evidence is accumulating that enteric glial cells ( egc ) are the morphological and functional equivalent of the astrocytes found in the central nervous system ( cns ) .recent studies have demonstrated that egc participate in homeostasis of the gastrointestinal tract . in transgenic mouse models
in which egc are selectively ablated , the loss of glia results in intestinal inflammation and disruption of the epithelial barrier . in vitro experiments showed that chronic egc activation causes the release of proinflammatory cytokines and nitric oxide ( no ) in the enteric neuroimmune network [ 4 , 5 ] .
no is a weak radical produced from l - arginine via the enzyme no synthase ( nos ) . because increased no production via inducible nos ( inos ) is involved in the pathogenesis of inflammatory bowel disease ( ibd ) , alterations in enteroglial structure have been studied extensively in patients with ibd [ 79 ] .
demonstration of the increased enteroglial - derived s100b protein expression together with a significant increase in no production in intestinal biopsy specimens of patients with celiac disease and ulcerative colitis ( uc )   suggests that egc participate directly in chronic mucosal inflammation via s100b upregulation stimulating no production .
these findings were confirmed using in vitro cultures of human egc exposed to proinflammatory stimuli , in which significant increases in the cell proliferation rate , expression of s100b protein , and no production consequent to the induction of egc - derived inos protein were seen when egc are activated [ 4 , 5 ] .
enteric glial cells express not only s100b , but also high levels of glial fibrillary acidic protein ( gfap ) .
several studies have shown that s100b immunoreactivity mostly colocalizes with the gfap - positive enteroglial mucosal network in tissue specimens from patients with intestinal inflammation [ 1 , 5 ] . similarly , in the cns , astrocytes induce endothelial cells to form the tight junctions of the blood - brain barrier ( bbb ) .
a peripheral blood - tissue barrier similar to the bbb exists in the intestine where the neuronal plexi are similarly impermeable to systemic macromolecules . in this sense ,
egc , besides releasing cytokines , likely control the integrity and permeability of the submucosal blood vessels to confer intestinal epithelial barrier functions , as demonstrated in animal studies in which ablation of the enteroglial network enhances intestinal vascular permeability , resulting in severe mucosal inflammation [ 3 , 7 ] .
cns astrocytic glial markers have been studied extensively in the peripheral blood of patients with either traumatic brain injury   or cerebral infarcts .
although suggestive , data on egc - derived glial markers are limited to the abovementioned experimental models of intestinal inflammation in the human gut [ 1 , 4 , 5 , 10 ] , while there is a lack of data on serum levels .
we hypothesized that a disruption of egc populations could lead to altered serum levels of egc - derived s100b and gfap via disruption of the peripheral blood - tissue barrier in patients with uc .
this study investigated whether these enteroglial markers could serve as noninvasive indicators of egc activation and the disease activity in patients with uc .
thirty - five uc patients from erciyes university medical school , gastroenterology department , and 40 age- and sex - matched controls from bozok university medical faculty , neurology department , ranging in age from 30 to 65 years , were enrolled in this cross - sectional prospective study between january 2012 and june 2013 .
the diagnosis of uc was based on standard clinical , radiological , endoscopic , and histological criteria .
clinical disease activity was evaluated using the modified truelove - witts severity index ( mtwsi ) [ 1315 ] .
clinical active disease was defined as an estimated mtwsi score of 4 or higher and patients with a score lower than 4 were considered to be in remission ( inactive ) .
the uc patients included were all on mesalazine , and 11 ( 31.4% ) were in remission with immunosuppressive agents including azathioprine or steroids .
patients with malignancy , chronic renal , hepatic , cardiovascular , or connective tissue diseases , thyroid disease , diabetes mellitus , chronic obstructive pulmonary disease , a history of local trauma or surgery , and acute or chronic infections were excluded from the study .
in addition , those who were pregnant , morbidly obese , current smokers , or current consumers of alcohol were excluded .
the study protocol was approved by the bozok university research ethics committee , and written informed consent was obtained from all participants . body mass index ( bmi ) was calculated as the weight in kilograms divided by the square of height in meters .
fasting venous blood samples were analyzed for measuring the erythrocyte sedimentation rate ( esr ) , c - reactive protein ( crp ) , and white blood cell count ( wbc ) using standard methods .
the specimens were centrifuged at 5000  rpm for 5  min , after which the supernatant was removed immediately and kept frozen at 80c until assayed . all serum samples were prepared within 30  min .
serum samples were analyzed for human gfap and s100b using commercial enzyme - linked immunosorbent assay ( elisa ) kits for human gfap and s100b ( biovendor research and diagnostic products ; heidelberg ; germany ) .
the samples were assayed following the manufacturer 's protocol in the laboratory of bozok university medical faculty . for the gfap elisa ,
100  l of diluted standard , quality controls , and serum samples were applied to an antibody - coated microtiter plate and incubated for 120  min , with shaking at 300  rpm on an orbital microplate shaker .
after washing , 100  l of biotin labeled antibody solution was added to each well and incubated for 60  min , with shaking at 300  rpm .
after washing , 100  l of conjugate was added and the plate was incubated for 60  min , with shaking at 300  rpm . after the third wash , 100  l of substrate solution was added and the plate incubated for a further 15  min in the dark .
then , 100  l of stop solution was added and the absorbance was measured at 450  nm on a microtiter plate reader .
the gfap levels in the culture medium were determined using a standard curve of gfap .
similarly , serum samples were used for s100b elisa . to minimize the assay variation ,
all serum samples were analyzed on the same day in the same laboratory batch and by the same analyst at bozok university medical faculty .
the limits of detection for human gfap and s100b were 0.045  ng / ml and 15  pg / ml , respectively . the serum gfap concentrations are expressed in ng / ml , whereas the s100b concentrations are expressed in pg / ml . the shapiro - wilk test , histograms , and q - q plots were used to test the normality of the data , and levene 's test was used to assess the variance in homogeneity .
independent - sample t - tests and mann - whitney u tests were used to compare differences between continuous variables , and chi - square (  ) analyses were used to assess differences between categorical variables .
pearson correlations were used to examine the relationships between s100b and uc disease activity and duration .
values are expressed as frequencies and percentages , means and standard deviations , or medians and interquartile ranges .
analyses were conducted using spss 15.0 ( spss ; chicago , il ; usa ) , and statistical significance was set at p < 0.05 .
the demographic and laboratory data for the control group and patients with uc are summarized in table 1 .
no significant differences were found between the groups in terms of age or sex ( p > 0.05 ) .
bmi was significantly lower in the patients with uc compared with the controls ( p < 0.05 ) .
the esr and crp were significantly higher in uc patients than in controls ( p < 0.001 ) , whereas the wbc was similar in both groups ( p > 0.05 ) .
no gfap was detected in the serum samples of either uc patients or controls ( p > 0.05 ; table 2 ) .
however , we found a significant decrease in the s100b serum levels in patients with uc compared with the controls ( p < 0.001 ; table 2 ; figure 1 ) .
no correlation between the serum s100b levels and the disease activity or duration was observed ( p > 0.05 ) .
twenty - four ( 68.6% ) patients were defined as having clinically active disease and 11 ( 31.4% ) patients were in remission .
the serum s100b levels did not differ between active and remission uc patients ( p > 0.05 ) .
the main findings of this study were as follows : ( 1 ) gfap was not detected in either the uc patients or controls , while the serum s100b levels were decreased markedly in the uc patients ; ( 2 ) there was no correlation between the serum s100b levels and the disease activity or duration in uc patients ; and ( 3 ) the serum s100b levels were similar in active and remission uc patients . similar to astrocytes in the cns ,
we hypothesized that disruption of intestinal barrier function or dysregulation of submucosal vascular function could lead to altered serum levels of egc - derived s100b and gfap in patients with uc .
the expression of gfap is modulated by glial cell differentiation , inflammation , and injury , and the gfap level corresponds to the functional state of glial cells . in animals ,
two classes of glial cells can be distinguished : gfap - positive and -negative groups .
von boyen et al . suggested that proinflammatory cytokines control gfap - positive enteric glia , which are in turn involved in modulating the integrity of the bowel during inflammation . in humans ,
gfap expression is altered in the mucosa of patients with ibd . in an animal model of crohn 's disease ,
the glial network was impaired in noninflamed regions of the intestinal mucosa , which was reflected in a significant decrease in gfap immunoreactivity .
thus , we might expect gfap to be detected in the serum of individuals with uc , mainly due to the disruption of egc populations via the disruption of the peripheral blood - tissue barrier .
however , no gfap was detected in the serum samples of either uc patients or controls .
a possible explanation is that gfap is strictly specific to cns - related astrocytic processes .
s100b belongs to a multigene family of calcium binding proteins and is expressed abundantly in the brain .
it is a small , diffusible neurotrophin that is situated in the cytoplasm or nucleus of cells in both nervous system and non - nervous system tissues . in the brain , as an intracellular regulator , s100b affects protein phosphorylation , energy metabolism , the dynamics of the cytoskeleton constituents , calcium homeostasis , and cell proliferation and differentiation [ 18 , 23 ] . at the extracellular level
, s100b has a dual role : it acts as a neuroprotective or a neurotoxic or neurodegenerative molecule depending on its concentration . at picomolar or nanomolar concentrations
, s100b promotes neuron survival , neurite outgrowth , and astrocyte proliferation and negatively regulates astrocytic responses to neurotoxic agents [ 18 , 23 ] .
micromolar amounts of s100b protein produce neuronal death , as seen in several neuropathologies , such as alzheimer 's disease and down syndrome .
in the human gut , among s100 proteins , only s100b protein is specifically and physiologically expressed by egc [ 1 , 5 , 10 ] , while other members , such as s100a8 , s100a9 , and s100a12 , are found in phagocytes and intestinal epithelial cells in patients with ibd . s100b interacts with target proteins within cells , altering their functions once secreted with the multiligand receptor for advanced glycation end - products ( rage ) .
this interaction with rage ultimately leads to the transcription of different cytokines , particularly inos .
therefore , s100b can be considered an easilydiffusible proinflammatory cytokine that gains access to the extracellular space , especially at sites of immune / inflammatory reaction in the gut [ 1 , 5 , 10 ] .
experimental models have indicated that egc are able to recognize inflammatory stimuli and that , once activated , they produce and release s100b in up to micromolar concentrations in the intestinal mucosa of patients with uc .
they thereby contribute to no production in the human gut , a hallmark of uc .
moreover , disruption of the peripheral blood - tissue barrier was shown to correlate positively with mucosal inflammation in uc patients , and a high serum level of s100b has been proposed as a marker for bbb disruption following exercise . however , we observed markedly decreased serum levels ( pg / ml ) of s100b in patients with uc .
this contrasts reports of experimental models showing increased s100b expression due to egc activation [ 1 , 4 , 5 , 10 ] . in other words ,
one is that its picomolar concentrations may suggest prosurvival effects of this protein in the enteroglial network in uc patients .
only newly diagnosed uc patients , while the median disease duration was 3 years in our patient group , who were all taking mesalazine and a small number were also taking immunosuppressive agents .
these agents might inhibit the inflammatory response related to the high s100b expression in our patients , as described in a previous study , displaying decreases in both the cerebrospinal fluid and serum concentrations of s100b under immunosuppressive treatment in patients with neuroinflammatory pathologies . in animal models of ibd , 5-aminosalicylic acid was found to inhibit the expression of inos , which is also involved in the pathogenesis of ibd .
it has already been demonstrated that enteroglial - derived s100b protein expression induces no production in experimental models [ 1 , 4 , 5 , 10 ] .
other than the effects of immunosuppressive treatment on no signaling targeting the vascular endothelium , mesalazine treatment could also inhibit inos via the inhibition of s100b , which might explain the decreased levels in our uc patients .
this raises the question : does mesalazine act via this pathway in the treatment of uc ?
alternatively , the persistent inflammation in uc could lead to severe , widespread damage to the enteroglial architecture in uc patients , leading to a reduction in the extracellular secretion of s100b protein .
furthermore , we hypothesized that s100b levels were further decreased in uc patients with high disease activity or duration .
however , we failed to identify a relationship between these variables , and larger cohorts are needed to produce more definitive results .
in addition , the serum s100b levels did not differ between uc patients with active disease and those in remission .
perhaps their ongoing medical treatment inhibits the egc - derived s100b release in uc patients with active or inactive disease .
first , it will be necessary to validate these findings in a larger cohort including untreated uc patients to clarify the exact role of s100b .
second , this study investigated only serum samples ; mucosal biopsy analyses should provide additional clarification . third , data on s100b gene expression profiles , posttranscriptional s100b protein levels determined by western blot analysis , and no measurements are lacking and might better clarify the mechanisms underlying uc - related enteroglial activation .
fourth , the control group should be well matched regarding bmi , which may be associated with serum s100b levels in humans .
some clinical studies have considered bmi to be an important confounding factor for examining the role of s100b protein [ 3133 ] , while others have not [ 34 , 35 ] . in conclusion , this is the first study to demonstrate that , unlike the markedly increased expression of gfap and s100b observed in experimental models , s100b levels were decreased significantly in patients ' serum samples .
after confirmation achieved by the inclusion of serum from the untreated uc patients in a larger cohort , s100b could be suggested to have an occult role , whereas gfap might be of no diagnostic value in patients with uc . despite the abovementioned limitations , we consider this to be a pioneer study that may guide subsequent research on this issue . indeed
, we wish to validate the present findings in a larger cohort , with additional research on no measurements and the inclusion of untreated patients , all of which we lacked due to limited funding .
future large - scale longitudinal studies that overcome the current study 's limitations will present a more detailed view of the underlying mechanisms of the regulation of s100b and its actual role as a noninvasive biomarker of egc activation in patients with uc .
 
 objective . 
 recent studies have demonstrated that enteric glial cells ( egc ) participate in the homeostasis of the gastrointestinal tract . 
 this study investigated whether enteroglial markers , including s100b protein and glial fibrillary acidic protein ( gfap ) , can serve as noninvasive indicators of egc activation and disease activity in uc patients 
 . 
 methods . this clinical prospective study included 35 patients with uc and 40 age- and sex - matched controls . 
 the diagnosis of uc was based on standard clinical , radiological , endoscopic , and histological criteria . 
 clinical disease activity was evaluated using the modified truelove - witts severity index . 
 serum samples were analyzed for human gfap and s100b using commercial enzyme - linked immunosorbent assay kits . 
 results . 
 gfap was not detected in the serum of either uc patients or controls 
 ( p > 0.05 ) . 
 however , we found a significant ( p < 0.001 ) decrease in the serum s100b levels in the uc patients . 
 no correlation between the serum s100b level and the disease activity or duration was observed 
 ( p > 0.05 ) . 
 the serum s100b levels did not differ between uc patients with active disease ( 24 patients , 68.6% ) or in remission ( 11 patients , 31.4% ) 
 ( p > 0.05 ) . 
 conclusions . 
 ulcerative colitis patients had significantly lower serum s100b levels , while gfap was of no diagnostic value in uc patients . 