the secretion of the disease-specific autoantibodies have been widely studied in organ culture of small-intestinal biopsy samples, but results hitherto have been contradictory. in the organ culture system conducted with biopsies derived from treated celiac disease patients, gliadin induced secretion of autoantibodies to culture supernatants, reduced epithelial cell height and increased the density of lamina proprial cd25+ cells. our results show that studies focusing on the toxic effects of gliadin in the organ culture system should be carried out with biopsy samples from short-term treated celiac disease patients who are likely still to have mucosal iga deposits present. in addition, during gluten consumption these autoantibodies can also be detected in patients' serum but disappear from the circulation on a gluten-free diet. interestingly, after adoption of a gluten-free diet the serum autoantibodies disappear from the circulation more rapidly than the small-intestinal mucosal autoantibody deposits. these autoantibodies are produced in the small-intestinal mucosa, where they can be found deposited extracellularly below the epithelial basement membrane and around mucosal blood vessels.
 in celiac disease gluten, the disease-inducing toxic component in wheat, induces the secretion of autoantibodies which are targeted against transglutaminase. these autoantibodies are produced in the small-intestinal mucosa, where they can be found deposited extracellularly below the epithelial basement membrane and around mucosal blood vessels. in addition, during gluten consumption these autoantibodies can also be detected in patients' serum but disappear from the circulation on a gluten-free diet. interestingly, after adoption of a gluten-free diet the serum autoantibodies disappear from the circulation more rapidly than the small-intestinal mucosal autoantibody deposits. the toxicity of gluten and the secretion of the disease-specific autoantibodies have been widely studied in organ culture of small-intestinal biopsy samples, but results hitherto have been contradictory. since the mucosal autoantibodies disappear slowly after a gluten-free diet, our aim was to establish whether autoantibody secretion to organ culture supernatants in treated celiac disease patient biopsies is related to the duration of the diet and further to the pre-existence of mucosal tg2-specific iga deposits in the cultured biopsy samples.  in the organ culture system conducted with biopsies derived from treated celiac disease patients, gliadin induced secretion of autoantibodies to culture supernatants, reduced epithelial cell height and increased the density of lamina proprial cd25+ cells. however, these changes could be demonstrated only in biopsies from short-term treated celiac disease patients, where the small-intestinal mucosal tg2-specific iga autoantibody deposits were still present. furthermore, in these biopsies autoantibody secretion could be stimulated fully only after a 48-hour gliadin challenge.  our results show that studies focusing on the toxic effects of gliadin in the organ culture system should be carried out with biopsy samples from short-term treated celiac disease patients who are likely still to have mucosal iga deposits present. in addition to providing an explanation for the discrepancies in previous publications, the present study also enables further validation of the organ culture method. 