Expression And Activity Of Ack1 And Mawbp In Inflammatory Bowel Disease

Background and aimsInflammatory bowel diseases (IBD) are chronic inflammatory disorders affecting the gastrointestinal tract. The two main forms of IBD are Crohn's disease (CD) and ulcerative colitis (UC). These conditions are thought to be genetically determined, overaggressive immune responses of the intestinal mucosa to normal resident bacterial constituents and environmental factors.The prevalence of IBD in the Western Britain and Northern Europe is high and it shows increasing trend in China. Recent studies were mainly focused on endoscopic diagnosis and treatment, while little is know about its molecular mechanism. Nowadays, the diagnosis of IBD is still established by the endoscopic, histologic, and radiologic methods and clinical manifestations.There is a close resemblance in clinical, radiological, endoscopic, surgical and histological features among IBD, intestinal tuberculosis (ITB) and intestinal lymphoma (IL). Patients with activity tuberculosis swallow sputum containing Mycobacterium tuberculosis through intestinal mucosa. Mycobacterium tuberculosis colonizated in the intestinal mucosa and ITB may occur. IL accounts for about 15% to 20% of primary gastrointestinal lymphoma. It is reported that nearly 20 years, the incidence of both IL and ITB has increased around the world. These diseases appear quite stereotyped including diarrhea, abdominal pain, fever and degradation of the general physical condition. The endoscopic features show they all have a predilection for the small bowel, particularly the terminal ileum, although any part of the gastrointestinal tract may be affected. And both ITB and CD are chronic granulomatous disorders.However, the treatments of these conditions are quite different, and the morbidity and mortality resulting from a delayed diagnosis or misdiagnosis is considerably high.Thus differential diagnosis of these conditions remains a major challenge to clinicians.There is currently no easy diagnostic tool as biomarks for these pathologies. The routine blood tests looking at the cell counts and fractionation of white blood cells and other conventional tests, such as the erythrocyte sedimentation rate and C-reactive protein, are too nonspecific which can change during inflammatory states. And do not aid in the work-up of these patients.The most reliable differentiation method of ITB is to find evidence of M. tuberculosis in the intestinal tissues. Unfortunately, acid-fast bacilli's staining lacks sensitivity and specificity. In addition, the biopsy culture for M. tuberculosis is time consuming (3-8 weeks) and results are frequently negative (accuracy ranging from 25 to 35%). The specific pathology of ITB is caseous granulomatous, and CD the whole wall inflammation, non-caseous granulomas. Primary intestinal lymphoma was more central lesions, lymphoma cells to the submucosa infiltrated. However, due to the Unreliable of endoscopic biopsy, typical pathological lesions are not always Available.And for PIL, sometimes surgery and immunohistochemistry is needed.The PPD skin test is not,because in areas with a very low incidence of active TB (<10 per 100,000 per year), a positive TST will more likely be a false positive, and the false-positive rate is very high in areas of the world where the BCG vaccination is still given. New finding such as "in situ PCR" is very promising, but there are many variables that may hamper the diagnostic value of the test, such as the tissue sample size and the duration of exposure of the sample to formalin and other technical aspects that need to be improved to gain a high yield.and "in situ PCR" is not sensitive enough and confusingly may occasionally detect mycobacterial signals from diagnosed CD tissues.More recently, the fecal protein marker calprotectin has been shown to closely correlate with macroscopic and histologic evidence of inflammation. Tibble et al. demonstrated that calprotectin is specific in differentiating between patients with irritable bowel syndrome and IBD.However, these measures are hampered by a low sensitivity and specificity for intestinal inflammation, and do not adequately reflect disease activity.As elevated concentrations can be due to other conditions, including colon cancer, colitis induced by nonsteroidal antiinflammatory agents, or intestinal infections. Notably, intestinal infections are a major issue, as acute exacerbations of IBD may require immunosuppressive therapy, which could be detrimental in patients with infectious diseases. Therefore, a marker that readily differentiates between acute IBD and intestinal infections is highly desirable for clinical decision-making. Wolfgang Tillinger recently demonstrated that CD64 could serve as a valuable tool to discriminate between IBD, infectious enterocolitis, and functional intestinal disorders.A recent series of studies showed that the use of different serological markers, such as the anti-neutrophil cytoplasmic antibodies, the peri-nuclear and the cytoplasmic variants (p-ANCA and c-ANCA), and the IgA and IgG subtypes ofAnti- Saccharomyces cerevisiae antibodies (ASCA).ASCA and ANCA are the only available commercial tests that can be helpful for CD and UC discrimination. Although, they show a quite good specificity, their sensitivity is rather low and they are therefore not recommended for broad clinical practice.However ASCA and ANCA had no significant diagnostic value in discriminating between ITB and CD. Taken together, laboratory markers are useful and should be part of the global management of our IBD patients. They are however not magic and until more data become available, the use of CRP and other laboratory markers should be seen as an additive tool to clinical observation and physical examination rather than a replacement.Therefore, initial diagnosis still relies on the combination of several biological and morphological tests, including gastrointestinal endoscopies and histology, and is based on standardized validated diagnostic criteria.Thus, a rapid test that is sensitive and specific in its ability to differentiate IBD, ITB, PIL and intestinal bacterial infections would be desirable.UC-cancer is different from the normal intestinal cancer, and they show different molecular mechanisms. UC-associated epithelial dysplasia is defined as unequivocally neoplastic epithelium. Currently, it is the most important marker of an increased risk of malignancy in UC. Dysplasia may grow as a flat lesion or as a dysplasia-associated lesion or mass (DALM). In patients with chronic UC, polypoid dysplastic lesions (PDLs) are morphologically similar to sporadic adenomas (SAs), but may be biologically distinct from them and are managed differently. One specific subtype of DALM that poses a diagnostic challenge to clinicians and pathologists is the isolated, discrete dysplastic nodule or polyp that pathologically resembles a sporadic colonic adenoma. Despite the histologic similarity between these two pathogenetically distinct neoplasms, the clinical distinction between UC- PDLs and SAs that arise in patients with UC is extremely important. The former lesion arises as a result of UC, and its presence is usually an indication for colectomy, whereas the treatment of SAs, whether UC-associated or not, is a simple polypectomy. On routine histologic evaluation, distinction between these two neoplasms is difficult. p53 mutations have been shown to occur at an earlier phase in the progression of UC-associated neoplasia when compared with sporadic colon carcinogenesis. In contrast, APC gene mutations are common and occur at an earlier stage in the development of SA.βcatenin is a cell membrane protein that accumulates in the nucleus of colon cancer cells in response to APC gene mutations. No differences were observed in p53 orβcatenin staining, between CUC-associated and non-CUC-associated SAs. Neither p53 norβcatenin expression correlated with any clinical or pathologic features, including size and degree of dysplasia of the polyps. CUC-associated PDLs and CUC-associated SAs may have a different molecular genotype. In patients with CUC, the combination of strong p53 expression and absent or weakβcatenin expression is evidence in favor of a UC-associated PDL in diagnostically difficult lesions. Furthermore, UC-associated and non-UC-associated SAs have a similar p53 andβcatenin immunophenotype and thus provide evidence that they are pathogenetically related neoplasms regardless of the presence or absence of colitis. Therefore, there is great interest in understanding more fully the molecular pathogenesis of these lesions and in detecting markers that may be used to differentiate them.Our search group has found several proteins present in UC unhealth mucosal that are differert from normal mucosal. activated cdc42 kinasel (Ackl) and Mawb binding protein (Mawbp) are two among them and they show significant difference than others.Nowadays, Ackl, a specific downstream effector of Cdc42, have show to be involved in cell growth, inhibit apoptosis, promoting angiogenesis, cell invasion and metastasis. Because it is conbined with Cdc42,the internal and external cellular signaling pathways core protein binding, which has involved with MAPK, NF-kB and other signaling pathways the inflammation associated with Which are not only tumor-related,but also inflammation-related, we guess it may play an important role in cells. The current literatures on the Mawbp are only three.By comparing protein expressions between the primary gastric cancer and the surrounding normal gastric mucosa of differences, Zhang J found that Mawbp are related with in gastric cancer. Westhoff TH found high expression of Mawbp in mice blood is associated with low blood pressure, suggesting its function of blood pressure regulater. Kurokawa analize the different expression genes of liver cancer and adjacent liver tisssues.Currently, there is no study about Ackl, Mawbp associated with IBD. The preliminary work of our group has found that the expression levers of Ackl and Mawbp in UC are significantly higher than that of normal intestinal mucosa. That the changes are UC specific or merely a general change in inflammation remains unclear. And just as noted above, the two proteins are associated with digestive tract tumors, we suspect wether they are involved in the formation of CUC-cancer and colon cancer.The aim of this study was to investigated the utility of Ackl,Mawbp as laboratory biomarkers of IBD to address:(1)whether these markers can differentiate IBD patients from non-IBD gastrointestinal inflammationthe(enterocolitis, chronic colitis);(2)whether they correlate with severity of inflammation; and (3)compare the performance of Ackl, Mawbp in different kind of polys. So we can identify the molecular basis for the development of IBD, and the difference between colorectal adenoma-carcinoma and UC-cancer.Materials and Methods1. A total of 212 colorectal specimens were collected randomly from individuals who underwent endoscopic resection under total colonoscopy. The specimens consisted of 78 UCs,27 CDs,15ITBs,6IL,20 Infectious colitis,26chronic colitis, 10 UC-associated SAs,10 non-UC-associated SAs,10 non-UC-associated inflammatory polyps,10 UC-associated inflammatory polyps and 60 healthy controls.2. The following indexes were obtained from all the patients: age, gender, course of disease, colitis location, serological tests(white blood cells, erythrocyte sedimentation rate, serum albumin, C-reactive protein,Platelet etc); endoscopic feature, and pathological feature.3. Expression of Ackl,Mawbp was examined by immunohistochemistry in all these specimens.4. The performance of each marker with reference to inflammatory activity and clinical activity was assessed by computing correlations, and sensitivity and specificity.Results1. The majority of UCs present with rectum colitis (43.4%) and CDs with small intestine and Ileum (74.1%);2. CRP of CDs was higher than that of UCs(10.95vs5, P<0.05); CRP of active UCs was higher than normal group, there was no significant difference of other indicators among normal, CDs and UCs. Both in the UCs or in the CDs, CRP and ESR were positively and closely correlated. In UCs, the clinical stage was positively and correlated with CRP and ESR. But in CDs, the clinical stage has nothing to do with CRP and ESR.3. In CDs and UCs, Ackl expression and clinical disease activity was positively correlated; in UCs, Ackl cytoplasm cytoplasmic expression and Mawbp were negative correlation; Mawbp and CEA in the CD group was negatively correlated with the cytoplasmic expression sex, Ackl the cytoplasm cytoplasmic expression and Mawbp expression.4. Mawbp, but not Ackl expression of Chronic colitis and infectious colitis was positively correlated with inflammatory pathology grading (P<0.01), but not closely correlated (correlation coefficient<0.5). In CDs and UCs, Ackl and Mawbp showed no correlated with inflammatory pathology grading.5. For differentiation between IBD and healthy controls,Ackl+ in UCs sensitivity (88.9%) and compliance rate (84.06%)was high, but specificity(78.8%)low; the CDs Ackl+ specifity(90%)and compliance rate(83.33%)was high, but sensitivity(66.6%)low.For differentiation between IBD, healthy controls and non-IBD gastrointestinal inflammationthe,UCs Ackl+ specificity(83.70%)was higher, but sensitivity (65.31%) slightly lower; in CDs,Ackl+specificity(92.30%) was higher,while sensitivity (32.00%) and compliance rate (68.75%)was low.6. In all samples of ITB (15/15) and IL (6/6) Ackl show high expression (3+) and Mawbp expression Medium (2+).7. The expression of Ackl in SAs (2+or3+) were significantly higher than that in inflammatory polyps (-or+).DALM, UC-associated-SAs, non-UC-associated-SAs staining had a similar Ackl immunophenotype.ConclusionsIn the present study, we have shown that Ackl, Mawbp protein expression is increased not only in IBD mucosa and ITB, IL but also in bacterial enterocolitis, chronic colitis, using Immunohistochemistry. None of these three markers is consistently superior in its ability to differentiate active IBD from other colitis.On the other hand, it may be noted in this study that colitic cancers develop in only a limited number of patients with UC, although Ackl is ubiquitously expressed in the inflamed UC mucosa. Thus Ackl, Mawbp may play a role in the UC-cancer sequence as one of the growth promoting as well as antiapoptotic factors, and function in colitic cancer development not as a inflammation/tumour initiator but as a inflammation/tumour promoter.