Colonic Epithelial Protein Research Articles

Resistin-like molecule beta, a colonic epithelial protein, exhibits antimicrobial activity against Staphylococcus aureus including methicillin-resistant strains.

Resistin-like molecule beta (RELMβ) is a small cysteine-rich protein secreted by colonic epithelial cells. RELMβ mRNA and protein expressions are dramatically induced by bacterial exposure in germ-free mice. We hypothesized that RELMβ has antimicrobial activity. The antimicrobial activity of RELMβ was screened by an agar spot test and confirmed by a liquid broth test. The amount of RELMβ in human stools was semi-quantified by Western blot analysis. The induction of RELMβ mRNA and protein expression by bacteria was measured by quantitative RT-PCR using LS174T cells. Electron microscopic immunohistochemistry was performed using polyclonal anti-RELMβ antibody. RELMβ showed antimicrobial activity against S. aureus and all MRSAs examined in a dose- and pH-dependent fashion. Western blot study showed that the amount of RELMβ in healthy human stools was comparable to that exhibiting antimicrobial activity in vitro. Both RELMβ mRNA and protein expression were induced by heat-inactivated S. aureus, but not by E. coli in LS174T cells. Electron microscopic immunohistochemistry showed that RELMβ bound to the cell surface of S. aureus, followed by destruction of the bacterial cytoplasm. RELMβ is a colonic antimicrobial protein and its antibacterial activity is species selective. Because RELMβ is abundant in healthy human stool, RELMβ may modulate gut flora.

Colonic Epithelial Surfactant Protein D Expression Correlates with Inflammation in Clinical Colonic Inflammatory Bowel Disease.

Inflammatory bowel diseases (IBD) are chronic disorders of the gastrointestinal tract. Surfactant protein D (SP-D) is expressed in the intestinal epithelium and is essential for innate host defense and regulation of inflammatory responses. Genetic variations of SP-D are associated with IBD, but the effects of SP-D in clinical disease development have not been clarified. We hypothesized that colonic epithelial SP-D expression is increased in parallel with intestinal inflammation with the capacity to dampen deleterious effects. Surgical specimens from IBD patients including Crohn's disease (n = 9) and ulcerative colitis (n = 18) were scored for expression of SP-D and inflammatory activity. Cohoused Sftpd+/+ and Sftpd-/- mouse littermates were subjected to dextran sodium sulfate (DSS) for 7 days to induce colitis. Colonic tissue was scored for histologic damage and analyzed for inflammatory markers and expression of SP-D. Surgical specimens from IBD patients showed a strong positive correlation between immunoscore for SP-D and inflammatory activity (R2 = 0.78, P < 0.0001). In mice, colonic epithelial SP-D expression was very low, and DSS-induced colitis was unaffected by SP-D deficiency, although DSS induced transcription of colonic SP-D to a mild degree. A strong positive correlation between inflammatory activity and epithelial expression of SP-D was observed in surgical specimens from IBD patients supporting a role for SP-D in clinical disease. The in vivo study was inconclusive due to very low intestinal SP-D expression in the mouse. Further studies are warranted to support that increased SP-D expression in the human colonic epithelium is protective against intestinal inflammation.

Alterations in the Immunohistochemical Expression of Das-1 and CG-3 in Colonic Mucosal Biopsy Specimens Helps Distinguish Ulcerative Colitis From Crohn Disease and From Other Forms of Colitis

Distinction between ulcerative colitis (UC) and Crohn disease (CD) in mucosal biopsies is often difficult. Das-1 and CG-3 are monoclonal antibodies directed against an unknown colonic epithelial protein and human tropomyosin isoform-5, respectively, both show altered expression in patients with UC. In this study, we evaluated the utility of Das-1 and CG-3 in distinguishing UC from CD and from other types of colitis. One colonic biopsy specimen from each of 85 patients with confirmed UC (n=25), CD (n=15), lymphocytic (n=15), collagenous (n=15), and ischemic (n=15) colitis, and also 10 samples from normal controls, were stained for Das-1 and CG-3 using standard techniques. Reactivity for Das-1 and CG-3 was noted to be absent or present, and the location (ie, surface+/-crypt epithelium) and degree (weak or strong) of CG-3 staining was recorded. Loss of Das-1 staining occurred more frequently in UC (96%) compared with CD (20%), lymphocytic (20%), collagenous (13%), and ischemic colitis (0%) cases, as well as controls (10%, P<0.001 for all comparisons). CG-3 positivity in crypt epithelium was significantly more common in UC (52%) compared with the other groups (P< or =0.02 for all comparisons). The combination of strong crypt CG-3 staining and loss of Das-1 staining was noted in 44% of UC cases, but not in any other type of colitis (P=0.003 for all comparisons). We conclude that the patterns of Das-1 and CG-3 staining in colonic mucosal biopsies may be clinically useful in distinguishing UC from CD and from other colitidies.

Ileal lesions in patients with ulcerative colitis after ileorectal anastomosis: Relationship with colonic metaplasia

To assess whether in ulcerative colitis (UC) patients with ileo-rectal anastomosis (IRA), ileal lesions may develop in the neo-terminal-ileum and their possible relation with phenotypic changes towards colonic epithelium. A total of 19 patients with IRA under regular follow up were enrolled, including 11 UC and 8 controls (6 Crohn's disease, CD; 1 familial adenomatous polyposis, FAP; 1 colon cancer, colon K). Ileal lesions were identified by ileoscopy with biopsies taken from the ileum (involved and uninvolved) and from the rectal stump. Staining included HE and immunohistochemistry using monoclonal antibodies against colonic epithelial protein CEP (Das-1) and human tropomyosin isoform 5, hTM5 (CG3). Possible relation between development of colonic metaplasia and ileal lesions was investigated. Stenosing adenocarcinoma of the rectal stump was detected in 1 UC patient. The neo-terminal ileum was therefore investigated in 10/11 UC patients. Ileal ulcers were detected in 7/10 UC, associated with colonic metaplasia in 4/7 (57.1%) and Das-1 and CG3 reactivity in 3/4 UC. In controls, recurrence occurred in 4/6 CD, associated with colonic metaplasia in 3/4 and reactivity with Das-1 and CG3 in 2/3. Present findings suggest that in UC, ileal lesions associated with changes towards colonic epithelium may develop also after IRA. Changes of the ileal content after colectomy may contribute to the development of colonic metaplasia, leading to ileal lesions both in the pouch and in the neo-terminal ileum after IRA.

Cloning and characterization of a new intestinal inflammation-associated colonic epithelial Ste20-related protein kinase isoform

Intestinal epithelial cells respond to inflammatory extracellular stimuli by activating mitogen activated protein kinase (MAPK) signaling, which mediates numerous pathophysiological effects, including intestinal inflammation. Here, we show that a novel isoform of SPS1-related proline alanine-rich kinase (SPAK/STE20) is involved in this inflammatory signaling cascade. We cloned and characterized a SPAK isoform from inflamed colon tissue, and found that this SPAK isoform lacked the characteristic PAPA box and alphaF loop found in SPAK. Based on genomic sequence analysis the lack of PAPA box and alphaF loop in colonic SPAK isoform was the result of specific splicing that affect exon 1 and exon 7 of the SPAK gene. The SPAK isoform was found in inflamed and non-inflamed colon tissues as well as Caco2-BBE cells, but not in other tissues, such as liver, spleen, brain, prostate and kidney. In vitro analyses demonstrated that the SPAK isoform possessed serine/threonine kinase activity, which could be abolished by a substitution of isoleucine for the lysine at position 34 in the ATP-binding site of the catalytic domain. Treatment of Caco2-BBE cells with the pro-inflammatory cytokine, interferon γ, induced expression of the SPAK isoform. Over-expression of the SPAK isoform in Caco2-BBE cells led to nuclear translocation of an N-terminal fragment of the SPAK isoform, as well as activation of p38 MAP kinase signaling cascades and increased intestinal barrier permeability. These findings collectively suggest that pro-inflammatory cytokine signaling may induce expression of this novel SPAK isoform in intestinal epithelia, triggering the signaling cascades that govern intestinal inflammation.

Dietary fiber decreases colonic epithelial cell proliferation and protein synthetic rates in human subjects

Although it has been proposed that high fiber consumption can prevent proliferative diseases of the colon, the clinical data to support this hypothesis have been inconsistent. To provide a more robust measure of the effects of fiber on colonic mucosal growth than previous studies, we evaluated both cell proliferation and colonic mucosal protein synthesis in nine healthy volunteers after they consumed a typical Western diet (<20 g fiber/day) or a Western diet supplemented with wheat bran (24 g/day) in a randomized crossover design. Biopsies taken from the sigmoid colon were used to assess mucosal proliferation by determining proliferating cell nuclear antigen (PCNA) in crypt cells and to assess mucosal protein synthetic rate using stable isotopically labeled leucine infusion. Fiber supplementation produced a 12% decrease in labeling index (%crypt cells stained with PCNA) (P < 0.001) and an 11% decrease in mucosal protein fractional synthetic rate (FSR; P < 0.05). Moreover, mucosal protein FSR correlated directly with labeling index (r2= 0.22, P < 0.05). These data demonstrate that increased wheat bran consumption decreases colonic mucosal proliferation and support the potential importance of dietary fiber in preventing proliferative diseases of the colon.

Predictors of recurrent specialized intestinal metaplasia after complete laser ablation.

The aim of this study was to determine whether specialized intestinal metaplasia recurs after complete laser ablation and to evaluate the persistence of colon epithelial protein in esophageal mucosa after laser ablation as a predictor of recurrence. A total of 31 patients with specialized intestinal metaplasia (Barrett's esophagus) underwent laser photoablation. Investigators without knowledge of treatment status evaluated serial hematoxylin and eosin-stained slides, Alcian blue-stained slides, and immunohistochemistry for the detection of colon epithelial protein (mAb Das-1). Endoscopic ablation of specialized intestinal epithelium was accomplished in 21 patients after 6.5 +/- 1.2 laser sessions. Complications included one perforation, one UGI bleed and one stricture. Of eight post-laser recurrences, seven were successfully re-ablated; one developed adenocarcinoma requiring esophageal resection. Cardia-type mucosa was present by biopsy at the time of complete ablation in all eight recurrent cases despite a normal endoscopic appearance. Colon epithelial protein was detected in all 31 patients before ablation, six of 21 completely ablated patients before they recurred and all eight recurrences. Only two of 15 patients, colon epithelial protein negative at the time of complete ablation, developed recurrent Barrett's esophagus. Thus, cardia-type mucosa and persistent colon epithelial protein staining after complete ablation of specialized intestinal epithelium were predictors of future recurrence (p < 0.001). Specialized intestinal epithelium was ablated by neodymium:yttrium-aluminum-garnet laser but recurred in eight of 21 (38%) of patients. Colon epithelial protein was present in all primary (31 of 31) and all recurrent (eight of eight) Barrett's esophagus. Recurrent specialized intestinal metaplasia may be deep to squamous epithelium. Replacement of specialized intestinal mucosa by cardia-type mucosa and persistence of colonic epithelial protein are predictors of recurrent specialized intestinal mucosa before its endoscopic or histological detection. Laser ablation of Barrett's epithelium is an investigational intervention that should be restricted to research protocols.

Predictors of recurrent specialized intestinal metaplasia after complete laser ablation

Objectives The aim of this study was to determine whether specialized intestinal metaplasia recurs after complete laser ablation and to evaluate the persistence of colon epithelial protein in esophageal mucosa after laser ablation as a predictor of recurrence. Methods A total of 31 patients with specialized intestinal metaplasia (Barrett’s esophagus) underwent laser photoablation. Investigators without knowledge of treatment status evaluated serial hematoxylin and eosin–stained slides, Alcian blue–stained slides, and immunohistochemistry for the detection of colon epithelial protein (mAb Das-1). Results Endoscopic ablation of specialized intestinal epithelium was accomplished in 21 patients after 6.5 ± 1.2 laser sessions. Complications included one perforation, one UGI bleed and one stricture. Of eight post-laser recurrences, seven were successfully re-ablated; one developed adenocarcinoma requiring esophageal resection. Cardia-type mucosa was present by biopsy at the time of complete ablation in all eight recurrent cases despite a normal endoscopic appearance. Colon epithelial protein was detected in all 31 patients before ablation, six of 21 completely ablated patients before they recurred and all eight recurrences. Only two of 15 patients, colon epithelial protein negative at the time of complete ablation, developed recurrent Barrett’s esophagus. Thus, cardia-type mucosa and persistent colon epithelial protein staining after complete ablation of specialized intestinal epithelium were predictors of future recurrence (p < 0.001). Conclusions Specialized intestinal epithelium was ablated by neodymium:yttrium-aluminum-garnet laser but recurred in eight of 21 (38%) of patients. Colon epithelial protein was present in all primary (31 of 31) and all recurrent (eight of eight) Barrett’s esophagus. Recurrent specialized intestinal metaplasia may be deep to squamous epithelium. Replacement of specialized intestinal mucosa by cardia-type mucosa and persistence of colonic epithelial protein are predictors of recurrent specialized intestinal mucosa before its endoscopic or histological detection. Laser ablation of Barrett’s epithelium is an investigational intervention that should be restricted to research protocols.

Expression of Das-1 in Primary Lung Adenocarcinomas Represents Reactivation of an Oncofetal Pulmonary Antigen

Objective: The monoclonal antibody (mAb) designated as mAb Das-1 was generated against a colonic epithelial protein. It reacts with bronchiolar epithelium and submucosal glands and weakly with alveolar lining cells of fetal lung. Adult alveolar epithelium is, however, nonreactive for mAb Das-1. The present study was designed to evaluate the immunoexpression of Das-1 and to correlate it with the histomorphology of primary lung adenocarcinomas and intestinal metaplasia. Methods: Eighty-four cases of primary lung adenocarcinomas were reviewed and categorized according to histologic grade and subtype. Immunohistochemical staining with mAb Das-1 was performed with normal colon as control. Results: Expression of Das-1 in the submucosal bronchial glands also served as internal control. Strong and diffuse staining was seen in 33 of the 84 cases of adenocarcinomas (39%). Conclusions: Expression of Das-1 was seen in primary lung adenocarcinomas even though adult alveolar epithelium is negative. Staining with mAb Das-1 was seen regardless of the grade or histological subtype of the lung adenocarcinomas. As most primary lung adenocarcinomas are not of bronchial gland origin, expression of Das-1 represents activation of oncofetal antigens.

GM-CSF regulates expression of a novel colonic epithelial protein (CEP) recognized by 7E12H12 monoclonal antibody

GM-CSF regulates expression of a novel colonic epithelial protein (CEP) recognized by 7E12H12 monoclonal antibody

ADENOCARCINOMA OF THE URACHUS AND BLADDER EXPRESSES A UNIQUE COLONIC EPITHELIAL EPITOPE: AN IMMUNOHISTOCHEMICAL STUDY

Purpose Primary adenocarcinoma of the bladder is a rare neoplasm whose histogenesis is poorly understood. Current data support the concept that adenocarcinoma of the bladder and urachus evolves from zones of intestinal metaplasia that become dysplastic and invasive. To address this hypothesis further we determined the immunoreactivity of benign and malignant epithelial tissue from the bladder and urachus with a monoclonal antibody that is reactive with colonic epithelium to evaluate the presence of a common reactive epitope. Materials and Methods The monoclonal antibody 7E 12 H 12 (IgM isotype), developed against a colonic epithelial protein, was used in an immunoperoxidase assay to survey formalin fixed, paraffin embedded archival tissue specimens. A total of 26 specimens obtained by endoscopic biopsy or extirpative surgery, including benign and malignant bladder and urachal epithelial abnormalities, was chosen for retrospective evaluation. Results All adenocarcinoma reacted positively regardless of the histological variant, differentiation, or bladder or urachal origin. In contrast, transitional cell and squamous cell carcinomas were nonreactive. Also, the pattern of reactivity in tissues that contained benign epithelial proliferations suggested a stepwise transition with no reactivity in normal urothelium or Brunn's epithelial nests, rare staining of cystitis cystica, and uniformly positive reactivity in cystitis glandularis and frank colonic intestinal metaplasia of the bladder and urachus. Conclusions The shared, aberrant phenotypic expression of a unique colonic epitope in benign epithelial metaplasia, and adenocarcinoma of the bladder and urachus suggests a common underlying pathway toward adenocarcinoma in cystic and urachal adenocarcinoma. The implications for diagnostic pathology are discussed.

Dietary fish oil blocks carcinogen-induced down-regulation of colonic protein kinase C isozymes.

In order to elucidate the influence of dietary constituents on colonic intracellular signal transduction, the effect of different fats on rat colonic epithelial protein kinase C (PKC) alpha (classical), delta (novel) and lambda-zeta (atypical) expression was determined in carcinogen-treated animals. Sprague-Dawley rats were provided with one of two fats (corn oil and fish oil); plus or minus the carcinogen azoxymethane (AOM) and killed at two time points (15 and 37 weeks) in a 2x2x2 factorial design. At 5 and 6 weeks of age, animals were injected s.c. with either AOM at a dose of 15 mg/kg body weight or saline once a week for 2 weeks and continued on the same diet until termination of the study. At 15 and 37 weeks after the second injection, 10 rats from each treatment group were killed. Colonic PKC alpha, delta and lambda-zeta steady-state protein and mRNA levels were determined using immunoblotting and relative quantitative polymerase chain reaction, respectively. Colonic mucosa from rats injected with AOM had significantly suppressed membrane and cytosolic PKC alpha and cytosolic lambda-zeta protein levels (P < 0.05) as compared to saline-injected control animals at both time points. In contrast, rats fed fish oil diets had significantly higher (P < 0.05) cytosolic PKC delta and lambda-zeta protein levels relative to animals fed corn oil diets. However, the effect of diet and AOM on the steady-state expression of PKC alpha, delta and zeta mRNA was not consistent with changes in the respective isozyme protein levels, suggesting regulation at the post-transcriptional level. These data demonstrate that dietary fish oil blocks the carcinogen-induced decrease in the steady-state levels of colonic mucosal PKC delta and lambda-zeta, which may in part explain why this fat source protects against colon cancer development.

Circulating immunoglobulin G1 antibody in patients with ulcerative colitis against the colonic epithelial protein detected by a novel monoclonal antibody.

Autoimmunity has been implicated in the pathogenesis of ulcerative colitis (UC). Several studies have shown amplified immunoglobulin G1 (IgG1) antibody response in UC; however the immunoreactive antigen(s) is unknown. To study this antigen(s), mucosal colonic extract was prepared by sonication, ultracentrifugation followed by ion exchange chromatography in fast protein liquid chromatography. The fraction (enriched colonic peptide), that was most reactive to a novel monoclonal antibody, 7E12H12 (IgM isotype), was isolated and used to examine the immunoreactivity against the patients' serum samples. Two hundred and thirteen coded samples from 111 patients with UC (symptomatic and untreated (63), symptomatic and treated (26), remission (22)); 47 with Crohn's disease (CD) (40 were symptomatic and untreated, and 30 had colonic disease); 29 with acute diarrhoea caused by specific pathogen(s); 10 with systemic lupus erythematosus, and 16 normal subjects were examined against the enriched colonic peptide by IgG subtype specific enzyme linked immunosorbent assays (ELISAs). Total IgG antibody reactivity was significantly (p < 0.01) higher only in symptomatic and untreated UC patients compared with each of the non-UC group, but the sensitivity was only 50%. IgG2 and IgG3 reactivities were not different among various groups. The IgG1 antibody reactivity against the enriched colonic peptide, however, differentiated UC patients from CD and each of the other non-UC groups. Seventy nine per cent of the patients with UC, treated or untreated, symptomatic or in remission, had significantly (p < 0.0001) higher IgG1 antibody against the enriched colonic peptide when compared with each of the other non-UC groups. Only 12% of CD serum samples and none of the other control serum samples reacted. Using purified serum IgG1 and 7E12H12-IgM, by 7E12H12 reactive peptide indeed reacts with UC-IgG1 antibody but not with control IgG1.

An Autoantigen in Psc? Whither or Whether? Mandal A, Dasgupta A, Jeffers L, Squillante L, Hyder S, Reddy R, Schiff E, Das Km. Autoantibodies in Sclerosing Cholangitis Against A Shared Peptide in Biliary and Colon Epithelium. Gastroenterology 1994;106:185-192

ABSTRACT Background/Aims: A strong association exists between ulcerative colitis and primary sclerosing cholangitis (PSC). Previously, the presence of a unique epitope shared by colon and biliary epithelial cells was shown by using the novel monoclonal antibody (MAb) 7E12H12 developed against a colonic epithelial protein. In the present study, the presence of circulating autoantibody in PSC against this peptide was examined. Methods: Sera from 16 patients with PSC, 13 with primary biliary cirrhosis, 6 with secondary biliary stricture, and 6 with chronic liver diseases and 10 normal subjects were used. An inhibition immunoperoxidase assay using the 7E2H12 MAb was developed against sections of bile duct and gallbladder. Sera were also examined in an enzyme–linked immunosorbent assay (ELISA) against the gallbladder extract enriched in 7E12H12–reactive protein. Results: About two thirds of the sera from patients with PSC blocked the binding of 7E12H12 MAb on the bile duct and gallbladder, whereas non–PSC sera did not. In the ELISA, 93% of PSC sera had circulating immunoglobulin G antibodies against the enriched gallbladder extract. The reactivity of sera from the PSC group was significantly (P &lt; 0.01 to P &lt; 0.0001) higher than in each of the non–PSC groups. Conclusions: Sera from patients with PSC contains autoantibodies against a cross–reactive peptide shared by colon and biliary epithelial cells.

Detection of a shared colon epithelial epitope on Barrett epithelium by a novel monoclonal antibody.

To determine if there is a reactive epitope common to colonic epithelium and Barrett epithelium, a premalignant metaplastic columnar-lined esophagus, usually arising as a complication of chronic reflux esophagitis. A monoclonal antibody, 7E12H12 (IgM isotype), developed against a colonic epithelial protein was used in a sensitive immunoperoxidase assay using formalin-fixed, paraffin-embedded tissue. One hundred sixteen tissue specimens from the esophagus, stomach, duodenum, and jejunum were examined. Twenty-two biopsy specimens were taken from 22 patients with benign Barrett epithelium, and 12 specimens were obtained from 12 patients with adenocarcinoma of the esophagus arising in Barrett epithelium. The remaining 85 tissue specimens were obtained from various parts of the upper gastrointestinal tract of patients with or without disease states. 21 of 22 (95%) Barrett epithelium specimens and all 12 adenocarcinomas arising from Barrett epithelium reacted with the 7E12H12 monoclonal antibody. Other tissues from esophagus, gastroesophageal junction, stomach, duodenum, or jejunum did not react. Squamous cell carcinoma of the esophagus also did not react. Barrett epithelium shares phenotypic expression of colonic epithelium. The 7E12H12 monoclonal antibody may provide insight into the origin and cellular lineage of Barrett epithelium.

Autoantibodies in sclerosing cholangitis against a shared peptide in biliary and colon epithelium

A strong association exists between ulcerative colitis and primary sclerosing cholangitis (PSC). Previously, the presence of a unique epitope shared by colon and biliary epithelial cells was shown by using the novel monoclonal antibody (MAb) 7E12H12 developed against a colonic epithelial protein. In the present study, the presence of circulating autoantibody in PSC against this peptide was examined. Sera from 16 patients with PSC, 13 with primary biliary cirrhosis, 6 with secondary biliary stricture, and 6 with chronic liver diseases and 10 normal subjects were used. An inhibition immunoperoxidase assay using the 7E12H12 MAb was developed against sections of bile duct and gallbladder. Sera were also examined in an enzyme-linked immunosorbent assay (ELISA) against the gallbladder extract enriched in 7E12H12-reactive protein. About two thirds of the sera from patients with PSC blocked the binding of 7E12H12 MAb on the bile duct and gallbladder, whereas non-PSC sera did not. In the ELISA, 93% of PSC sera had circulating immunoglobulin G antibodies against the enriched gallbladder extract. The reactivity of sera from the PSC group was significantly (P < 0.01 to P < 0.0001) higher than in each of the non-PSC groups. Sera from patients with PSC contains autoantibodies against a cross-reactive peptide shared by colon and biliary epithelial cells.

Gastrin's trophic effect in the colon: Identification of a signaling pathway mediated by protein kinase C

In previous studies we have reported that gastrin exerts a trophic effect on rat colonic epithelial cells in vitro. The effect of gastrin appeared to be mediated through a protein kinase C mechanism. In this study, we have characterized the role of protein kinase C in the gastrin-induced stimulation. Gastrin, in a time- and dose-dependent manner, increased protein kinase C translocation from the cytosol to the membrane, an index of enzyme activation. Maximum translocation occurred in 1 to 2 min following exposure to gastrin (10 −8 M), before declining back to baseline level within 5 min. Gastrin did not change total protein kinase C activity in the colonic cells. Staurosporine, an inhibitor of protein kinase C, totally abolished the basal as well as the gastrin-stimulated activity of protein kinase C. The tumor promoter phorbol 12-myristate 13-acetate also stimulated colonic epithelial protein kinase C. However, prolonged treatment of cells with phorbol inhibited their subsequent response to gastrin stimulation. The response to gastrin was also prevented by the gastrin receptor antagonist proglumide. These observations suggest that protein kinase C mediates the stimulatory effect of gastrin on colonic epithelial cells, possibly through a receptor mechanism.

Mr 40,000 human colonic epithelial protein expression in colonic mucosa and presence of circulating anti-Mr 40,000 antibodies in cotton top tamarins with spontaneous colitis.

Saguinus oedipus, Callithrix jacchus, and Saguinus fuscicollis are three species of New World monkeys which develop a form of colitis that is similar to human ulcerative colitis. Only S oedipus, however, develop colon cancer. We examined intestinal tissues from these animals for the presence of an antigen cross reacting to the Mr 40,000 human colonic epithelial protein that acts as an autoantigen in ulcerative colitis. Using an anti-Mr 40,000 monoclonal antibody (7E12H12, IgM isotype), by an immunoperoxidase assay we showed that all colon specimens from S oedipus reacted with 7E12H12; however, the colonic tissue from C jacchus and S fuscicollis did not. In immunotransblot analysis eluted IgG antibody bound to human ulcerative colitis colon (CCA-IgG) reacted with Mr 40,000 protein(s) present in the extracts of colon from S oedipus animals and humans. Small intestinal tissue reacted neither with 7E12H12 nor with CCA-IgG. In S oedipus, the Mr 40,000 protein was localised exclusively to colonic epithelial cells. Preincubation of seven S oedipus colon specimens with eight of 10 sera from animals with acute or chronic colitis and 0 of four sera from animals without colitis almost completely inhibited the binding of 7E12H12 to the colonic epithelium. Four of these 10 sera inhibited the binding of 7E12H12 to the autologous colon. These results show the presence of circulating autoantibodies in S oedipus with colitis against an epitope(s) on Mr 40,000 protein shared by human and S oedipus colon.

The presence in experimental animals of a colon specific Mr 40,000 protein(s) with relevance to ulcerative colitis.

In patients with ulcerative colitis a colon tissue bound IgG and serum antibodies against an Mr 40,000 colonic protein(s) has been identified. Using an anti-Mr 40,000 protein monoclonal antibody, 7E12H12, by an immunocytochemical method, the protein was localised in human tissue exclusively to colonic epithelial cells. In this study the presence of the Mr 40,000 protein was assessed in experimental animals by the direct and inhibition enzyme linked immunosorbent assays (ELISAs) using the anti-Mr 40,000 protein monoclonal antibody, 7E12H12 (IgM isotype). In addition, a total of 129 specimens including colon, small intestine, gall bladder, biliary tract, and kidney from nine strains of rats and mice, and from human tissue were studied by the immunocytochemical method using 7E12H12. All colon specimens from both humans and animals reacted with 7E12H12 in the immunocytochemical and ELISA assays. None of the non-colonic organs reacted with 7E12H12. While in human colon 7E12H12 recognised the absorptive epithelial cells, in all the animals it recognised mainly the colonic goblet cells. Extracts of animal colon but not of small intestine inhibited the binding of 7E12H12 to the human colon extract. This study shows the presence of an organ specific Mr 40,000 colonic epithelial protein(s) in humans and experimental animals. A differing cellular localisation of the Mr 40,000 protein(s) in human v animal tissue was also shown. Further characterisation of the Mr 40,000 protein(s) may provide important clues regarding the autoimmune mechanisms in ulcerative colitis.

A shared and unique epitope(s) on human colon, skin, and biliary epithelium detected by a monoclonal antibody

An immune-complex-mediated inflammation has been suggested in the pathogenesis of skin and biliary complications in ulcerative colitis, although specific antigen-antibody complexes have not been found. We recently identified an Mr 40,000 colonic protein that reacts with tissue-bound immunoglobulin G eluted from colon specimens of patients with ulcerative colitis and not with immunoglobulin G eluted from colon specimens of patients with other diseases (J Clin Invest 1985;7:311). Murine monoclonal antibodies to the purified Mr 40,000 colonic protein are developed. The presence of the Mr 40,000 protein was examined with a monoclonal antibody (7E12H12, immunoglobulin M isotype) by an immunoperoxidase assay against various human epithelial tissues (74 specimens) including colon, ileum, jejunum, duodenum, stomach, esophagus, pancreas, liver, respiratory, urinary, and genital tracts. In addition, gall bladder, bile duct, hepatic ducts, skin, synovial membrane, and eye tissue were also examined for any cross-reactivity with 7E12H12 and another anti-Mr 40,000 monoclonal antibody (7E6A5, immunoglobulin G1 isotype). 7E12H12 reacted only with colonicepithelial cells mainly along the basolateral domains of plasma membrane and apical areas. The reactivity was present in both adult and fetal colon including the appendix. Among all the noncolonic tissue specimens, 7E12H12 reacted only with the gall bladder, the bile duct, the hepatic ducts, and the skin. All other epithelial tissues from 15 different organs, including the small intestine, synovium, and eye tissue, did not react. In the skin and biliary tract the immunoreactivity was exclusively present in the epidermal epithelial cells and mucosal epithelium respectively. 7E6A5 stained colonic mucosal cells but did not react with skin, biliary epithelium, synoyium and eye tissue. These results indicate the presence of a unique epitope or epitopes on Mr 40,000 colonic epithelial protein that is shared by the skin and biliary tract epithelial cells. Future studies of this shared epitope or epitopes and its immune recognition may provide further understanding in the pathogenesis of extraintestinal complications involving these organs in patients with ulcerative colitis.