BACKGROUND
inflammation processes are essential for the immune system of a host organism attacked by bacteria, viruses or other immunogenic molecules.
however, persistent inflammation is a pathologic indication.
the intestine, being the largest barrier of the human body to the environment, is under a state of persistent controlled inflammation because of its permanent contact with the gut microbiota.
intestinal epithelial cells release cytokines and chemokines upon external stimulation, e.g.
by bacteria and their surface structures  <cit> .
the factors which trigger inflammation and the release or suppression of cytokines and chemokines have been investigated thoroughly over the last decade, but the process is still not fully understood.
clearly, cytokine secretion can be triggered by lipopolysaccharide  on the surface of gram-negative bacteria  <cit>  or capsular polysaccharides and lipoteichoic acid from gram-positive species  <cit> .
oligo- and polysaccharides containing fructose have been known for several years as prebiotics  <cit> .
fructose recently was described as a signaling molecule and lead structure for carbohydrates with enhanced antigenicity in hiv vaccination  <cit> .
the extent of the fructan oligo- and polymerization was described as controllable in an enzymatic synthesis process  <cit> .
fructosyltransferases like inulosucrases and levansucrases which synthesize fructans of various chain lengths are common in many different bacteria including the gut microbiota  <cit> .
the challenges to access large, branched oligosaccharides using chemical synthesis, may be overcome using chemo-enzymatic approaches  <cit> .
sucrose analogues synthesized by sacb from b. megaterium were used as precursors for the synthesis of oligofructosides with the fructosyltransferase suc <dig> from a. niger <cit> .
for the present study, the enzymatic synthesis process was scaled up to yield biological test amounts.
the tailored oligofructosides tested in this study are capped by the monosaccharides d-glucose, d-mannose, d-galactose, d-fucose or d-xylose, elongated with fructosyl units under tight control of the degree of polymerization.
these oligofructosides are supposed to mimic the structural characteristics of immunogenic carbohydrate patterns of antigens, thus triggering the release of cytokines and/or chemokines.
RESULTS
chemo-enzymatic synthesis of novel oligofructosides by substrate engineering of fructosyltransferases
tailored oligofructosides were synthesized, purified and characterized regarding their composition and stereochemistry.
substrate engineering of two fructosyltransferases from gras organisms  leads to novel tailor-made oligofructosides of defined fructosyl backbone lengths .
the first step in their enzymatic synthesis is the formation of the α- linked disaccharide .
the fructosyltransferase sacb from b. megaterium provides access to the efficient synthesis of sucrose analogues  under appropriate reaction conditions.
the synthesis reaction was performed according to the process of sucrose analogue synthesis by sacb from b. subtilis <cit> .
however, sacb from b. megaterium proved to be much more efficient in terms of chemo-enzymatic synthesis with an increased substrate affinity  and turnover number   <cit> .
each chemo-enzymatic synthesis process was analyzed by hpaec.
as an example, the process for fuc-fru synthesis is shown in figure 3a.
the distinct addition of the second and third unit of the fructosyl backbone was performed by the fructosyltransferase suc <dig> from a. niger.
suc <dig> is highly specific for the synthesis of defined tri- and tetrasaccharides, 1-kestose  and 1-nystose  and their analogues depending on the reaction conditions.
trisaccharide kestose analogues are gal- <dig>  man- <dig>  xyl- <dig>  and fuc- <dig>  tetrasaccharide nystose analogues containing three fructosyl moieties are gal- <dig>  man- <dig>  xyl- <dig> and fuc- <dig> .
the previously described products  <cit>  as well as the novel fucosyl-capped tri- and tetrasaccharides were identified and analyzed by tlc and hpaec.
scale-up of the synthesis process yields pure oligofructosides in cell culture test amounts .
as an example, the synthesis process for fuc-fru <dig> and fuc-fru <dig> is shown in figure 3b.
reaction conditions were suc <dig> enzyme dilution 1: <dig> ,  <dig> mm sucrose analogue to be converted in sörensen`s phosphate buffer  at 45°c and  <dig> rpm.
the reaction time depending on the oligosaccharide to be synthesized is indicated.
oligofructoside-stimulated caco- <dig> cells differentially secrete cxcl <dig> and ccl2
the novel synthesized oligofructosides were tested in terms of immunostimulating properties on human epithelial caco- <dig> cells.
this cell line is a model for the absorption of pharmacological products in the intestinal region  <cit> .
<dig> cytokines and chemokines were analyzed in the growth medium during co-cultivation with the novel oligofructosides .
caco- <dig> cells were grown for  <dig> h in 24-well dishes containing the oligofructosides to be investigated in a concentration of  <dig> μm.
five-fold repeats of the experiment for each carbohydrate  ensured the reproducibility of the results.
cxcl <dig>  and ccl <dig>  were the only cytokines/chemokines in the media supernatants which led to a significant signal detected by the luminex system.
both standard curve fits show a correlation of over 99% .
the measured fluorescence intensities are in the linear part of the standard curve indicating the reliability of the data.
the other  <dig> cytokines and chemokines analyzed showed no significant response under these conditions.
ccl <dig> generally shows a higher signal-to-background enhancement compared to the cultivation of caco- <dig> cells without added carbohydrates.
the ccl <dig> release is increased up to a concentration of  <dig> pg ml- <dig> .
the characteristics of a longer fructosyl backbone seem to enhance this effect.
for the tetrasaccharides 1-nystose , man-fru <dig> , fuc-fru <dig>  and xyl-fru <dig> , the release of ccl <dig> is clearly triggered .
the stimulation of cxcl <dig> is also observable when the fucose-containing 1-nystose analogue fuc-fru <dig> is added .
also in the case of cxcl <dig>  longer fructosyl chains seem to enhance the release of this cytokine.
in the samples containing man-fru <dig> , fuc-fru <dig>  and xyl-fru <dig>  the highest concentrations of cxcl <dig> were detectable .
cxcl <dig> and ccl <dig> release was consistently not enhanced when incubated with the trisaccharides 1-kestose and man-fru <dig> .
discussion
co-incubation of human epithelial caco- <dig> cells with certain types of pure, unconjugated oligofructosides leads to enhanced secretion of cxcl <dig> and ccl <dig>  cxcl <dig> is a potent inflammation marker recruiting neutrophils to sites of infection.
it is secreted by various cell types including epithelial cells  <cit> .
ccl <dig> is described as an effective chemoattractor for monocytes from the blood stream  <cit> .
our results show that ccl <dig> release can be clearly triggered by the tetrasaccharide 1-nystose and even more enhanced by the nystose analogues man-fru <dig> and fuc-fru <dig>  the observed differential cytokine secretion pattern raises questions: is this stimulating effect dependent on the carbohydrate structure and if so, which structural elements trigger or suppress the release of cytokines and chemokines?
because of the differential secretion pattern and only two significant signals , it was shown that cytokine secretion by caco- <dig> cells in this assay is dependent on the oligofructoside type.
but why are just  <dig> significant signals of secreted proteins detectable?
one possibility is, that epithelial cells in the intestine only have a restricted repertoire of cytokines to be synthesized and secreted.
another point is, that this cell type is in constant contact with ubiquitous nutrients and commensal gut bacteria.
hence, it may have evolved tolerance against certain alien structures.
the intestine being the largest barrier of the human body to the environment, has a special set of immunologically active cells.
this area is under a state of persistent controlled inflammation because of its permanent contact with the gut microbiota.
special intestinal macrophages  mediate tolerance to beneficial gut bacteria.
perturbations of these processes, like the release of ccl <dig>  inhibit the differentiation of macrophages to imacs thus leading to  inflammatory bowel diseases   <cit> .
intestinal epithelial cells release cytokines and chemokines upon external stimulation, e.g.
by bacteria and their surface structures  <cit> .
the factors which trigger inflammation and the release or suppression of cytokines and chemokines have been investigated thoroughly over the last decade, but the process is still not fully understood.
in this study, mannose- and fucose-capped oligofructosides generally evoke the highest increase in ccl <dig> and cxcl <dig> release .
this might be due to their participation in natural cell-cell communication processes.
fucose often is a branching carbohydrate unit e.g.
in the lewis x motif.
this motif is known as immunogenic under certain conditions, e.g.
incomplete sialylation.
mannose is part of the core n-glycan structure.
its exposition often leads to the release of cytokines, e.g.
ccl <dig> in mannosidase knock-out mice  <cit> .
interestingly, the different monosaccharide cap structure of the fructosyl backbone is not the only factor influencing the release of cxcl <dig> and ccl <dig>  but also the length of the fructosyl backbone.
for example, ccl <dig> secretion is triggered by 1-nystose and its tetrasaccharide analogues man-fru <dig> and fuc-fru <dig> but suppressed by kestose and its analogue man-fru <dig> .
thus, stereochemical and spatial aspects of oligosaccharides obviously have to be considered in terms of cell signalling processes.
recently, it was described that the different shape of bacterial lipopolysaccharide  determines which receptor is targeted and thus how cell signalling is processed  <cit> .
the potential target receptors which are known to act competitively are shown in additional file 1: figure s <dig>  the differential secretion of cytokines and thus the induction of an inflammatory response by the interaction of these receptors is still a scientific area with many long-standing questions.
CONCLUSIONS
carbohydrates are ubiquitious structures on the surface of a plethora of different cell types including potentially pathogenic and beneficial gut bacteria.
auto-immune diseases like crohn`s disease are linked to persistent, pathologic inflammation.
as abundant surface structures of host and pathogen cells, carbohydrates may play an important role in the induction of inflammation and tolerance, respectively.
advances in carbohydrate research in combination with cell biology and immunology methods may lead to a detailed understanding of inflammation processes.
the pure, tailored carbohydrate structures examined in this study induce such a differential secretion of cytokines in endothelial cells in vitro.
further advances in oligosaccharide synthesis lead broadened possibilities to investigate in vivo inflammation mechanisms of carbohydrate-cell receptor crosstalk.
controlled stimulation of the immune system may be one component towards a successful treatment of auto-immune diseases.

 inflammation processes are essential for the immune system of a host organism attacked by bacteria, viruses or other immunogenic molecules. however, persistent inflammation is a pathologic indication. the intestine, being the largest barrier of the human body to the environment, is under a state of persistent controlled inflammation because of its permanent contact with the gut microbiota. intestinal epithelial cells release cytokines and chemokines upon external stimulation, e.g. by bacteria and their surface structures. the factors which trigger inflammation and the release or suppression of cytokines and chemokines have been investigated thoroughly over the last decade, but the process is still not fully understood. clearly, cytokine secretion can be triggered by lipopolysaccharide on the surface of gram-negative bacteria or capsular polysaccharides and lipoteichoic acid from gram-positive species. oligo- and polysaccharides containing fructose have been known for several years as prebiotics. fructose recently was described as a signaling molecule and lead structure for carbohydrates with enhanced antigenicity in hiv vaccination. the extent of the fructan oligo- and polymerization was described as controllable in an enzymatic synthesis process. fructosyltransferases like inulosucrases and levansucrases which synthesize fructans of various chain lengths are common in many different bacteria including the gut microbiota. the challenges to access large, branched oligosaccharides using chemical synthesis, may be overcome using chemo-enzymatic approaches. sucrose analogues synthesized by sacb from b. megaterium were used as precursors for the synthesis of oligofructosides with the fructosyltransferase suc from a. niger. for the present study, the enzymatic synthesis process was scaled up to yield biological test amounts. the tailored oligofructosides tested in this study are capped by the monosaccharides d-glucose, d-mannose, d-galactose, d-fucose or d-xylose, elongated with fructosyl units under tight control of the degree of polymerization. these oligofructosides are supposed to mimic the structural characteristics of immunogenic carbohydrate patterns of antigens, thus triggering the release of cytokines and/or chemokines.  chemo-enzymatic synthesis of novel oligofructosides by substrate engineering of fructosyltransferases tailored oligofructosides were synthesized, purified and characterized regarding their composition and stereochemistry. substrate engineering of two fructosyltransferases from gras organisms leads to novel tailor-made oligofructosides of defined fructosyl backbone lengths. the first step in their enzymatic synthesis is the formation of the α- linked disaccharide. the fructosyltransferase sacb from b. megaterium provides access to the efficient synthesis of sucrose analogues under appropriate reaction conditions. the synthesis reaction was performed according to the process of sucrose analogue synthesis by sacb from b. subtilis. however, sacb from b. megaterium proved to be much more efficient in terms of chemo-enzymatic synthesis with an increased substrate affinity and turnover number. each chemo-enzymatic synthesis process was analyzed by hpaec. as an example, the process for fuc-fru synthesis is shown in figure 3a. the distinct addition of the second and third unit of the fructosyl backbone was performed by the fructosyltransferase suc from a. niger. suc is highly specific for the synthesis of defined tri- and tetrasaccharides, 1-kestose and 1-nystose and their analogues depending on the reaction conditions. trisaccharide kestose analogues are gal- man- xyl- and fuc- tetrasaccharide nystose analogues containing three fructosyl moieties are gal- man- xyl- and fuc-. the previously described products as well as the novel fucosyl-capped tri- and tetrasaccharides were identified and analyzed by tlc and hpaec. scale-up of the synthesis process yields pure oligofructosides in cell culture test amounts. as an example, the synthesis process for fuc-fru and fuc-fru is shown in figure 3b. reaction conditions were suc enzyme dilution 1:, mm sucrose analogue to be converted in sörensen`s phosphate buffer at 45°c and rpm. the reaction time depending on the oligosaccharide to be synthesized is indicated. oligofructoside-stimulated caco- cells differentially secrete cxcl and ccl2 the novel synthesized oligofructosides were tested in terms of immunostimulating properties on human epithelial caco- cells. this cell line is a model for the absorption of pharmacological products in the intestinal region. cytokines and chemokines were analyzed in the growth medium during co-cultivation with the novel oligofructosides. caco- cells were grown for h in 24-well dishes containing the oligofructosides to be investigated in a concentration of μm. five-fold repeats of the experiment for each carbohydrate ensured the reproducibility of the results. cxcl and ccl were the only cytokines/chemokines in the media supernatants which led to a significant signal detected by the luminex system. both standard curve fits show a correlation of over 99%. the measured fluorescence intensities are in the linear part of the standard curve indicating the reliability of the data. the other cytokines and chemokines analyzed showed no significant response under these conditions. ccl generally shows a higher signal-to-background enhancement compared to the cultivation of caco- cells without added carbohydrates. the ccl release is increased up to a concentration of pg ml-. the characteristics of a longer fructosyl backbone seem to enhance this effect. for the tetrasaccharides 1-nystose, man-fru, fuc-fru and xyl-fru, the release of ccl is clearly triggered. the stimulation of cxcl is also observable when the fucose-containing 1-nystose analogue fuc-fru is added. also in the case of cxcl longer fructosyl chains seem to enhance the release of this cytokine. in the samples containing man-fru, fuc-fru and xyl-fru the highest concentrations of cxcl were detectable. cxcl and ccl release was consistently not enhanced when incubated with the trisaccharides 1-kestose and man-fru. discussion co-incubation of human epithelial caco- cells with certain types of pure, unconjugated oligofructosides leads to enhanced secretion of cxcl and ccl cxcl is a potent inflammation marker recruiting neutrophils to sites of infection. it is secreted by various cell types including epithelial cells. ccl is described as an effective chemoattractor for monocytes from the blood stream. our results show that ccl release can be clearly triggered by the tetrasaccharide 1-nystose and even more enhanced by the nystose analogues man-fru and fuc-fru the observed differential cytokine secretion pattern raises questions: is this stimulating effect dependent on the carbohydrate structure and if so, which structural elements trigger or suppress the release of cytokines and chemokines? because of the differential secretion pattern and only two significant signals, it was shown that cytokine secretion by caco- cells in this assay is dependent on the oligofructoside type. but why are just significant signals of secreted proteins detectable? one possibility is, that epithelial cells in the intestine only have a restricted repertoire of cytokines to be synthesized and secreted. another point is, that this cell type is in constant contact with ubiquitous nutrients and commensal gut bacteria. hence, it may have evolved tolerance against certain alien structures. the intestine being the largest barrier of the human body to the environment, has a special set of immunologically active cells. this area is under a state of persistent controlled inflammation because of its permanent contact with the gut microbiota. special intestinal macrophages mediate tolerance to beneficial gut bacteria. perturbations of these processes, like the release of ccl inhibit the differentiation of macrophages to imacs thus leading to inflammatory bowel diseases. intestinal epithelial cells release cytokines and chemokines upon external stimulation, e.g. by bacteria and their surface structures. the factors which trigger inflammation and the release or suppression of cytokines and chemokines have been investigated thoroughly over the last decade, but the process is still not fully understood. in this study, mannose- and fucose-capped oligofructosides generally evoke the highest increase in ccl and cxcl release. this might be due to their participation in natural cell-cell communication processes. fucose often is a branching carbohydrate unit e.g. in the lewis x motif. this motif is known as immunogenic under certain conditions, e.g. incomplete sialylation. mannose is part of the core n-glycan structure. its exposition often leads to the release of cytokines, e.g. ccl in mannosidase knock-out mice. interestingly, the different monosaccharide cap structure of the fructosyl backbone is not the only factor influencing the release of cxcl and ccl but also the length of the fructosyl backbone. for example, ccl secretion is triggered by 1-nystose and its tetrasaccharide analogues man-fru and fuc-fru but suppressed by kestose and its analogue man-fru. thus, stereochemical and spatial aspects of oligosaccharides obviously have to be considered in terms of cell signalling processes. recently, it was described that the different shape of bacterial lipopolysaccharide determines which receptor is targeted and thus how cell signalling is processed. the potential target receptors which are known to act competitively are shown in additional file 1: figure s the differential secretion of cytokines and thus the induction of an inflammatory response by the interaction of these receptors is still a scientific area with many long-standing questions.  carbohydrates are ubiquitious structures on the surface of a plethora of different cell types including potentially pathogenic and beneficial gut bacteria. auto-immune diseases like crohn`s disease are linked to persistent, pathologic inflammation. as abundant surface structures of host and pathogen cells, carbohydrates may play an important role in the induction of inflammation and tolerance, respectively. advances in carbohydrate research in combination with cell biology and immunology methods may lead to a detailed understanding of inflammation processes. the pure, tailored carbohydrate structures examined in this study induce such a differential secretion of cytokines in endothelial cells in vitro. further advances in oligosaccharide synthesis lead broadened possibilities to investigate in vivo inflammation mechanisms of carbohydrate-cell receptor crosstalk. controlled stimulation of the immune system may be one component towards a successful treatment of auto-immune diseases. 
the type and amount of cytokine secretion depends on the type of co-cultivated oligosaccharide. substrate engineering of these glycosyltransferases generally acting on sucrose leads to the controlled formation of novel tailored di-, tri- and tetrasaccharides. a differential secretion of cxcl and ccl was observed upon oligosaccharide co-cultivation with colorectal epithelial caco- cells.
 it is well known that carbohydrates play fundamental roles in cell signaling and infection processes as well as tumor formation and progression. however, the interaction pathways and cellular receptors targeted by carbohydrates and glycoconjugates remain poorly examined and understood. this lack of research stems, at least to a major part, from accessibility problems of large, branched oligosaccharides.  to test glycan - cell interactions in vitro, a variety of tailored oligosaccharides was synthesized chemo-enzymatically. glycosyltransferases from the gras organisms bacillus megaterium and aspergillus niger were used in this study. substrate engineering of these glycosyltransferases generally acting on sucrose leads to the controlled formation of novel tailored di-, tri- and tetrasaccharides. already industrially used as prebiotics in functional food, the immunogenic potential of novel oligosaccharides was characterized in this study. a differential secretion of cxcl and ccl was observed upon oligosaccharide co-cultivation with colorectal epithelial caco- cells.  pure carbohydrates are able to stimulate a cytokine response in human endothelial cells in vitro. the type and amount of cytokine secretion depends on the type of co-cultivated oligosaccharide. oligofructosideglycosyltransferasesuc1aspergillus nigersacbbacillus megateriumcxcl ccl caco-2 