Mucin Core Peptide Research Articles

Mucin-Related Molecular Responses of Bronchial Epithelial Cells in Rats Infected with the Nematode Nippostrongylus brasiliensis.

Although mucins are essential for the protection of internal epithelial surfaces, molecular responses involving mucin production and secretion in response to various infectious agents in the airway have not been fully elucidated. The present study analysed airway goblet cell mucins in rats infected with the nematode Nippostrongylus brasiliensis, which migrates to the lungs shortly after infection. Goblet cell hyperplasia occurred in the bronchial epithelium 3–10 days after infection. The high iron diamine-alcian blue staining combined with neuraminidase treatment showed that sialomucin is the major mucin in hyperplastic goblet cells. Immunohistochemical studies demonstrated that goblet cell mucins were immunoreactive with both the major airway mucin core peptide, Muc5AC, and the major intestinal mucin core peptide Muc2. Reverse transcription real-time PCR studies demonstrated upregulation of gene transcription levels of Muc5AC, Muc2, the sialyltransferase St3gal4, and the resistin-like molecule beta (Retnlb) in the lungs. These results showed that nematode infection induces airway epithelial responses characterised by the production of sialomucin with Muc5AC and Muc2 core peptides. These mucins, as well as Retnlb, might have important roles in the protection of mucosa from migrating nematodes in the airway.

Contact Lens Materials, Mucin Fragmentation and Relation to Symptoms

Mucins adhere to contact lenses (CLs), reflecting the renewal of the preocular fluid and enzymatic activity at the ocular surface. In this study, we aimed to analyze mucin fragmentation on materials new to the ocular surface and investigate whether this correlates with wearing comfort. Lenses were obtained from new CL wearers after 2 weeks each of wearing vifilcon A, followed by senofilcon A, and then by vifilcon A lenses. Symptoms were evaluated using the Ocular Surface Disease Index (OSDI). CLs were extracted in a mixture of guanidinium hydrochloride and radioimmunoprecipitation assay buffer. Mucin mobility was analyzed after electrophoresis, Western blotting, and visualization with antibodies against mucin peptide core. Mobilities, normalized to total reactivity in the lane, were compared between visits for each subject and were expressed as shifts. Mucin (MUC)5AC polymers exceeding 260 kDa were observed in agarose gels; NuPAGE resolved polymers from 260 to 3.5 kDa: when large mucins were detected, the smallest fragments were missing. Fragmentation patterns were significantly different between lens types for MUC1 (analysis of variance, P = 0.006) and MUC4 (P < 0.001) but not for MUC5AC or MUC16 (P > 0.293). Mobility shifts of MUC1 and MUC4 were significantly negatively correlated (Pearson, r = -0.908; P = 0.002). For OSDI scores >15, mucin fragmentation was unchanged, whereas for OSDI scores <15, MUC4 and MUC5AC fragments were longer on vifilcon A than on senofilcon lenses (unpaired t test, P = 0.046), irrespective of the direction of change (analysis of variance, P > 0.366). Changes in MUC1 breakdown were significantly negatively correlated to the overall OSDI score (r = -0.891, P = 0.001). In asymptomatic CL wearers, only changes in mucin fragmentation in response to a new material were consistent and fast, irrespective of CL order. Lack of change seems, therefore, to be connected with discomfort during CL wear.

Comparison of intestinal metaplasia in gastric cardia and Barrett's esophagus

To evaluate and compare the pathological features and immunostaining pattern (cytokeratin 7 (CK-7), mucin core peptide 1 (Muc-1)) in Barrett's esophagus (BE) and cardiac intestinal metaplasia (CIM). According to endoscopic diagnosis, patients with gastric cardiac inflammation and BE were selected from March 2008 to February 2009 in Renji Hospital, Shanghai Jiaotong University School of Medicine. Those patients who had histological findings of intestinal metaplasia (82 cases of CIM and 64 special type BE) were enrolled in our study. Hematoxylin-eosin, periodic acid-Schiff and Alcian blue staining and an immunohistochemical examination (CK-7, Muc-1) were undertaken in all of them. Squamous mucosa overlying the columnar crypts with intestinal metaplasia, also called buried metaplasia, was often found in the BE group (56.2%), mainly as an incomplete type (85.9%). Inflammation in the gastric antrum was more severe in the CIM group (45.1% vs 26.6%), in contrast, esophagitis was more severe in the BE group (53.1% vs 35.4%). CK-7 was highly expressed in the BE group (84.4%) in contrast to the CIM group (37.8%). There was no difference in the expression of Muc-1 in these two kinds of intestinal metaplasia (14.1% vs 19.5%). Buried intestinal metaplasia, mainly as an incomplete type, is the major predominant type of BE. The degree of inflammation in the gastric antrum and esophagus can differentiate BE from CIM to some extent. CK-7 immunohistochemical staining can help identify BE and CIM but Muc-1 cannot.

Direct effects of IL-4/IL-13 and the nematode Nippostrongylus brasiliensis on intestinal epithelial cells in vitro

Nematode infections induce the upregulation of mucin- and glycosylation-related genes in intestinal epithelial cells in vivo. However, the factor(s) that induce these changes in epithelial cells have not been fully elucidated. In the present study, we analysed the effects of the Th2 cytokines IL-4 and IL-13 and the excretory-secretory (ES) product of the nematode Nippostrongylus brasiliensis on the gene expression of the major mucin core peptide MUC2, the sialyltransferase ST3GalIV (Siat4c) and the sulphotransferase HS3ST1 in intestinal epithelium-derived IEC-6 cells by quantitative reverse transcription (RT)-PCR. The administration of IL-4 and IL-13 resulted in a significant upregulation of ST3GalIV and HS3ST1 gene transcription, but had no effect on MUC2, in IEC-6 cells. RT-PCR studies also demonstrated the constitutive expression of IL-13Ralpha1 and IL-4R in IEC-6 cells. On the other hand, the ES product induced upregulation of ST3GalIV, but not HS3ST1 or MUC2, while coadministration of IL-13 and the ES product induced a slight but significant upregulation of MUC2. Co-incubation of live N. brasiliensis adult worms with IEC-6 cells resulted in the upregulation of ST3GalIV and MUC2. These results suggested that HS3ST1 gene expression is strictly regulated by IL-4/IL-13, while ST3GalIV and MUC2 gene expressions are regulated by redundant mechanisms.

A Case of Extrahepatic Bile Duct Wall Recurrence of Gastric Carcinoma that Was Treated with Pancreaticoduodenectomy

We report on a patient with obstructive jaundice caused by recurrence of gastric carcinoma in the wall of an extrahepatic bile duct more than 5 years after gastrectomy who was treated with pancreaticoduodenectomy. Histopathologic examination of the surgically resected specimen revealed a poorly differentiated adenocarcinoma with focal signet ring cells in the wall of the common bile duct which was histologically similar to the primary gastric carcinoma. To confirm the diagnosis, immunohistochemical staining was performed with antibodies against cytokeratins (CK7, CK20) and mucin peptide core antigens (MUC5AC, MUC6, MUC2). Based on the expression patterns of this monoclonal antibody panel, the final diagnosis of the common bile duct tumor was an isolated local recurrence of the gastric carcinoma. The patient has survived for more than 26 months after pancreaticoduodenectomy without recurrence.

The isolated MUC5AC gene product from human ocular mucin displays intramolecular conformational heterogeneity

Atomic force microscopy (AFM) has been used to show that human ocular mucins contain at least three distinct polymer conformations, separable by isopycnic density gradient centrifugation. In this work we have used affinity purification against the anti(mucin peptide core) monoclonal antibody 45M1 to isolate MUC5AC gene products, a major component of human ocular mucins. AFM images confirm that the affinity-purified polymers adopt distinct conformations that coidentify with two of those observed in the parent population, and further reveal that these two different conformations can be present within the same polymer. AFM images of the complexes formed after incubation of 45M1 with the parent sample reveal different rates of binding to the two MUC5AC polymer types. The variability of gene products within a mucin population was revealed by analyzing the height distributions along the polymer contour and periodicities in distances between occupied antibody binding sites. AFM analysis of mucin polymers at the single molecule level provides new information about the genetic origins of individual polymers and the contributions of glycosylation to the physicochemical properties of mucins, which can be correlated with information obtained from biochemistry, antibody binding assays, and molecular biology techniques.

Altered expression of goblet cell‐ and mucin glycosylation‐related genes in the intestinal epithelium during infection with the nematode Nippostrongylus brasiliensis in rat

Intestinal nematode infection induces marked goblet cell hyperplasia and mucus secretion, but the mechanisms of regulation of the changes still remain to be elucidated. In the present study, epithelial cells were isolated from the rat small intestine at various times after Nippostrongylus brasiliensis infection, and the levels of expression of goblet cell- and mucin glycosylation-related genes were estimated by semi-quantitative reverse transcription (RT)-PCR. Among the genes investigated, mucin core peptide (MUC) 2, sialyltransferase (Siat) 4c and trefoil factor family (TFF) 3 were upregulated as early as 2-4 days post-infection, suggesting that they are associated with an early innate protective response. Seven days post-infection and thereafter, when the nematodes reached maturity, significant upregulation of MUC3, MUC4, resistin-like molecule beta (Relmbeta) and 3O-sulfotransferase (3ST)1 was observed, while 3ST2 expression levels increased after the majority of the worms were expelled from the intestine. Similar alterations of glycosylation-related gene expression were also observed in mast-cell-deficient Ws/Ws rats, suggesting that mast cells in the epithelium are not relevant to the upregulation of these genes. The present finding that the expression level of each goblet cell- or glycosylation-related gene was altered differently during the time course of infection indicates the progression of sequential qualitative changes in the mucus layer after infection.

Human lacrimal gland mucins.

The objective of this study was to determine whether the lacrimal gland synthesizes mucins and whether they are changed with age or in cases of dry eye. Expression of mucins in human lacrimal glands was monitored by reverse transcription-polymerase chain reaction analysis. Furthermore, the presence and distribution of MUC1, -2, -4, -5AC, -5B, -6 and -7 in epithelia of the human lacrimal gland and its excretory duct system were assessed with antisera to mucin peptide cores. Thirty normal tissues from cadavers of different ages were tested, plus four with dry eye treated with artificial tears. Expression studies detected mRNAs for mucins MUC1, -4, -5AC, -5B, -6 and -7; whereas the MUC2 message was absent. The message for MUC4 was present in all four cases of dry eye, but only in six out of the 30 normal glands from individuals who did not receive artificial tears. MUC6 mRNA was detected only in about half of the investigated samples. Immunohistochemistry revealed membrane-bound MUC1 at the apical surface of acinar cells, absence of MUC2, MUC5AC associated with goblet cells of excretory ducts, MUC5B and -7 in the cytoplasm of acinar cells, and MUC7 also in epithelial cells of excretory ducts. MUC4 mucin was detected only in those individuals in which message was identified. In dry eyes, MUC5AC and -5B were localized in the same acinar cells; whereas MUC2 and MUC6 were not detectable. Dot-blot analysis clearly revealed increased amounts of MUC4, -5AC, and -5B in the glands of elderly women who received treatment for dry eyes. These results confirm that the human lacrimal gland synthesizes a spectrum of mucins; part of them might be correlated with age.

Immunoreactivity of cytokeratins (CK7, CK20) and mucin peptide core antigens (MUC1, MUC2, MUC5AC) in adenocarcinomas, normal and metaplastic tissues of the distal oesophagus, oesophago-gastric junction and proximal stomach.

Adenocarcinomas of the distal oesophagus and especially the oesophago-gastric junction have shown an increasing incidence during the last decade. Definition of subgroups according to different sites of development, histogenesis or aetiology may prove to be valuable for clinical diagnosis and treatment. Previous studies have shown differences in cytokeratin patterns between Barrett's metaplasia of the oesophagus and intestinal metaplasia in the stomach. The aim of our study was to investigate whether the expression of certain cytokeratins (CK7, CK20) and mucins (MUC1, MUC2, MUC5AC) exhibit clear-cut patterns, thus allowing a subclassification of adenocarcinomas of the oesophago-gastric junction. The possibility of a relationship between antigen expression and the presence or absence of Barrett's metaplastic epithelium was also studied. CK7, CK20, MUC1, MUC2 and MUC5AC were visualized in six adenocarcinomas of the distal oesophagus, 29 adenocarcinomas of the oesophago-gastric junction and eight adenocarcinomas of the proximal stomach. CK7, CK20 and MUC1 were strongly expressed in the great majority of all neoplasms under study, whereas MUC2 and MUC5AC were absent or only faintly detectable. CK20 exhibited a significantly stronger expression in poorly differentiated tumours (G3) and MUC1 immunoreactivity correlated with tubular and papillary versus signet-ring cell histopathology. Other statistically significant correlations between antigens and histopathological features (pTNM stage, grading, histopathological subtype, presence/absence of Barrett's epithelium) were not observed. According to our results, most adenocarcinomas of the oesophago-gastric junction show a CK7+, CK20+, MUC1+ phenotype irrespective of the presence or absence of Barrett's epithelium. The immunohistochemical data suggest a similar histogenesis of these tumours.

Correlation of MUC5AC immunoreactivity with histopathological subtypes and prognosis of gastric carcinoma.

MUC5AC represents a mucin peptide core expressed in normal gastric epithelia. Its presence in gastric carcinomas was previously described as a characteristic of gastric differentiation. MUC5AC reactivity was investigated by immunohistochemistry and correlated with clinicopathological variables in a large series (n = 200) of gastric carcinomas. A statistically significant association between MUC5AC positivity and parameters of cancer progression (pTNM staging and grading) could not be observed. However, MUC5AC exhibited correlations with certain subtypes of histopathological differentiation. A significant reduction of MUC5AC expression was evident in mucinous and undifferentiated carcinomas according to the World Health Organization classification, as well as in type III cancers according to the Goseki classification system. Furthermore, reduced MUC5AC reactivity (confined to up to 35% of the tumor area) was significantly correlated with an unfavorable prognosis of all patients in univariate and multivariate analysis. The same association could be observed in the subgroup of pTNM stage I patients (n = 60). A significant reduction of gastric differentiation as reflected by MUC5AC immunoreactivity represents a marker of worse survival probability in gastric cancer. Finally, reduced MUC5AC positivity defines a high-risk subgroup of pTNM stage I patients.

Immunoreactivity of Lewis blood group and mucin peptide core antigens: correlations with grade of dysplasia and malignant transformation in the colorectal adenoma-carcinoma sequence.

Previous studies on the immunoreactivity of various mucin peptide and carbohydrate antigens in neoplastic colorectal tissues led to at least in part contradictory results. Therefore, we investigated a series of 42 adenomas and 44 carcinomas applying monoclonal antibodies (mabs) directed against Lewis blood group antigens (sialyl-Le(a), Le(x), sialyl-Le(x), Le(y)) as well as mucin peptide cores (MUC1, MUC2 and MUC5AC) by immunohistochemistry. A statistically significant positive correlation between the development of high-grade dysplasia in colorectal adenomas and the immunoreactivity of Le(y) and MUC1 epitopes was observed, whereas MUC2 exhibited a significant negative correlation. The reactivity of the other epitopes did not show an association with the progression of malignant transformation. Colorectal carcinomas were subdivided according to their histopathological subtype. The immunohistochemical staining resulted in a significantly stronger MUC2 reactivity of mucinous vs. tubular adenocarcinomas. Immunoreactivity of the MUC1-specific mab, which does not react with the fully glycosylated peptide core, showed a statistically non-significant inverse tendency, whereas all carbohydrate antigens displayed a strong expression in both tumor subtypes. Furthermore, correlations between mucin peptide and carbohydrate epitope labelling were evaluated. Progression of the adenoma-carcinoma sequence was accompanied by an increase of Le(y) as well as MUC1 antigen and an increase of all Lewis antigens compared to MUC2 immunoreactivity. On the other hand, mucinous carcinomas exhibited an inverse pattern. In conclusion, these results demonstrate that Le(y) and MUC1 immunoreactivity correlate with malignant transformation in the colorectum, whereas MUC2 represents a marker for low-grade dysplasia and the subtype of mucinous carcinomas.

Immunoreactivity of Monoclonal Antibody BW835 Represents a Marker of Progression and Prognosis in Early Gastric Cancer

The Thomsen-Friedenreich (TF) antigen is a well-known human pan-carcinoma antigen. It represents a carbohydrate core disaccharide (Galβ1-3GalNAc) which is predominantly bound to mucin peptide cores. Its immunoreactivity depends on changes in glycosylation which lead to a reduction in the carbohydrate chain length and the exposure of core carbohydrates. In the present study, we investigated 208 gastric adenocarcinomas with respect to their immunohistochemical reactivity applying two monoclonal antibodies (MAbs). MAb specifically detecting TF antigen (A78-G/A7) and MAb BW835 were included. The latter reacts with a certain glycoform of the MUC1 peptide core, characterized by core-type glycans like TF. A78-G/A7 epitopes were detected in 68.8% and BW835 epitopes in 57.7% of the carcinomas. BW835 immunoreactivity correlated with the presence of lymph node metastases. Both A78-G/A7 and BW835 staining were significantly stronger in tubular/papillary cancer (WHO classification) and intestinal-type cancer according to Laurén. In univariate survival analyses of all patients studied, BW835 immunoreactivity was a marker of an unfavorable prognosis (p < 0.05). The presence of A78-G/A7 and BW835 epitopes exerted a negative effect on the subgroup of pTNM stage I carcinomas. These results indicate that TF and MUC1-TF immunoreactivity defines a ‘high-risk’ subgroup of stage I patients in gastric cancer.

Two novel monoclonal antibodies against the MUC4 tandem repeat reacting with an antigen overexpressed by lung cancer.

In this study we investigated the immunochemical and cytochemical reactivity of two monoclonal antibodies against the 16-amino acid tandem repeat of MUC4 to demonstrate a possible variation of the mucin core peptide expression related to lung cancer. The immunocytochemical anti-MUC4 reactivity was analyzed in four lung cancer cell lines (Calu-1, Calu-3, H460, SKMES) and in other tumor cell lines, as well as in frozen materials from 21 lung adenocarcinomas (ACs), including five bronchioloalveolar carcinomas (BACs), and 11 squamous cell lung carcinomas (SqCCs). A weak fluorescence anti-MUC4 positivity (range: 10.3-16.2) was observed only in acetone-fixed lung cancer cell lines Calu-1, Calu-3 and H460. These three lung cancer cell lines also showed a cytoplasmic immunoperoxidase reactivity. The immunostaining in lung cancer tissues showed a granular cytoplasmic reactivity: 15/21 (71%) and 17/21 (80%) ACs were positive with BC-LuC18.2 and BC-LuCF12, respectively. All BACs were positive. Moderate to strong reactivity was present in well-differentiated ACs. In the normal lung parenchyma counterparts weak reactivity was found only in bronchiolar cells. All SqCCs were negative. Anti-MUC4 reactivity was also observed in the alveolar mucus. In conclusion, our anti-MUC4 MAbs detect a secretion product present in mucus and this product is elaborated by lung cancer cells and overexpressed in well-differentiated lung ACs.

MUC-6 mucin is a major component of ‘blood group substance’ from human ovarian cyst fluid

Ovarian cyst fluid has been a valuable source of the mucins (traditionally termed ‘blood group substances’) that were used for the elucidation of the structures of the ABO Lewis blood group determinants, but the identity of the mucin peptide core(s) carrying these carbohydrate specificities is not known. An ovarian cyst fluid mucin was purified, deglycosylated with HF and digested with trypsin or chymotrypsin to yield a number of peptides. Amino acid sequencing of these peptides yielded five different sequences which showed complete or partial homology to the MUC-6 apomucin deduced from DNA sequencing. As no other sequences were identified, it is concluded that MUC-6 is the major mucin core structure of ovarian cyst fluid mucin.

Biochemistry and pathological importance of mucin-associated antigens in gastrointestinal neoplasia

Publisher Summary This chapter focuses on mechanisms of alterations of Mucin-Associated Antigens in gastrointestinal neoplasia and their implications in clinical diagnostic and therapeutic oncology. The chapter focuses on real mucins (MUC), taking MUC1 and MUC2 as models. In addition, the chapter defines mucins and discusses the biochemistry and molecular biology of mucins, such as structural aspects of mucins, biosynthesis of mucins, glycosylation site specificity and biosynthetic routes of O-glycosylation, and antigenicity of mucins. During carcinogenesis, alterations of mucin peptide and mucin glycosylation occur. Although glycosylation of proteins should be dependent on the rate of synthesis, and overexpression of mucin species in certain cancers is associated with reduced glycosylation, there is no clear-cut evidence for a causal relationship between these events. The chapter provides an account for the expression of mucin-asssociated antigens in gastric tissues (in non-neoplastic and neoplastic gastric tissues); and expression in colorectal tissues (in non-neoplastic colorectal tissues and colorectal adenoma-carcinoma sequence). Mucin-associated peptide and carbohydrate antigens play an important role in different aspects of proliferation, local invasion, and metastasis of cancer cells. Thus, the carbohydrate antigens gained clinical importance as prognostic markers in a great variety of human carcinomas. Serum tumor markers (circulating antigens) also help in clinical tumor diagnostics. The chapter concludes with the perspectives in tumor immunology and cancer therapy. Mucin peptide cores, in addition to substituting carbohydrate structures, are involved in different types of cellular and humoral immune response in cancer. Biosynthesized glycopeptides containing clustered saccharide domains and peptides that mimic carbohydrates may be applied in future studies on therapy of gastrointestinal carcinomas.

Altered mucin core peptide expression in acute and chronic cholecystitis.

Human mucin genes include membrane-bound mucins (MUC1, MUC3, MUC4) and secretory mucins (MUC2, MUC5AC, MUC5B, MUC6). Our aim was to determine mucin gene expression in human gallbladder cell lines, normal gallbladder from liver donors (N = 7) and surgical specimens with mild chronic cholecystitis (N = 29), chronic cholecystitis (N = 48), and acute and chronic cholecystitis (N = 27). MUC1 mRNA was ubiquitous; however, only rare MUC1 immunoreactivity was detected. MUC3, MUC5AC, MUC5B, and MUC6 mRNA were present in all gallbladder specimens and cell lines examined. Prominent MUC3, MUC5AC, MUC5B, and MUC6 immunoreactivity was present in 86-100% of normal gallbladders. The frequency of MUC5AC reactivity was decreased in specimens with acute cholecystitis (P < 0.05). In contrast, MUC2-reactivity was absent in normal gallbladder and present in 53.8% of acute cholecystitis specimens (P < 0.05). Surface epithelium is characterized by MUC3, MUC5AC, and MUC5B, whereas deeper mucosal folds display MUC5B and MUC6 immunoreactivity. Gallbladder epithelium demonstrates a unique and diverse pattern of mucin core proteins that becomes altered with increasing degrees of inflammation.

MUC1 Expression in Intramucosal Colorectal Neoplasms

Purpose: The mucin core peptide MUC1 often is detectable in colorectal carcinoma (CRC) tissue and cell lines. However, whether MUC1 in CRC correlates with tumor histogenesis and progression is unclear. We studied the relationship between MUC1 expression in intramucosal CRC and clinicopathologic features, expression of Ki-67, and p53 protein, and apoptosis. Methods: The intramucosal CRC we studied included 140 endoscopically or surgically resected lesions, including 106 low-grade carcinomas and 34 high-grade carcinomas. De novo carcinoma, defined as carcinoma with no adenomatous component, represented 9 of 140 tumors. Three macroscopic types were identified: 57 lesions were polypoid, 55 were superficial and flat, and 28 were granular-type laterally spreading tumors (G-LST). MUC1, Ki-67, and p53 expression were examined immunohistochemically. Apoptotic cells were identified by in situ DNA nick end labeling. Results: MUC1 expression in high-grade carcinomas was significantly more frequent (p < 0.01) than in low-grade carcinomas; expression in adenomas was almost nil. MUC1 expression in polypoid carcinomas was significantly more frequent (p < 0.05) than in superficial carcinomas or G-LST. MUC1 expression in carcinomas with p53 expression was significantly more frequent (p < 0.01) than in carcinomas not expressing p53. No significant correlation was found between expression of MUC1 and Ki-67 labeling index. MUC1 was expressed more frequently in carcinomas with relatively high apoptotic index (p < 0.01). MUC1 expression did not differ between de novo carcinomas and those developing from adenomas. Conclusions: The results suggest that MUC1 is likely to be expressed in the course of colorectal carcinoma development when p53 protein is overexpressed and apoptosis is prominent.

Anti-tumor activity and immune responses induced by human cancer-associated mucin core peptide

Mucin molecules are displayed on most human cancer cell surface, and are different from that expressed on normal epithelial cells. The molecular structure of mucin core peptide (apomucin) was identified recently. However, the function of apomucin is only poorly understood. To further elucidate the role of apomucin in the modulation of cancers, this study was to investigate the immune responses induced by mucin core peptide in mice. The mucin core peptide was isolated from pancreatic cancer cell line SW1990. When mice immunized with this apomucin (10 micrograms/time x 6) plus DETOX, all mice developed delayed-type hypersensitivity (DTH) after challanged with apomucin or synthetic mucin core peptide MUC-2 or MUC-3, while the mice immunized with only apomucin did not develop DTH. No antibodies were detected by ELISA after immunization. When the splenic cells of vaccinated mice were cocultured with this apomucin (10-50 micrograms/ml) and rhIL-2 (50 U/ml) in vitro, the proliferated lymphocytes showed cytotoxicity against human cancer cells, including colon cancer, gastric cancer, pancreatic cancer and leukemia as measured by Cr-51 release assay. The cytotoxicity could be blocked by antibodies against MUC-2 and MUC-3. These results provide a rational basis for the use of apomucin as a vaccine to stimulate anti-tumor immunity.

Correlation of the immunohistochemical reactivity of mucin peptide cores MUC1 and MUC2 with the histopathological subtype and prognosis of gastric carcinomas.

The expression of MUC1 and MUC2 mucin peptide core antigens in gastric carcinomas was studied by immunohistochemistry to determine correlations with TNM stage and histo-pathological classifications as well as a possible prognostic impact. Paraffin-embedded specimens from 128 gastric carcinomas with a minimal follow-up of 5 years were immunostained. In addition to a polyclonal antiserum generated against polymorphic epithelial mucin (MUC1) from human milk, 2 monoclonal antibodies (MAbs), HMFG2 (anti-MUC1) and 4FI (anti-MUC2), were applied. Reactivity of carcinomas was correlated with the classifications of the UICC (TNM), WHO and Laurén. Correlations with overall survival were analyzed using the Kaplan and Meier product limit method. MUC1 immunoreactivity was associated with an advanced pTNM stage. The demonstration of both mucin species (MUC1, MUC2) displayed a statistically significant correlation with tubular/papillary vs. signet-ring cell differentiation as well as with intestinal-type vs. diffuse-type of tumor growth according to Laurén. In particular, MUC2 was only rarely detectable in signet-ring cell and diffuse-type tumors. MUC1 correlated with poor prognosis in all cases and the subgroup of stage I tumors. According to the histopathological classifications, a similar result was observed in signet-ring cell and diffuse-type carcinomas. In contrast, MUC2 reactivity was associated with a favourable prognosis of intestinal-type carcinomas. In the non-neoplastic gastric mucosa, both peptide cores were recognized in the superficial epithelium, whereas parietal cells contained only MUC1, and intestinal metaplasia almost exclusively MUC2 antigens. We conclude that the mucin peptide core antigens are suitable markers for the tubule-rich gastric carcinomas, which may in part be derived from intestinal metaplasia. In addition, MUC1 may exert a prognostic relevance and appears to be involved in the progression of diffuse-type tumors.

Coexpression of MUC1 mucin peptide core and the Thomsen-Friedenreich antigen in colorectal neoplasms.

Controversial findings have been reported regarding the expression of the Thomsen-Friedenreich (TF) antigen in colorectal neoplasms when different monoclonal antibodies (MoAbs) have been used. Moreover, there is no information available regarding the carrier protein(s) of this antigen. Forty-five colorectal adenomas and 48 carcinomas were studied by avidin-biotin complex-peroxidase immunohistochemistry. The immunohistochemistry employed the MoAb BW835, which was reactive to a carrier specific and site specific TF antigen on MUC1 mucin, as well as reference antibodies directed to MUC1 (HMFG2) or MUC2 core peptides (4F1) and directed to TF antigen irrespective of its carrier (A78-G/A7, peanut agglutinin). To evaluate the coexpression of different epitopes by the same antigen, sandwich enzyme-linked immunoadsorbent assays were performed. Although MUC1 peptide antigen and MUC1-bound TF antigen were not detectable in normal or transitional mucosa surrounding colorectal neoplasms, expression of these antigens in adenomas accompanied the development of high grade dysplasia. By contrast, MUC2 expression detected by the MoAb 4F1 was inversely correlated with the progression of the adenoma-carcinoma sequence. In well- and moderately differentiated colorectal carcinomas, the neo-expressed TF antigen is predominantly bound to MUC1. This feature could be demonstrated by antigen coexpression using peptide and the TF antigen specific MoAbs. However, in mucinous carcinomas exhibiting a weak MUC1 peptide expression in most specimens, the presence of TF antigen on the MUC2 peptide core cannot be ruled out. TF antigen is strongly coexpressed with MUC1 mucin peptide core in the colorectal adenoma-carcinoma sequence, resulting in well- and moderately differentiated carcinomas. Only in mucinous carcinomas may it be coexpressed with MUC2 antigen.