Immunohistochemical demonstration of IgA and secretory component in relation to epithelial cell differentiation in normal colorectal mucosa and metaplastic polyp: a semiquantitative study

Summary The metaplastic polyp is a non-neoplastic epithelial lesion found within the human colorectum. Although not regarded as precancerous, recent studies have demonstrated the expression of multiple cancerassociated phenotypes. This might indicate a possible indirect relationship between metaplastic polyps and colorectal cancer. Epithelial secretory component and IgA were demonstrated by the immunoperoxidase technique and staining intensities were assessed semiquantitatively. The findings were related to cellular differentiation in normal colorectal epithelium as compared to the metaplastic polyp. The crypt base cells and also the surface epithelial cells stained with similar intensity in both types of epithelium. However, the expected increase in staining characterizing normal lower and upper crypt columnar cells and reduction in staining associated with the switch from crypt to surface columnar cell was not observed in the metaplastic polyp. Metaplastic crypt columnar cells showed significantly reduced staining for both IgA and secretory component as compared to their normal counterparts. There was also a significant reduction in the number of IgA-secreting plasma cells in the lamina propria of the metaplastic polyp. These findings are consistent with the concept of a premature switch to mature surface cell characteristics within the metaplastic polyp. They are discussed in the light of other changes in phenotype associated with this lesion.     

Cathepsin E in follicle associated epithelium of intestine and tonsils: localization to M cells and possible role in antigen processing

A specific rabbit anti-human serum was used selectively to localize the aspartic proteinase cathepsin E to follicle associated epithelium (FAE) of human and rat intestine, including jejunum, ileum, appendix, colon and rectum, as well as of human palatine, pharyngeal and lingual tonsils. Coexpression of class II histocompatibility antigen HLA-DR antigen has been observed in some of the cathepsin E-positive epithelial cells. In addition, cathepsin E has been detected in a few mononuclear cells of intestinal lymphoid structures and tonsils resembling interdigitating reticulum cells of lymph nodes. Another aspartic proteinase, cathepsin D, has been found to be poorly represented in FAE and intensely expressed by macrophages. Electron immunocytochemistry localized cathepsin E to endosomal vesicles and endoplasmic reticulum of M cells in rat and human ileum as well as of M-like cells in human palatine tonsil. The results suggest a possible role of endosomal cathepsin E in the processing of macromolecules and microorganisms transported by M cells and related epithelial cells to mucosal associated lymphoid tissue (MALT).     

Detection of cancer clones in human colorectal adenoma as revealed by increased DNA instability and other bio-markers

An immunohistochemical differential staining of cancerous cells with anti-cytidine antibody after denaturation of nuclear DNA by acid hydrolysis with 2N HCl at 30 degrees C for 20 min (DNA-instability test) has been used as a marker for malignancy. The test was applied to bioptic tissues of human colorectal polyps assessed histopathologically as hyperplastic polyp (11 cases), tubular adenoma of mild (68 cases), moderate (102 cases), and severe (46 cases) dysplasia, and adenocarcinoma (30 cases). The serial sections of the same tissues were also subjected to immunohistochemical staining for Ki67, p53, DNA-fragmentation factor 45 (DFF45) and vascular endothelial growth factor (VEGF). The DNA-instability test was positive in 30 (100%) adenocarcinoma cases, 46 (100%) severe dysplasia adenoma cases, 36 (35.29%) moderate dysplasia adenoma cases, and 8 (11.76%) mild dysplasia adenoma cases, indicating malignancy. All hyperplastic polyps were negative to the DNA-instability test. Furthermore, the percentage of glands positive in the DNA-instability test steadily increased in going from mild (10%), to moderate (35%), to severe (100%) dysplasia, and adenocarcinoma (100%). All other biological markers tested in the present study showed significantly higher values in those adenoma glands that were positive to the DNA-instability test, irrespective of the dysplasia grade, as compared to the markers in the adenoma glands that were negative to DNA instability testing. Furthermore, the former values were comparable to those in adenocarcinoma. The results indicate that cancer cell clones are already present at the adenoma stages showing positivity to DNA instability testing, enhanced proliferative activity, p53 mutation and induction of DFF45 and VEGF, at a time when the degree of morphological atypia are not yet large enough for them to be identified as cancer. These factors promote cancer cell proliferation, produce heterogeneous subclones due to DNA instability, enhance their survival by escaping apoptosis, and provide abundant nutrients by neovascularization during the early-stage progression of colorectal cancer.     

An electron microscopic study of the behavioral characteristics of human lung tumor cells cultured on soft agar]

The ultrastructure of cells from seven human lung cancers and from the colonies formed by these cells in soft agar was investigated. Tumor cells developed to display the morphofunctional potentials of the initial tumors. Cultured cells of squamous-cell carcinomas contained numerous tonofilaments, those of adenocarcinomas developed microvilli on their apical surfaces and intracellular lumens. On the other hand, cells of squamous-cell carcinomas showed features specific of adenoma epithelium, i.e. well developed microvilli and intracellular lumens. Besides, cells of adenocarcinoma often contained large quantities of tonofilaments considered to be characteristic of epidermoid epithelium. The results obtained suggest a possibility of metaplastic transformation of the lung epithelium.     

Peri-epithelial origin of prostanoids in the human colon

The biology of prostanoids in the normal human colon is only beginning to be understood. We used in situ and in vitro techniques to define the lineage, number, per cell enzyme content, and epithelial functional effect of prostaglandin-generating cells, identified by the presence of cyclooxygenase 1 (COX 1). Immunohistochemical results were quantitated densitometrically, and cell surface staining in situ was verified by flow cytometry of isolated cells and by Western blotting. Three populations of COX 1(+) mucosal cells were identified, based on their morphology and local distribution in human mucosa; these were in the intra-epithelial, crypt apical, and lamina propria regions, with each containing a similar amount of COX 1 protein on a per cell basis. The most numerous were COX 1(+) mononuclear cells in the lamina propria, identified as CD3(+) T lymphocytes, both in situ and ex vivo. In toto, 21% of lamina propria mononuclear cells were COX 1(+), and over 50% of these cells were CD3(+) T cells. Findings were similar in the colon with mild-moderate inflammation due to ulcerative colitis. Using established surface markers, intra-epithelial and crypt apical COX 1(+) cells were non-lymphoid CD45(+) leukocytes; neither IgA (B-lymphocytes) nor alpha-smooth muscle actin (myelofibroblasts) was co-expressed on these COX 1(+) cells. Examining the effect of a major product of COX 1 in an in vitro system of human colonic epithelial monolayers, prostaglandin E(2) (PGE(2)) in low concentration (10(-6) M) enhanced epithelial barrier function and partially protected epithelia from the barrier-disruptive consequences of a pro-inflammatory cytokine, IFN-gamma. We conclude that the human colon contains three tiers of cell types for local synthesis of prostanoids, distinguishable by their location, morphology, and cell lineage. Further, maintenance of the barrier function of colonic epithelium may be added to other cell functions in mucosa regulated, in part, by prostanoids.     

ERBB3 Positively Correlates with Intestinal Stem Cell Markers but Marks a Distinct Non Proliferative Cell Population in Colorectal Cancer

Several studies have suggested ERBB3/HER3 may be a useful prognostic marker for colorectal cancer. Tumours with an intestinal stem cell signature have also been shown to be more aggressive. Here, we investigate whether ERBB3 is associated with intestinal stem cell markers in colorectal cancer and if cancer stem cells within tumours are marked by expression of ERBB3. Expression of ERBB3 and intestinal stem cell markers (LGR5, EPHB2, CD44s and CD44v6) was assessed by qRT-PCR in primary colorectal tumours (stages 0 to IV) and matched normal tissues from 53 patients. The localisation of ERBB3, EPHB2 and KI-67 within tumours was investigated using co-immunofluorescence. Expression of ERBB3 and intestinal stem cell markers were significantly elevated in adenomas and colorectal tumours compared to normal tissue. Positive correlations were found between ERBB3 and intestinal stem cell markers. However, co-immunofluorescence analysis showed that ERBB3 and EPHB2 marked specific cell populations that were mutually exclusive within tumours with distinct proliferative potentials, the majority of ERBB3+ve cells being non-proliferative. This pattern resembles cellular organisation within normal colonic epithelium where EPHB2 labelled proliferative cells reside at the crypt base and ERBB3+ve cells mark differentiated cells at the top of crypts. Our results show that ERBB3 and intestinal stem cell markers correlate in colorectal cancers. ERBB3 localises to differentiated cell populations within tumours that are non-proliferative and distinct from cancer stem cells. These data support the concept that tumours contain discrete stem, proliferative and differentiation compartments similar to that present in normal crypts.     

Comparison of different techniques for detection of Gal-GalNAc, an early marker of colonic neoplasia

The tumor marker, D-galactose-beta [1-3]-N-acetyl-D-galactosamine (Gal-GalNAc, also known as T-antigen) can be identified by a very simple galactose oxidase-Schiff's (GOS) reaction either on tissues or on rectal mucus samples from patients with colorectal neoplasms. Gal-GalNAc is expressed in the neoplastic mucosa as well as the remote non-neoplastic mucosa. It is, however, not expressed in colonic mucosa of normal subjects. We studied the expression of Gal-GalNAc by GOS reaction, lectin reactivity and immunocytochemistry in 10 normal, .45 precancerous [5 Crohn's disease, 15 ulcerative colitis (5 without dysplasia and 10 with dysplasia), 25 tubular adenomas], and 25 adenocarcinoma cases. Normal mucosa remote from tubular adenoma and adenocarcinoma was also studied. The GOS method was compared with reactivity of the lectin jacalin and immunostaining with antibody to T antigen (Anti-Tag Ab). GOS reaction was negative in all of the 10 normal specimens. Of the 5 Crohn's disease specimens, 2 were positive and 3 negative. In the 5 ulcerative colitis cases without dysplasia, positive reaction was seen in 2 cases and negative in 3. Of the 10 cases of ulcerative colitis with dysplasia, 5 showed positivity in dysplastic areas, and 3 of these were also positive in remote non dysplastic mucosa. Twenty of 25 tubular adenomas yielded a positive reaction in the adenoma, 14 of them showing positivity also in remote mucosa; 3 cases showed a positive reaction only in remote mucosa. Of the 25 adenocarcinomas, 21 showed a positive reaction in the adenocarcinoma as well as the remote mucosa. GOS reaction was intense in well differentiated adenocarcinoma and weak in poorly differentiated adenocarcinoma. Intense reaction was also seen in the intracellular mucus of some aberrant crypts and morphologically normal crypts remote from adenocarcinoma and tubular adenoma. GOS reaction showed an overall sensitivity of 75.7% and specificity of 100% for cancer and precancerous lesions. Jacalin reactivity was slightly more sensitive (84.3%) but less specific (80%) and Tag Ab reactivity even less sensitive (50%) but as specific (100%) for neoplastic and dysplastic mucosa. We conclude that the detection of the carbohydrate moiety Gal-GalNAc varies with the technique used. Compared to other techniques, GOS reaction is extremely simple and has a high degree of sensitivity and specificity. It can be used for detection of this tumor marker in remote non-neoplastic mucosa of patients with neoplasia or at risk of developing neoplasia. It, therefore, could be used as a cost effective screening test in rectal biopsy specimens of such patients.     

Cathepsin D expression in colorectal adenocarcinomas and adenomas

The aim of this study was to investigate the role of cathepsin D in colorectal cancer. For this purpose cathepsin D expression was evaluated by means of immunohistochemistry in stromal and tumor cells of 31 colorectal carcinomas and 29 adenomas. Cytoplasmic cathepsin D expression of tumor cells was present in 90.3% of the carcinoma cases and various degrees of stromal cell cathepsin D expression were present in all cases. In the adenomas, the epithelial cells and stromal cells expressed cathepsin D in 68.96% and 96.55% of cases, respectively. The staining intensity was always weaker in the adenomas. When the stromal and tumor cell cathepsin D expression in the adenocarcinoma and adenoma cases were compared, a statistically significant difference was observed in the staining of stromal cells. Furthermore, stromal cathepsin D expression in the adenocarcinomas was related to tumor stage when the carcinomas were divided into low and high stage. Cathepsin D expression in stromal cells may be an important indicator of poor prognosis in colorectal adenocarcinomas.     

The pathogenesis of ulcerative colitis-associated colorectal cancer

The fact that there is an increase in cases of ulcerative colitis-associated dysplasia and colitic cancer raises some problems for clinical practice and pathological diagnosis. How to detect ulcerative colitis-associated dysplasia and early cancer endoscopically? How to discriminate inflammatory regenerative epithelium from UC-associated dysplasia and aberrant crypt foci histologically? How to distinguish dysplasia-associated lesion or mass from sporadic adenoma pathologically? UCAC has established risk factors including, among others, long duration of disease, large extent and low activity of disease, and the lack of adequate surveillance and therapy. Colonoscopic and histological evidence of low grade and high grade dysplasia means the possible evidence of coexisting carcinoma. Carcinoma typically may occur in the entire colon, is often multiple and has undifferentiated histology. Important factor is the effectiveness of dysplasia surveillance as a population based strategy to decrease colorectal cancer mortality in inflammatory bowel disease patients. Colitis-related cancer may have distinct pathogenesis to sporadic colorectal cancer.     

The spectrum of cytogenetic changes in the colorectal polyps with different histologic structure

A cytogenetic study on colorectal polyps with different histological structures from 54 patients was carried out. Diploid karyotypes dominated in these polyps. Abnormal karyotypes were found in 17 (31.5%) polyps: the most frequent anomalies were in adenomas, in hamartomatous polyps they were absent. The typical features of the karyotypes were numeric changes, such as additional copies of chromosomes 13, 7, 20, 18 and 16 in some combinations. The polyps with high-grade dysplasia in all cases had abnormal karyotypes. One adenoma with dysplastic changes had specific chromosomal anomalies, such as additional copies of chromosomes 13, 18, and 20; this adenoma localized near adenocarcinoma. Unlike the previous studies of the mucous membrane of intestine from the patients suffering from ulcerative colitis and Cronh’s disease, we have studied the peculiarities of the karyotype exactly in inflammatory polyps, and found the chromosomal anomalies in 21.4% of cases.     

Early lesions induced in rat colon epithelium by N-methyl-N′-nitro-N-nitrosoguanidine

The development of ACF (aberrant crypt foci), adenoma and cancer following intrarectal administration of the alkylating agent N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) has been described. However, microscopic lesions not previously reported were observed as soon as two weeks following carcinogen treatment. These lesions protrude slightly over the epithelial lining of the colon, with a micropolyp-like appearance. Oriented sections show that the centre of these lesions present pseudo-“cystic” appearance, with disorganized crypts made of normal cells. The chorion of the lesion is invaded by numerous inflammatory cells and some ACF may be present nearby. The epithelium lining the cysts and the distorted crypts shows expression of gastric mucin M1/MUC5AC, an early marker of colonic carcinogenesis which is not present in normal colon. This mucin is retained within the “cysts” together with some inflammatory cells.The micropolyps observed contain in a minute form some histological elements described in ulcerative colitis or short-term radiotherapy (distortion of crypts, crypt abscesses, increase of chorion cellularity, infiltration by immune cells). In addition, the presence of bifid crypts nearby suggests mucosal regeneration.Our hypothesis is that these modifications are steps in a normal healing pathway that may in some cases degenerate into precancerous lesions and cancer.     

Proper E‐cadherin membrane location in colon requires Dab2 and it modifies by inflammation and cancer

We reported that Disabled‐2 (Dab2) is located at the apical membrane in suckling rat intestine. Here, we discovered that, in colon of suckling and adult mouse and of adult human, Dab2 is only at lateral crypt cell membrane and colocalized with E‐cadherin. Dab2 depletion in Caco‐2 cells led to E‐cadherin internalization indicating that its membrane location requires Dab2. In mice, we found that 3 days of dextran sulfate sodium‐induced colitis increased Dab2/E‐cadherin colocalization, which was decreased as colitis progressed to 6 and 9 days. In agreement, Dab2/E‐cadherin colocalization increased in human mild and severe ulcerative colitis and in polyps, being reduced in colon adenocarcinomas, which even showed epithelial Dab2 absence and E‐cadherin delocalization. Epithelial Dab2 decrement preceded that of E‐cadherin. We suggest that Dab2, by inhibiting E‐cadherin internalization, stabilizes adherens junctions, and its absence from the epithelium may contribute to development of colon inflammation and cancer.     

Proteomic screening of a cell line model of esophageal carcinogenesis identifies cathepsin D and aldo-keto reductase 1C2 and 1B10 dysregulation in Barrett's esophagus and esophageal adenocarcinoma

Esophageal adenocarcinoma (EA) incidence is increasing rapidly and is associated with a poor prognosis. Identifying biomarkers of disease development and progression would be invaluable tools to inform clinical practice. Two-dimensional polyacrylamide gel electrophoresis was used to screen 10 esophageal cell lines representing distinct stages in the development of esophageal cancer. Thirty-three proteins were identified by MALDI-TOF-MS which demonstrated differences in expression across the cell lines. Western blotting and qRT-PCR confirmed increased cathepsin D and aldo-keto reductases 1C2 and 1B10 expression in metaplastic and dysplastic cell lines. Expression of these proteins was further assessed in esophageal epithelium from patients with nonerosive (NERD) and erosive gastro-esophageal reflux disease, Barrett's esophagus (BE) and EA. When compared with normal epithelium of NERD patients, (i) cathepsin D mRNA levels demonstrated a stepwise increase in expression (p<0.05) in erosive, metaplastic and EA tissue; (ii) AKR1B10 expression increased (p<0.05) 3- and 9-fold in erosive and Barrett's epithelium, respectively; and (iii) AKR1C2 levels increased (p<0.05) in erosive and Barrett's epithelium, but were reduced (p<0.05) in EA. These proteins may contribute to disease development via effects on apoptosis, transport of bile acids and retinoid metabolism and should be considered as candidates for further mechanistic and clinical investigations.     

Alterations in the composition of the supramucosal defense barrier in relation to disease severity of ulcerative colitis.

Mucin glycoproteins and trefoil peptides play an important role in protection and repair of the gastrointestinal epithelium. This study investigates alterations in mucin and trefoil peptide gene expression and product localization in ulcerative colitis (UC). Product localization and message expression of mucin MUC1 to 6 and trefoil peptide TFF1 to 3 genes was analyzed in rectosigmoid tissue from a cohort of patients with active UC and compared with that of normal colorectal mucosa. MUC1 expression was upregulated in severe UC at the site of rupture of crypt abscesses. Reduction in MUC2 expression occurred in UC adjacent to ulceration. No alteration in MUC3 or MUC4 gene expression was detectable in UC compared with normal colorectal mucosa. No ectopic expression of MUC5AC, MUC5B, or MUC6 was identified in UC. Ectopic TFF1 expression was identified in tissues eliciting histological features of severe disease. Decreased TFF3 localization was demonstrated in UC tissues, but no TFF2 expression was detected in any colorectal specimens. Subtle alterations in composition of the supramucosal defense barrier exist in UC and vary in relation to clinical severity of disease. There is upregulation in mucin MUC1 at crypt abscesses and neo-expression of TFF1 trefoil peptide in severe disease.     

Colorectal cytology in chronic ulcerative colitis

During a period of ten years, 109 colorectal cytology specimens were obtained from 41 patients with chronic ulcerative colitis. There were 28 male and 13 female patients, whose average age was 45 years. All patients were symptomatic and had ulcerative colitis with repeated attacks of profuse diarrhea on the average for 12 years prior to their first cytologic examination. Eight cytology specimens were positive for malignant cells, 35 had atypia, 58 were negative and 8 were unsatisfactory. The malignant cells showed marked anisocytosis, pleomorphism and nuclear hyperchromasia and appeared in loosely cohesive clusters or in single forms in an inflammatory and necrotic background. Subsequent colectomy revealed invasive carcinoma in five patients and carcinoma in situ in two. Smears that were negative or showed atypia contained abundant chronic inflammatory cells. The atypical colonic epithelial cells contained prominent nuclei and formed cohesive clusters. Surgical biopsy and/or segmental resection revealed the presence of polyps, pseudopolyps, polypoid hyperplasia, mucosal atypia and crypt abscesses in patients with atypical and negative cytologic findings. It is concluded that cytologic examination of the colon can play an important role in the examination of patients with ulcerative colitis and allows for the detection of malignant transformation of the colonic mucosa.     

Evidence that hydrogen sulfide is a genotoxic agent

Hydrogen sulfide (H2S) produced by commensal sulfate-reducing bacteria, which are often members of normal colonic microbiota, represents an environmental insult to the intestinal epithelium potentially contributing to chronic intestinal disorders that are dependent on gene-environment interactions. For example, epidemiologic studies reveal either persistent sulfate-reducing bacteria colonization or H2S in the gut or feces of patients suffering from ulcerative colitis and colorectal cancer. However, a mechanistic model that explains the connection between H2S and ulcerative colitis or colorectal cancer development has not been completely formulated. In this study, we examined the chronic cytotoxicity of sulfide using a microplate assay and genotoxicity using the single-cell gel electrophoresis (SCGE; comet assay) in Chinese hamster ovary (CHO) and HT29-Cl.16E cells. Sulfide showed chronic cytotoxicity in CHO cells with a %C1/2 of 368.57 micromol/L. Sulfide was not genotoxic in the standard SCGE assay. However, in a modified SCGE assay in which DNA repair was inhibited, a marked genotoxic effect was observed. A sulfide concentration as low as 250 micromol/L (similar to that found in human colon) caused significant genomic DNA damage. The HT29-Cl.16E colonocyte cell line also exhibited increased genomic DNA damage as a function of Na2S concentration when DNA repair was inhibited, although these cells were less sensitive to sulfide than CHO cells. These data indicate that given a predisposing genetic background that compromises DNA repair, H2S may lead to genomic instability or the cumulative mutations found in adenomatous polyps leading to colorectal cancer.     

M6 membrane protein plays an essential role in Drosophila oogenesis

We had previously shown that the transmembrane glycoprotein M6a, a member of the proteolipid protein (PLP) family, regulates neurite/filopodium outgrowth, hence, M6a might be involved in neuronal remodeling and differentiation. In this work we focused on M6, the only PLP family member present in Drosophila, and ortholog to M6a. Unexpectedly, we found that decreased expression of M6 leads to female sterility. M6 is expressed in the membrane of the follicular epithelium in ovarioles throughout oogenesis. Phenotypes triggered by M6 downregulation in hypomorphic mutants included egg collapse and egg permeability, thus suggesting M6 involvement in eggshell biosynthesis. In addition, RNAi-mediated M6 knockdown targeted specifically to follicle cells induced an arrest of egg chamber development, revealing that M6 is essential in oogenesis. Interestingly, M6-associated phenotypes evidenced abnormal changes of the follicle cell shape and disrupted follicular epithelium in mid- and late-stage egg chambers. Therefore, we propose that M6 plays a role in follicular epithelium maintenance involving membrane cell remodeling during oogenesis in Drosophila.     

Evidence for Follicle Wall Involvement in Ovulation and Progesterone Production by Frog (Rana pipiens) Follicles in vitro

Involvement of different cellular investments of the amphibian ovarian follicle wall in the ovulatory process, progesterone production, and oocyte maturation was investigated. Following microdissection, to selectively remove one or more layers (surface epithelium, theca, follicle cells) of the follicle wall, dissected and undirected ovarian follicles were treated with frog pituitary homogenate (FPH) or progesterone. Intact follicles ovulated in response to pituitary homogenate and this was associated with contractions of the follicle wall. Ovulation and follicular contractions were not observed following removal of the surface epithelium without removing the thecal layer. Oocyte maturation occured in response to FPH following removal of the surface epithelium alone or together with the theca, but not in the absence of the follicle cells. Intact follicles were most responsive to FPH with respect to progesterone production, and removal of all somatic cells from oocytes obliterated FPH stimulated progesterone production. Oocytes, regardless of wether any or all follicular wall layers were removed, matured but did not ovulate following exposure to progesterone. The results suggest that the surface epithelium, but not the theca, is required for FPH-induced extrusion (ovulation) of the oocyte from ovarian follicle wall. Additionally, the somatic tissue rather than the oocyte appears to be the cells producing progesterone following FPH treatment. The results indicate that separate cellular layers (individually and/or as a result of interactions) of the follicle wall carry out different functions during follicular differentiation and mediation of ovulation. Data provide functional evidence for a role of the surface epithelium in controlling the process of ovulation and follicular contraction.