The Efficacy Of Collagen Fibers And Fibrosis Related Proteins For Discriminating ITB From CD

BackgroundIntestinal tuberculosis was mainly caused by the infection of mycobacterium tuberculosis, less caused by mycobacterium tuberculosis through drink of polluted milk. On the other hand,Crohn’s disease was an idiopathic inflammatory bowl disease with a genetic background and affected by multiple environmental factors. It can involve all the digestive tract from oral to the rectum.Though the etiology was totally different between this two disease,both ITB and CD a granulomatous disease with a lot of similarities in clinical,endoscopic and pathological features,which make differentiation between the two disease remain a challenging but at the same time critical work,since treatment strategies was completely different and might lead to deadly consequence to patients.When intestinal tuberculosis was misdiagnosed as crohn’s disease, using immunosuppressor will cause the spread of TB; When patients with crohn’s disease was misdiagnosed as intestinal tuberculosis, unnecessary anti-tuberculosis treatment will cause side effects of drugs, at the same time delay in the treatment of primary disease might cause complications which can be avoided if the primary disease be recognized earlier. Accounting for the lack of specific indicator to discriminate ITB from CD, misdiagnosis rate was remain high and reported at about 50-70%.Fibrosis has been found to be important in the pathological process of CD and ITB. Current hypothesis supposed that the initiating factor for intestinal fibrosis is a tissue damage caused by a chronic inflammatory state. Secondly,activated fibroblasts are recruited to the inflammation area and induced healing and finally,fibrosis results due to the disposition of excessive extracellular matrix(ECM). Study of the mechanism for fibrosis mainly focus on CD nowadays,it was shown that CD related fibrosis might be the result of epithelial-to-mesenchymal transition(EMT),which was marked by the decreased expression of epithelial markers and increased expression of mesenchymal markers like vimentin. At the same time,TGF-β was recognized as the most important protein for fibrosis in both intestinal and other organs.Increased expression of TGF-β in stenosed part of the bowl was related to early surgery in adult CD.Of note,there was a distinct difference of fibrosis in different disease. Transmural fibrosis was observed in CD,while only mucosa and sub-mucosa was affected in UC. Those differences indicated that the degree and mechanism for fibrosis of different disease might be different as well,quantification for fibers content and proteins that were associated with fibrosis may help to discriminate different etiology like ITB and CD.Our research here attempts to find a new clue to discriminate ITB from CD through the comparison of fibrosis and fibrosis related proteins differences between this two disease,which was evaluated by quantification of collagen fibers as well as the expression level of TGF-β and vimentin in Colonoscopic biopsies of this two disease.ObjectiveWe compared the differences of collagen fibers and fibrosis related proteins in patients with intestinal tuberculosis(ITB) and Crohn’s disease(CD) in order to find out a new clue to discriminate the two diseases.MethodColonoscopic biopsies from healthy controls and patients diagnosed as ITB and CD in Zhujiang hospital from 2006 to 2015 were collected, including 15 patients diagnosed as ITB,19 patients diagnosed as CD and 15 normal control.The content of total collagen were evaluated by masson’s trichrome staining. The content of type I and type III collagen were evaluated by double label immunofluorescence staining.Immunohistochemistry was used to detected the expression level of TGF-β and vimentin.Image-Pro Plus 6.0 Software was used to analyze the image and average optical density (IOD) was used to describe the expression level of those indicators.Receiver Operator Characteristic curve(ROC curve) was established to evaluate the value of those indicators for discriminating ITB from CD. Area under the curve (AUC)describe the accuracy of those indicators for discriminating this two disease,there was no diagnostic value when AUC was less than 0.5,diagnostic value was low when AUC was between 0.5-0.7 and relatively high when AUC was larger than 0.7.ResultsComparison of clinical,endoscopy and pathology features between ITB and CD the comparison of clinical manifestation and complication between ITB and CD showed that abdominal pain, diarrhea and weight loss were more common in CD group than ITB group,while fever and bloody stools were more common in ITB group than CD group. But those manifestations were all of no significant different between the two groups (P> 0.05). Extra-intestinal tuberculosis was statistically more frequent in ITB group than CD group(P=0.028). Intestinal stenosis, obstruction and fistula were seen more frequent in CD group than ITB group,while there were no significant different between two groups (P> 0.05). As for endoscopy and pathology features,pseudopolyp, and cobblestone appearance were significantly more common in CD group than ITB group (P<0.05).Caseating granuloma was specific feature for ITB,which was seen only in ITB group (P<0.05). Whlie the rest features like Intestinal mucosa inflammation, erosion, ulceration, longitudinal ulcer, mucous bridge,lesion of ileocecal valve,circular ulcer,granuloma formation, non caseating granuloma and crack ulcer were all of no significant different between two groups (P >0.05).Quantification of total collagen fibers in masson staining we used Massonstaining to evaluate the total collagen fibers content in CD, ITB and healthy people. Collagen fibers were stained blue in Masson staining. The content of total collagen fiber was significantly different among ITB group (22093.53± 1643.12), CD group (15200.42±1232.63) and normal control group (6964.20±819.98) (P＜0.05), and those in ITB group was significantly higher than the other two groups(PCD vs ITB=0.007, PCD vs normal=0.000, PITB vs CD=0.000).Quantification of type I and type III collagen fibers by double label immunofluorescence staining Double label immunofluorescence staining was applied to observed type I collagen (abeam 1:50) and type III collagen (abcam 1:50).Type I collagen was stained red,while type III collagen was stained green and nucleus stained blue. The content of type I and type III collagen were both significantly different among ITB group (type I collagen quantitative 7086.±948.45; type III collagen quantitative 17030.47±1697.28), CD group (type I collagen quantitative 4304.47±454.17;type III collagen quantitative 11292.11±758.46) and normal control group (type I collagen quantitative 3475.60±397.32;type III collagen quantitative 2684.80±328.82) (P＜0.05), and those in ITB group was significantly higher than the CD groups(type I collagen quantitative:PCD vs rrB=0.045, P CDvs normal=0.440, PITB vs normal=0.007;type III collagen quantitative:PCDvsITB=0.017, PCD vs normal=0.000, P ITB vs normal=0.000).Quantification of TGF-β、vimentin expression level by immunohistochemistry Immunohistochemistry was used to observed the expression of TGF-β (WuHan boster,1:50) and vimentin (WuHan boster,1:50). Positively stain TGF-P was mainly located in the cytoplasm of gland cells and mesenchymal cells, while vitmentin was mainly located in the cytoplasm of mesenchymal cells,both of them appeared as light brown and brown particles.The expression level of TGF-P was significantly different among ITB group (70327.80±15523.75), CD group (67823.21±6302.86) and normal control group (21871.±3229.77) (P<0.05),while there was no significant difference between ITB group and CD group (P CD vs TTB=0.998, P CD vs normal=0.000, P ITB vs normai=0.023). The expression level of vimentin was significantly different among ITB group (63583.40±.80), CD group (96885.84±7073.05) and normal control group (30959.80±3650.68) (P＜0.05), and those in CD group was significantly higher than the other two groups (P CD vs ITB=0.046, P CD vs normal=0.000, P ITB vs normal=0.030).ROC curve analyze for the value of total collagen fibers,type Icollagen, type Ⅲ collagen and vimentin quantitative in discriminating ITB from CDAs it was showed above, total collagen fibers,type I collagen, type III collagen and vimentin quantitative of colonoscopic biopsies in ITB and CD were significantly different. Receiver operator characteristic curve (ROC curve) was established to evaluate the efficacy of those indicators for discriminating ITB from CD. It was showed by ROC curve analyze that except for quantification of vimentin,whose area under the curve was lower than 0.5(AUC 0.235,95% CI:0.059-0.411),the other three indicators were all valuable for discriminating ITB from CD. Area under the curve of total collagen fibers,type I collagen,type III collagen quantitative were 0.807 (P=0.002,95%CI 0.661-0.953),0.751 (P=0.013,95% CI 0.589-0.913),0.814 (P =0.002,95% CI 0.672-0.956) respectively. Quantification of total collagen fibers,collagen type I,collagen type III achieve a sensitivity and specificity of 80.0% and 78.9%,86.7% and 52.6%,73.3% and 73.7% respectively in the optimal cut off point in ROC curve analyses.ConclusionThere existed a lot of similarities of ITB and CD in clinical,endoscopy and pathology features,which make discriminate one from the other very difficult. Fibrosis was characteristic of both disease. We found that the amount of total collagen,the amount of type I collagen and type III collagen were significantly higher in ITB group than CD group,those difference were valuable for discriminating ITB from CD,which might become a new clue for discriminating the two disease in the future.