inflammatory bowel disease ( ibd ) is a group of chronic conditions of the colon and small intestine , consisting of crohn 's disease ( cd ) and ulcerative colitis ( uc ) , characterized by acute pain , vomiting , and diarrhea symptoms followed by remission .
a single etiology has not been identified , but rather the pathogenesis of ibd is very complex and involves the external environment , genetic makeup , intestinal microbial flora , and immune system .
although new and powerful medical treatments are available , many are biological drugs or immunosuppressants , which are associated with significant side effects , in particular infection and increased risk of malignancy , and elevated costs which require optimal medical treatment adjustment . as a result ,
major attempts have been made at identifying clinical characteristics , concurrent medical therapy , and serological and genetic markers as predictors of response to biological agents .
only few reports exist on how mucosal / tissue markers are capable of predicting clinical behaviour of the disease or its response to therapy .   due to its ability of interfering with intestinal barrier function and stimulating local and systemic inflammation , dextran sodium sulfate ( dss )
is often used as a mouse model of colitis which can mimic clinical and histological features of ibd with uc characteristics .
most commonly , experimental colitis is induced by heparin - like polysaccharide dss because of its capacity of inducing colonic lesions .
.   showed that oral administration of 5% of dss in drinking water of balb / c mice was able to induce a chronic colitis after several cycles of dss .
this study was followed by a report from cooper et al .   who induced chronic colitis by ( a ) 7 days of oral dss followed by 7 days of h2o ( for 1 , 2 , and 3 cycles ) and ( b ) 7 days of oral dss followed by 14 and 21 days of h2o .
the results of this study showed that chronic colitis induced after only 7 days of dss may serve as a useful model to study the effects of pharmacologic agents in human inflammatory disease and mechanisms of perpetuation of inflammation and gave an extensive description of histological lesions , showing that the main histological changes consisted of focal crypt loss , which was followed by signs of both acute and chronic inflammation .  
the present study aimed to investigate the effect of fr-91 on the attenuation of the chronic experimental colitis induced by dss in swiss mice .
furthermore , we investigated if the chronic phase was characterized by a regulation in the expression of apoptotic genes and by a dysregulation of t helper 1 ( th1)/t helper 2 ( th2 ) balance and how this would relate to mucosal regeneration .
twenty - six specific pathogen - free swiss cd1 female mice ( 7 weeks old ; santiago de compostela 's university animal breeding core , spain ) were maintained ( two or three per cage ) in isolator plastic cages with shavings under standard laboratory conditions ( sterilizable diet , 50% humidity , 23 - 24c temperature , and 12-h light / dark cycle ) .
all mice were quarantined 3 weeks after arrival and then randomized by body weight into experimental and control groups .
all mice were permitted free access to a commercial diet and treatment or normal drinking tap water in individual bottles .
all procedures conformed to the guidelines established by the european communities council directive of 24 november 1986 ( 86/609/eec ) and by the spanish royal decree 1201/2005 for animal experimentation and were approved by the ethical committee of ebiotec .  
the design of the present study was focused primarily on inducting colitis - associated dysplasia and/or ulcerative hallmarks by administering synthetic dextran sulfate sodium ( dss ) to mice and then treating them with different fr91 ( standardized lysate of microbial cells belonging to the bacillus genus ) dilutions as shown in figure 1 . at the age of 7 weeks
, the animals were divided into two control groups ( 1 and 2 , n = 4 each group ) and three experimental groups ( 35 , n = 6 each group ) . along the entire experimental procedure , distilled water containing different fr91 dilutions ( 5% in group 3 , 10% in 1 and 4 , and 20% in 5 )
onwards , distilled water containing 20  g / l ( 2% ) synthetic dextran sulfate sodium ( dss ; mol mass 5000 ; d4911 , sigma - aldrich ; mo , usa ) was also administrated to animals of groups 25 . for comparison , control groups 1 and 2 received dss or the fr91 treatment alone , respectively , as untreated control .
all mice were sacrificed at the end of the experiment ( 8th week ) , at the age of 15 weeks .  
experimental colitis was induced to mice of groups 2 , 3 , 4 , and 5 , by repeated administrations of 2% ( wt / vol ; 20 
this dose was empirically reported to induce moderate to severe colitis while minimize mortality in mice .
none of the mice died before the termination of this experiment study at day 50 .
mice were deeply anesthetized with ether and intracardially perfused with saline buffer and then fixed by 4% paraformaldehyde in 0.1  m phosphate buffer ( ph 7.4 ) .
the entire colorectum ( from colocecal junction to the anal verge ) was removed , measured , examined macroscopically , washed with saline buffer , and immediately fixed by immersion in the same fixative for 48  h. part of the colon was divided into three equal segments ( proximal , middle , and distal ) , and portions were determined under a dissecting stereomicroscope ( leica , m125 ) .
intestine portions were cryoprotected with 30% sucrose in 0.1  m phosphate buffer , embedded in oct compound ( tissue tek , torrance , ca ) , and frozen with liquid - nitrogen - cooled isopentane .
parallel series of transverse sections of 1416  m thick were obtained on a cryostat ( starlet 2212 , bright , uk ) and mounted on superfrost plus ( menzel - glser ) slides .
routine histological examination was performed on hematoxylin and eosin ( h&e)-stained sections , where different morphological alterations that occur during inflammation- associated colorectal carcinogenesis ( such as cryp abscess , mucosal dysplasia , adenomas , and adenocarcinomas ) , were identified and diagnosed according to previous published studies , see keohane et al . . to detect the expression of colorectal histopathological markers such as catenin- , p53 , bcl-2 , mlh1 , and apc , we used immunohistochemical techniques .
the sections were pretreated with h2o2 to eliminate endogenous peroxidase , rinsed twice in phosphate - buffered saline ( pbs ) at ph 7.4 ( 10 minutes each ) , and then sequentially treated with nonspecific binding blocked solution ( 0.1  m pbs containing 0.2% tween 20 and 15% normal goat serum from dako ; glostrup , denmark ) for 1  h , primary rabbit policlonal antibodies such as anti - catenin- , anti - p53 , anti - bcl-2 , anti - mlh1 , and anti - apc antibodies ( rabbit antibodies that were affinity purified from rabbit antiserum by affinity chromatography using mouse epitope - specific immunogen ; bioworld technology , mn , usa ; cat .
bs3603 , bs3736 ; bs1511 , bs2418 , bs1017 respectively , dilution 1  :  200 ) overnight , pbs ( two 10-min rinses ) , goat anti - rabbit igg serum biotinylated ( dako , dilution 1  :  100 ) for 1  h , pbs ( two 10-min rinses ) , vectastain abc kit ( vector laboratories , burlingame , ca , usa ) for 1  h , and pbs ( two 10-min rinses ) . as a negative control , omission of the primary , secondary or tertiary antibodies were used , and no immunostaining was observed . at the last step ,
the immunoreaction was developed with 0.005% diaminobenzidine ( dab ; sigma - aldrich ) and 0.003% h2o2 .
all dilutions were made in pbs containing 0.2% tween 20 , and incubations were made in a humid chamber at room temperature . finally , the sections were dehydrated , mounted , and coverslipped .   antibody characterization and specificity . according to the technical information supplied by the manufacturer ( bioworld technology , mn , usa ) , the primary antibodies used were raised against denatured mouse epitopes from rabbit antiserum , and they were affinity purified by chromatography using epitope - specific immunogen with purity higher than 95% ( by sds - page ) .
its specificity has been assessed by western blot ; it recognizes a single - protein band of approximately 8690  kd ( -catenin ) , 4345  kd ( p53 ) , 2628  kd ( bcl-2 ) , 8486  kd ( mlh1 ) , and 270280  kd ( apc ) .
moreover , antibodies have wide species cross - reactivity and were used for demonstrating their expression in mouse , rat , and human .
lesions were classified as positive for catenin-/bcl-2/mlh1/apc if cytoplasmic / nuclear staining was detected , and p53 was considered positive if nuclear expression was detected .
two different observers evaluated individually and independently the experimental group slides in a double - blind manner and achieved a high level of concordance .
three sections of each mouse colorectal segment were coded and scored for lesions according to the extent ulceration ( 0 , not present and 1 present ) , severity of lesions , hyperplasia , and area involved , graded as follow : 0 , normal ; 1 , mild ; 2 , moderate ; 3 , severe .
the sections were photographed with an olympus microscope ( bx50 ) equipped with a color digital camera ( dp10 ) .
the photographs were converted to gray scale and adjusted for brightness and contrast with corel draw ( corel , ottawa , canada ) , and the plates composed with corel photo paint .
quantitative detection of gm - csf , ifn- , il-1 , il-2 , il-4 , il-5 , il-6 , il-10 , il-17 , and tnf- was performed by using a flowcytomix mouse th1/th2 10plex ( bms820ff ) from bender medsystems which allowed to measure multiple analytes in a single 50 microliter aliquot of mice sera . in brief
, microparticle beads were dyed with differing concentrations of two fluorophores to generate distinct bead sets .
captured analyte was detected using a biotinylated detection antibody and streptavidin - phycoerythrin ( s - pe ) .
a facscan flow cytometer from becton dickinson was used to acquire samples . for calculation of results ,
the mice that received repeated administrations of 2% dss and lower or absent levels of fr91 ( groups 2 and 3 ) showed bloody stools during the second half of the experiment , ( from the fifth week onwards ) , whereas no such internal inflammation feature was observed in the other mice groups .
macroscopically , we have identified numerous gross inflammatory polypoid lesions in mice of groups 2 ( 6/6 ; 100% ) and 3 ( 5/6 ; 83.3% ) , mainly on the middle and distal portions of the colorectal segment , and very few were observed in group 4 ( 1/6 ; 16.6% ) .
remarkably , none of the mice group 5 ( dss/20%fr91 ) showed any ulcer formation in the colorectal segment analyzed , as well as the treatment control mice group 1 ( 10%fr91 ) that was also free of colitic ulcerations .
these macroscopical observations were confirmed by histological analysis of the intestinal morphology emphasizing the alterations regarding the integrity and inflammation of colonic mucosa and submucosa , dysplastic epithelium , and the presence of ulcers .
this histological examination of transverse sections staining with h&e ( figure 2 ) showed ulcers with moderate - to - severe morphological alterations ( multifocal areas of inflamation in the submucosa or ulcers that covered large mucosal areas ) , mild - to - severe crypt hyperplasia ( lining epithelium was two to three times normal thickness , marked hypercromasia of cells , and multiple crypts with arborizing pattern ) , epithelial dysplasia ( alteration in the differentiation of epithelial cells that may progress to invasive carcinoma ) , and large affected areas of crypt loss .
however , severity of these colorectal lesions differed significantly among mice groups , being the mice group 2 ( dss ) the one with a high severity level , while the mice group 3 ( dss/5%fr91 ) showed a mild - to - moderate severe level , and the mice group 4 ( dss/10%fr91 ) presented a few mild scattered lesions .
no colitis characteristic lesions were observed in the colorectal segment of mice group 5 ( dss/20%fr91 ) or in the treatment control mice group 1 ( 10%fr91 ) , ( figure 2 ) , as described previously in the macroscopical exam .
histological scoring data obtained from the colorectal lesions examination are presented in table 1 .  
immunohistochemical techniques used to identify cell markers of the colorectal lesions showed expression of catenin- ( cellular adhesion regulator ) , bcl-2 ( apoptotic regulator ) , mlh1 ( dna - mismatch repair ) , apc , and p53 ( tumour suppressor proteins ) in all colonic mice lesions observed and described above .
catenin- antibody detected endogenous expression levels of catenin- protein mainly in the cytoplasm of adenocarcinoma and dysplastic cells .
intense catenin- expression was observed in the proximal and middle ( figure 3(f ) ) colon of mice group 2 ( dss ) whereas a moderate - to - intense immunoreactivity was also observed in the colorectal segments ( figure 3(k ) ) of mice group 3 ( dss/5%fr91 ) .
catenin--immunoreactive ( -ir ) cells were localized in the dysplastic criptal cells and adenocarcinoma cells , at the internal criptal layers ( figures 3(f ) and 3(k ) ) .
colorectal portions of mice groups 1 , 4 , and 5 showed a weak or absent catenin--positive reaction in their cryptal cells ( figures 3(a ) , 3(p ) , and 3(u ) ) , considered as catenin- cellular basal expression .
bcl-2-ir and p53-ir cells were observed at colorectal portions of mice groups 2 ( figures 3(g ) and 3(j ) ) and 3 ( figures 3(l ) and 3(o ) ) , showing an intense staining in adenocarcinoma and cryptal cells , particularly strong in mice group 2 .
immunoreactivity to bcl-2 and p53 was absent in colorectal section of mice groups 1 , 4 , and 5 studied ( figures 3(b ) , 3(q ) , 3(v ) , 3(e ) , 3(t ) , and 3(y ) ) .
strong staining mlh1-ir cells were observed at colorectal segments of mice groups 2 ( figure 3(h ) ) and 3 ( figure 3(m ) ) , showing an intense staining in adenocarcinoma and cryptal cells of the dysplastic epithelium , particularly intense in mice group 2 .
mlh1-ir cells were absent in colorectal section of mice groups 1 , 4 , and 5 studied ( figures 3(c ) , 3(r ) , and 3(w ) ) .
apc immunoreactivity was intensely observed in all colorectal portions of mice groups 2 ( figure 3(i ) ) and 3 ( figure 3(n ) ) although cryptal cells of mice group 2 showed a stronger positivity .
colorectal portions of mice groups 1 , 4 , and 5 showed a weak apc immunoreactivity in their cryptal cells ( figures 3(d ) , 3(s ) , and 3(x ) ) .
the incidence of colon lesions showed by these cell markers in the group 5 was 8.2%  ( 4% ) , whereas it was 38.2%  ( 6% ) , 33.3%  ( 6% ) , and 20.4%  ( 3% ) in groups 2 , 3 , and 4 , respectively ( see figures 4 and 5 ) .
similar histological incidence was observed in mice group 5 when compared with negative control group 1 ( 7.8% ,  2% ) .
two - factor anova of data for each immunohistological markers in the colorectal portion showed significant differences among experimental groups 1/5 and 2/3/4 , ( figure 5 ) .
group 5 was the most resistant mice group to dss - induced lessions in the colorectal portion , as indicated by the lowest value for each marker , similar to that observed in the control group 1 .
groups 2 , 3 , and 4 showed more susceptibility to dss - induced lessions , consistent with high values .
significant group differences were found between group 1/5 and 2/3/4 in dss susceptibility , being correlated with differences in the fr91 mean consumption per group .
we next considered potential mechanisms which might underlie the colitis exhibited by dss treatment and the effect of fr-91 in the treated mice groups .
as shown in table 2 , dss treatment increased only the production of ifn- proinflammatory cytokine .
no significant changes were detected in il-1 , tnf- , il-6 , il-10 , and il-17 in both treated and untreated groups .
more than 20,000 bioactive compounds synthesized by microorganisms have been identified , and over 10,000 of these secondary metabolites are produced by actinomycets , representing 45% of all bioactive metabolites discovered . among actinomycetes , around 8,000 compounds are synthesized by streptomyces species .
in addition , many of these compounds , such as anthracyclines ( aclarubicin , daunomycin , and doxorubicin ) , peptides ( bleomycin and actinomycin d ) , aureolic acids ( mithramycin ) , enediynes ( neocarzinostatin ) , antimetabolites ( pentostatin ) , carzinophilin , mitomycins , and others [ 9 , 10 ] have also been tested for the inhibition of chemically induced carcinogenesis in both in vitro and in vivo animal models . in the present study
, we investigated the effect of fr91 , a standardized lysate of microbial cells belonging to the bacillus genus which has been previously shown to have significant immunomodulatory effects tested on human tumor cell lines , on colonic inflammation induced by 8-week exposure of 2% dss in the drinking water and , in particular , whether fr91 affects colorectal inflammation .
we found that the six - week treatment resulted in a slightly reduction of colorectal lesions at lower dosis ( 5% fr91 ) and a moderate - to - complete reduction at higher dosis ( 1020% fr91 ) .
moreover , histopathological data showed that fr91 has no pathological effect on the morphological organization of the mice colon tissues , as observed when administering fr91 alone during the entire experimental study .
the data obtained suggests that fr91 may be an important chemopreventive agent against intestinal inflammation in mice colon . in our experiment
, we induced a wide range of colorectal lesions to better evaluate the effects of an anticancer agent ( fr91 ) on chronic ulcerative colitis in mice .
the repeated administration of dss as an inductor of ulcerative lesions in mice models of colitis was extensively reported [ 1318 ] , being essential in advancing our understanding of the complex interactions between the environment , genetics , and epithelial barrier dysfunction in the human - related inflammatory bowel disease [ 1922 ] . in this study ,
the use of dss in drinking water during five weeks resulted in epithelial damage and a robust inflammatory response , obtaining a valid mice model acute injury colon to test an eight - week treatment with fr91 , as reported in previous studies [ 2326 ] .
our results demonstrated that the optimal dose response was the 20% fr91 concentration tested in mice group 5 , where no histological alterations or mild lesions were observed .
routinary histological staining [ 20 , 23 , 24 , 27 ] and pretumoral cell markers [ 18 , 20 , 23 , 28 ] used to evaluate the severity of lesions confirmed the protective effect of fr91 against the inflammation effect of dss .
these pretumoral cell markers such as apc , p53 , mlh , bcl-2 , catenin- , and cytokines , among others , take part in the molecular pathogenesis pathway of chronical colorectal inflammation that has been reported to derive at further stages in the development of sporadic colorectal carcinoma and colitis - associated colon cancer [ 22 , 2931 ] .
the present results show the same significant interaction of these genetic markers in the pathologic characterization of the dss - induced lessions observed .
as reported in similar studies , a dosis - dependent effect is normally associated with the chemoprevention process in dss - induced colitis [ 25 , 26 ] .
therefore , we tested other lower fr91 concentrations in drinking water , obtaining gradual colorectal lesions as the fr91 concentrations decreased .
moreover , we also tested the effects of fr91 in mice during a large period of time ( 8 weeks ) , obtaining similar histological results as the nontreated mice , which indicates a chemopreventive action against carcinogenesis without interfering with healthy epithelial structures of the mice colon .  
chemopreventive effects of a wide range of compounds on colonic tumors induced in mice have been reported previously , such as organosulfur compounds , n acetylcysteine , tetrandrine , phytosteryl ferulates , and dibenzoylmethane derivatives .
the dss - induced lessions reported in these studies are similar to that observed in the preset work , where generally predominated in the midcolon [ 7 , 35 ] and distal [ 24 , 36 ] intestinal portions .
these particular locations have been attributed to several factors such as the selective uptake of dss in certain colonic portions , the presence of weak intestinal barrier regions , and different distribution of macrophages populations along the mice colon .  
although the real impact of these findings in the pathogenesis of ibd remains a controversial issue , as it is unclear whether they are primary or secondary factors involved in the regulation of the mucosal intestinal immune system , they can be considered as markers in the differentiation of groups of patients . in active ibd
, an unbalance between regulatory and effector cells has been described , which mainly involves effector t cells ( th1 and th2 ) and regulatory t cells ( tregs , th3 ) .
cd is associated with a th1 t - cell cytokine profile , including ifn- , tnf- , and il-12 , whereas uc is associated with a modified th2 type response cytokine profile including il-15 and il-10 .
in addition , these findings have been recently complemented by the discovery of the il-23/il-17 axis , that is , part of the effector t - cell immunological response and seems to be involved in ibd .
levels of expression of il-23 and il-17 are increased in patients with active ibd .  
although specific determination of the fr91 metabolic action in the colitis prevention and treatment will require further investigations , we showed that fr91 prevented ulcerative lesions in mice models of colitis , inhibiting the development of colorectal tumors .
fr91 has proved to be an interesting and promising investigational agent for studying chemoprevention of carcinogenesis .
 one of the main treatments currently used in humans to fight cancer is chemotherapy . 
 a huge number of compounds with antitumor activity are present in nature , and many of their derivatives are produced by microorganisms . 
 however , the search for new drugs still represents a main objective for cancer therapy , due to drug toxicity and resistance to multiple chemotherapeutic drugs . in animal models , 
 a short - time oral administration of dextran sulfate sodium ( dss ) induces colitis , which exhibits several clinical and histological features similar to ulcerative colitis ( uc ) . 
 however , the pathogenic factors responsible for dss - induced colitis and the subsequent colon cancer also remain unclear . 
 we investigated the effect of fr91 , a standardized lysate of microbial cells belonging to the bacillus genus which has been previously shown to have significant immunomodulatory effects , against intestinal inflammation . 
 colitis was induced in mice during 5 weeks by oral administration 2% ( dss ) . 
 morphological changes in the colonic mucosa were evaluated by hematoxylin - eosin staining and immunohistochemistry methods . 
 adenocarcinoma and cryptal cells of the dysplastic epithelium showed cathenin- , mlh1 , apc , and p53 expression , together with increased production of ifn-. in our model , the optimal dose response was the 20% fr91 concentration , where no histological alterations or mild dss - induced lesions were observed . 
 these results indicate that fr91 may act as a chemopreventive agent against inflammation in mice dss - induced colitis . 