The gut microbiota, bile acids and their correlation in primary sclerosing cholangitis associated with inflammatory bowel disease.

BACKGROUND: Patients with primary sclerosing cholangitis associated with inflammatory bowel disease (PSC-IBD) have a very high risk of developing colorectal neoplasia. Alterations in the gut microbiota and/or gut bile acids could account for the increase in this risk. However, no studies have yet investigated the net result of cholestasis and a potentially altered bile acid pool interacting with a dysbiotic gut flora in the inflamed colon of PSC-IBD. AIM: The aim of this study was to compare the gut microbiota and stool bile acid profiles, as well as and their correlation in patients with PSC-IBD and inflammatory bowel disease alone. METHODS: Thirty patients with extensive colitis (15 with concomitant primary sclerosing cholangitis) were prospectively recruited and fresh stool samples were collected. The microbiota composition in stool was profiled using bacterial 16S rRNA sequencing. Stool bile acids were assessed by high-performance liquid chromatography tandem mass spectrometry. RESULTS: The total stool bile acid pool was significantly reduced in PSC-IBD. Although no major differences were observed in the individual bile acid species in stool, their overall combination allowed a good separation between PSC-IBD and inflammatory bowel disease. Compared with inflammatory bowel disease alone, PSC-IBD patients demonstrated a different gut microbiota composition with enrichment in Ruminococcus and Fusobacterium genus compared with inflammatory bowel disease. At the operational taxonomic unit level major shifts were observed within the Firmicutes (73%) and Bacteroidetes phyla (17%). Specific microbiota-bile acid correlations were observed in PSC-IBD, where 12% of the operational taxonomic units strongly correlated with stool bile acids, compared with only 0.4% in non-PSC-IBD. CONCLUSIONS: Patients with PSC-IBD had distinct microbiota and microbiota-stool bile acid correlations as compared with inflammatory bowel disease. Whether these changes are associated with, or may predispose to, an increased risk of colorectal neoplasia needs to be further clarified.

Expression of LewisX and sialylated LewisX antigens in human colorectal polyps.

The LewisX (LeX) antigen [characterized by trisaccharide Gal beta 1----4 (Fuc alpha 1----3)N-acetylglucosamine] is an oncodevelopmental antigen in the human colon. Monoclonal antibodies (MoAbs), anti-SSEA-1 and AH8-183, which recognize LeX antigen either on short oligosaccharide side chains or as a terminal immunodeterminant on longer carbohydrate side chains of glycoconjugates, bind to most colon cancer tissues but also to some normal colon mucosae. However, the monoclonal antibodies FH1, FH4, FH6, and IB9, which recognize extended difucosylated and trifucosylated LeX structures or their sialylated derivatives, are more cancer-associated because they rarely bind to normal colon mucosa. In the present study, these MoAbs were used to compare the expression of various LeX-related antigens in premalignant (adenomatous) and nonpremalignant (hyperplastic) colorectal polyps. Antigen expression in polyps was also compared to antigen expressions of normal colon mucosa and colon cancer tissues. The four MoAbs recognizing extended LeX antigens bound to adenomatous polyps (APs) significantly more than to hyperplastic polyps (HPs). In contrast, anti-SSEA-1 and AH8-183 recognizing monofucosyl LeX were less able to distinguish between APs and HPs. In APs, staining with the four MoAbs recognizing extended LeX antigens correlated with the premalignant parameters of larger polyp size, more severe dysplasia, and increased villose component. However, staining with AH8-183 correlated only with polyp size, and anti-SSEA-1 correlated only with polyp size and degree of dysplasia. In general, the staining frequency of HPs was similar to that of normal colon mucosa, although FH6, which did not stain any specimens of normal mucosa, stained a few HPs. The staining frequency of APs was less than that of colon cancer tissues, but these differences were generally not statistically significant. In conclusion, extended LeX antigens and their sialylated derivatives are cancer-associated antigens that are expressed preferentially in premalignant colon polyps, that tend to correlate with malignant potential in these polyps, and that may eventually help to define mechanisms involved in the polyp-to-cancer sequence.

Cancer-associated alterations of blood group antigen expression in the human pancreas.

Certain alterations of blood group substance (BGS) expression have been observed in gastrointestinal cancer tissues. However, in the pancreas little is known about BGS expression by normal or malignant tissue. The present immunohistochemical study analyzed simultaneously the expression of A, B, H, Lewisa (Lea), and Lewisb (Leb) antigens in specimens of normal pancreas, chronic pancreatitis, and pancreatic carcinoma (primary and metastatic). In normal pancreas all five antigens were expressed in ducts, ductules, and acini, but not in islets. Acinar cells expressed A, B, H, and Leb in supranuclear cytoplasm, whereas Lea was found mainly on centroacinar cells. Only BGSs that were appropriate for the host's blood type were expressed, except for one case of Lea deletion. BGS expression by chronic pancreatitis tissue closely resembled that by normal tissue. In primary pancreatic cancer two cancer-associated alterations were noted that were not found in either normal pancreas or chronic pancreatitis. Deletion of an expected A, B, H, or Leb antigen occurred in approximately 25% of cases, particularly in more poorly differentiated cancers. Incompatible expression of an unexpected A or B antigen occurred in 33% of cases, regardless of degree of differentiation. Metastatic pancreatic cancers also exhibited BGS deletion and incompatibility. In both primary and metastatic cancers the incidence of incompatible A or B expression was higher in cancers from the United States than in cancers from Japan, but the incidence of BGS deletion was similar between the two countries. It was concluded that deletion of A, B, H, or Leb antigens and incompatible expression of A or B antigens are cancer-associated events in the pancreas.

Use of model cell lines to study the biosynthesis and biological role of cancer-associated sialosyl-Tn antigen.

Sialosyl-Tn (STn) is a mucin-associated carbohydrate antigen that is not expressed by most normal epithelial cells but becomes expressed in several types of adenocarcinomas, where it is often associated with a poor prognosis. Little is known about the regulation of the STn phenotype in tumor cells and the immune response to STn antigen. In the present study, we established clonal cell lines in which virtually all of the cells were STn positive (designated LS-C) or STn negative (designated LS-B). These two cell lines, derived from a single parental cell line, LS174T, have the same total protein electrophoretic profiles but carry markedly different oligosaccharide structures on their mucin; the mucin from LS-C cells has only the Tn and STn structures, whereas LS-B cell mucin lacks these simple structures but carries more complex oligosaccharides. These results indicate that lack of STn expression by cells can be due to the lack of STn synthesis rather than inaccessibility of antibodies to bind to STn by steric hindrance. Both clones were similar in their growth rates, response to gamma-interferon, and sensitivity to lysis by lymphokine-activated killer cells. These cells may be important models for understanding the regulation of glycosylation at the cellular level and for further studies of tumor biology and immune response to STn antigen.

Mucins bearing the cancer-associated sialosyl-Tn antigen mediate inhibition of natural killer cell cytotoxicity.

The sialosyl-Tn (STn) antigen is a mucin-associated carbohydrate antigen expressed by a variety of adenocarcinomas. In the colon, expression of this antigen has been associated with a poor prognosis, independent of tumor stage or histology. The present study was performed to determine whether this adverse clinical outcome might be due to an interaction between STn-positive mucin and natural killer (NK) cell cytotoxicity. Ovine submaxillary mucin (OSM), a mucin highly rich in STn antigen, partially inhibited NK cell cytotoxicity against K562 target cells, but only at high concentrations. Low concentrations of OSM were not inhibitory but became markedly inhibitory in the presence of ammonium ions. Two other STn-positive submaxillary mucins also markedly inhibited NK cytotoxicity when combined with ammonium ions. Removal of sialic acid from OSM reversed the OSM/ammonium-mediated inhibition of NK cell activity. Unlike the submaxillary mucins, two mucins derived from human breast and lung cancer cells which lack the STn antigen, did not inhibit NK cell activity in this system. Likewise, four other non-mucin glycoproteins which lack STn expression did not inhibit NK cells despite having levels of sialic acid that were, in some cases, comparable to submaxillary mucin. These results indicate that mucins bearing the cancer-associated STn antigen can effectively inhibit NK cell cytotoxicity in the presence of ammonium ions. While this NK cell inhibition is likely to be caused by ammonium, mucin markedly enhances this effect, thereby implicating a novel immunomodulatory property of mucin.

Mucin gene expression in colonic tissues and cell lines.

Complementary DNA clones encoding four different mucin core peptides have been isolated. However, the expression of these mucin genes in the colon has not been systematically studied. The present investigation used Northern blot analysis to study the expression of MUC1, MUC2, MUC3, and MUC4 mRNA in paired normal and cancerous colonic tissues, and nine colon cancer cell lines. Results were correlated with the clinicopathological features of the tumors and with the immunohistochemical expression of several carbohydrate tumor-associated antigens that may reside on mucins. MUC1 mRNA was expressed in all colonic tissues, and levels in paired normal and cancer tissues were similar in most cases. MUC2 and MUC3 mRNAs were expressed in both normal and cancer tissues, but levels were often decreased in the cancers. MUC4 mRNA was present in normal mucosa with comparable or sometimes greater expression in cancers. There was no apparent correlation between the expression of any particular mucin gene or pattern of mucin genes and the site, stage, or histological type of tumor. In addition, the expression of mucin-associated carbohydrate antigens did not correlate with any individual mucin gene or group of mucin genes. In colon cancer cell lines all four MUC genes were expressed rather weakly or not at all. These results indicate that the human colon expresses a broad repertoire of mucin genes which are differentially regulated in malignancy. Whether this differential regulation of mucin genes affect the behavior of the tumor and results in the altered glycosylation commonly seen in these requires further investigation.

ABH blood group antigen expression, synthesis, and degradation in human colonic adenocarcinoma cell lines.

The synthesis of blood group ABH antigens is under genetic control, where the primary gene products are glycosyltransferases. Several studies have demonstrated cancer-associated alterations in ABH antigen expression in human colon cancer tissues. However, the mechanism(s) responsible for these alterations has not been elucidated. Therefore, experiments were conducted using nine established colon cancer cell lines (four type O, three type A, and two type B) to examine ABH antigen expression by immunocytochemistry and correlate this with activities of ABH biosynthetic (glycosyltransferase) and degradative (glycosidase) enzymes. The products of the glycosyltransferase enzymes were characterized by high performance liquid chromatography and paper chromatography, and substrate affinities (apparent Km values) of the cancer cell-derived glycosyltransferases were analyzed. The present data demonstrate: (a) all cell lines except H-498 (blood type A) expressed the appropriate ABH glycosyltransferase as well as all three glycosidases; (b) product characterization and substrate dependence experiments suggested that the cancer cell-derived ABH glycosyltransferase enzymes had properties that were similar to those of the ABH enzymes in human serum; (c) H-498 cells exhibited A antigen deletion with accumulation of H precursor substance, most likely due to insufficient A transferase activity; (d) SW1417 cells (blood type B) demonstrated B antigen deletion without precursor accumulation, despite adequate levels of B transferase and low alpha-galactosidase activity; and (e) weak incompatible A antigen expression occurred in LoVo (type B) and SW1116 (type O) cells, and weak incompatible B antigen expression occurred in H-498 (type A) and SW1116 cells. However, since these cells lacked incompatible A or B transferase activity, these incompatible antigens are probably not the true A or B antigens. Thus, the colon cancer cell lines used in this study exhibit all of the ABH alterations previously described in colon cancer tissues and appear to be useful experimental models for studying the molecular events involved in cancer-associated ABH expression.

Lex and Ley antigen expression in human pancreatic cancer.

Carbohydrate antigens are useful markers for the serological detection of pancreatic cancer. However, data concerning the expression of structurally well-defined carbohydrate antigens in normal and malignant pancreatic tissue is quite limited. The Lex and Leg antigens are closely related carbohydrate antigens synthesized on type 2 blood group oligosaccharide side chains of glycolipids and glycoproteins. Monoclonal antibodies anti-SSEA-1 and AH6 recognize "simple" Lex and Ley epitopes, respectively, regardless of the length of the carrier carbohydrate. Other monoclonal antibodies recognize Lex (FH4), sialyl Lex (FH6, IB9) or Ley (KH1, CC-1, CC-2) carried only by elongated type 2 side chains with or without internal alpha 1,3 fucosyl substitution. The present comparative immunohistochemical study used tissues of normal pancreas, chronic pancreatitis, and pancreatic cancer to determine the normal expression of Lex and Ley antigens in the pancreas and to elucidate any cancer-associated alterations. Lex-related antigens were not expressed in normal pancreas, expressed in only 10-20% of chronic pancreatitis tissues, but expressed in 50-70% of pancreatic cancer tissues. The frequency of Lex-related antigen expression in pancreatic cancer tissues was lowest in poorly differentiated cancers. Within a given specimen, at least three or all four of the Lex recognizing monoclonal antibodies were simultaneously expressed. Unlike Lex antigens, Ley-related antigens were expressed in 32-77% of specimens of normal pancreas, with similar frequencies in specimens of chronic pancreatitis and pancreatic cancer. In normal pancreas, simple Ley was expressed by both ductal and acinar cells, but extended Ley antigens were expressed only by acinar cells. In pancreatic cancer, extended Ley antigen expression was found in less than 10% of poorly differentiated tumors. Coexpression among the Ley-related antigens was less common than with the Lex-related antigens. Also in cancer specimens, simple Lex and simple Lex antigens were often concordantly expressed, whereas extended Lex and extended Ley antigen expression was often discordant. Hyperplastic ducts and ductules associated with pancreatic cancer expressed Lex-related antigens more frequently than morphologically similar lesions associated with chronic pancreatitis. These results demonstrate that Lex-related antigens are cancer-associated determinants in the human pancreas. The discrepant expression between Lex and Ley antigens in these tissues implies altered regulation of fucosyltransferase activity associated with the malignant state.

Cancer-associated alterations of blood group antigen expression in human colorectal polyps.

Human colorectal carcinoma tissues may exhibit several patterns of altered blood group substance (BGS) expression: reappearance of A, B, H, or Lewisb antigens in distal colon; deletion of BGS in the proximal colon with or without precursor substance accumulation; and incompatible BGS expression in proximal or distal colon. The present study evaluated these cancer-associated alterations in colorectal polyps with different malignant potential. With respect to ABH antigens, hyperplastic polyps (HPs), considered to have no malignant potential, did not exhibit incompatibility and only a few cases demonstrated BGS reappearance or deletion. Adenomatous polyps (APs) however, frequently reexpressed ABH antigens or expressed incompatible BG-A or B in 27% of polyps; one specimen demonstrated BG-B deletion. Precursor expression was not found in HPs but was frequently observed in APs. Reappearance of ABH in distal polyps was significantly correlated with increasing grade of dysplasia, but was not significantly correlated with polyp size or histological type. With respect to Lewis antigen expression, Lewisb reappearance occurred in almost every distal polyp, and Lewisa-Lewisb coexpression was also quite common. Lea deletion was frequently noted, especially in HP, but the significance of this finding is unclear. This study indicates that several antigenic alterations that occur in colorectal cancer tissues also appear in premalignant polyps, and often in early stages of the neoplastic process. The observation that incompatible expression of BG-A or B occurs only in AP and cancer tissues (as well as mucosa adjacent to cancer) but not in fetal colonic mucosa, adult normal colonic mucosa, or HP, suggests that this may be a cancer-specific phenomenon.

Tumor-associated sialylated antigens are constitutively expressed in normal human colonic mucosa.

Immunohistochemical studies have indicated that sialylated carbohydrate antigens such as sialyl-Tn, sialyl-Le(a), and sialyl-Le(x) are expressed in a tumor-associated fashion in human colon. Since sialic acid residues are O-acetylated more extensively in normal colonic epithelium than in colon cancer cells, we examined whether deacetylation of colonic tissues might enable monoclonal antibodies to recognize these tumor-associated sialylated antigens. In normal colon, deacetylation turned most cases (82%) positive with anti-sialyl-Tn mAb TKH2; and in colon cancers, it increased the number of TKH2-positive cells. Sialyl-Le(a) and sialyl-Le(x) detection was also increased after deacetylation of normal and malignant colonic tissues so that the frequency of positive cases in normal tissues was similar to that in the cancers. However, in the stomach and pancreas, the same treatment rarely increased the detection of the sialylated epitopes in normal or cancerous tissues. Thus, the same sialylated epitopes can be expressed in a tumor-associated fashion by different mechanisms in different gastrointestinal organs; in the colon, these antigens are constitutively expressed and O-acetylated, whereas in the upper gastrointestinal tract, they are rarely O-acetylated, suggesting that other mechanisms such as differences in glycosylation account for the cancer-associated expression.

Immunohistochemical comparison of Lea, monosialosyl Lea (CA 19-9), and disialosyl Lea antigens in human colorectal and pancreatic tissues.

The CA 19-9 antigen is a monosialosyl Lea blood group antigen which has been shown to be a useful tumor-associated antigen for the diagnosis of gastrointestinal cancers. Recently, a sialylated derivative of this antigen, disialosyl Lea, was isolated from a colon cancer liver metastasis and a monoclonal antibody (FH7) recognizing this novel determinant was developed. The present study simultaneously compared the expression of Lea, monosialosyl Lea, and disialosyl Lea antigens in a variety of nonmalignant, premalignant, and malignant tissues of the colorectum and pancreas with an aim toward elucidating whether disialosyl Lea is expressed as a tumor-associated antigen. In normal colonic mucosa, disialosyl Lea expression closely resembled Lea expression in overall frequency, segmental distribution, and cellular localization whereas monosialosyl Lea (CA 19-9) was essentially absent. Along the crypt axis, Lea was more often expressed in goblet cells of the upper crypt whereas disialosyl Lea was found in goblet cells along the entire crypt. Fetal colonic mucosa expressed all three antigens, as did most colorectal cancers regardless of location within the colon or degree of differentiation. The majority of hyperplastic polyps and practically all adenomatous polyps also expressed these three antigens, and in adenomas, antigen expression was independent of polyp size, villous morphology, or degree of dysplasia. In the normal pancreas, the three antigens were expressed on ductal, ductular and centroacinar cells of all specimens. The majority of pancreatic cancers expressed all three antigens. Thus, in the normal colon, the absence of monosialosyl Lea (CA 19-9) in the presence of disialosyl Lea suggests that an alpha 2,6 sialyltransferase is active, which results in the masking of CA 19-9 antigen expression. These results further support the concept that specific sialyltransferases play a role in regulating the expression of tumor-associated antigens.

Distribution of blood group antigens A, B, H, Lewisa, and Lewisb in human normal, fetal, and malignant colonic tissue.

In humans, most blood group substances (BGS) are expressed throughout the fetal colon but are absent from the distal portion of adult colon. Cancers of the distal colon frequently reexpress BGS thereby suggesting that these antigens behave as oncofetal antigens at this organ site. We used a sensitive immunoperoxidase method with monoclonal antibodies directed against blood groups A, B, O (H), Lewisa and Lewisb to systematically evaluate BGS expression in fetal colon, normal adult colon from immediate autopsies of kidney donors, mucosa adjacent to cancer (transitional mucosa) and colorectal cancer tissues. In normal colon, BG-A, B, H, and Lewisb were expressed in proximal but not distal colon, whereas Lewisa was distributed uniformly throughout the colon. In colon cancer, and fetal colon, the proximal-distal gradient of BG-A, B, H, and Lewisb expression was abolished because of enhanced distal expression of these antigens. In cancer tissues, three patterns of altered BGS expression emerged: (a) incompatible expression of BG-A or BG-B (over 50% of patients); (b) deletion of BGS; and (c) precursor BG-H accumulation (80% of 25 tumors). BGS staining of transitional mucosa closely resembled that of the adjacent tumor except that no examples of BGS deletion were encountered in transitional mucosa. The goblet cell secretory vacuole accounted for most of the BGS expression in normal colon, but cancer cells demonstrated differentiation-dependent antigenic expression such that well-differentiated tumors expressed BGS on cell apical membranes and glandular contents, but poorly differentiated cancers exhibited diffuse cytoplasmic staining. These findings confirm the oncofetal nature of BGS in distal colon cancer, and provide immunohistochemical evidence for a diverse repertoire of altered antigen expression in colon cancer. Further investigation is needed to elucidate the possible genetic and biochemical mechanisms involved.

Expression of LeY and extended LeY blood group-related antigens in human malignant, premalignant, and nonmalignant colonic tissues.

The LeY determinant, a difucosylated type 2 blood group-related antigen, is a positional isomer of the Leb blood group antigen and a fucosylated derivative of the LeX antigen. The LeX antigen behaves like an oncodevelopmental tumor-associated antigen in human colon cancer, and extended polyfucosyl LeX antigens are more specific for colon cancer tissues than are simple, monofucosyl LeX antigens. The present investigation compared the expression of simple and extended LeY antigens in a variety of malignant and nonmalignant human colonic tissues to gain insight into the normal distribution and cancer-associated expression of these antigens. Monoclonal antibody AH-6, which recognizes the LeY epitope irrespective of its carrier carbohydrate chain, stained the majority of specimens regardless of malignant potential or location within the colon. In contrast, CC-1 and CC-2 monoclonal antibodies, which recognize extended LeY structures, and KH-1, which is specific to trifucosyl LeY, preferentially stained malignant colonic tissues and rarely stained normal colonic mucosae. Mucosa immediately adjacent to cancer usually stained with AH-6 but not with KH-1, CC-1, or CC-2. Extended or trifucosyl LeY antigen expression was limited exclusively to premalignant (adenomatous) polyps and was invariably absent from nonpremalignant (hyperplastic) polyps. Moreover, among adenomatous polyps, extended LeY antigen expression tended to correlate with three parameters of malignant potential: larger polyp size; villous histology, and severe dysplasia. AH-6 failed to distinguish between hyperplastic and adenomatous polyps. In second-trimester fetal colonic mucosa, AH-6 bound to both proximal and distal segments whereas KH-1, CC-1, and CC-2 bound only to proximal segments. We conclude that in human colon, the LeY hapten is an oncodevelopmental cancer-associated antigen and extended LeY antigens are highly specific markers for malignancy and premalignancy.

An animal model for colon cancer metastasis: establishment and characterization of murine cell lines with enhanced liver-metastasizing ability.

A detailed understanding of the pathogenesis of colon cancer metastasis has been hindered by the lack of appropriate animal models which accurately reflect events in this complex process. An animal model for colon cancer metastasis is described in which spontaneously metastasizing colonic tumors are formed after injection of murine colon cancer cells into the cecal wall of BALB/c mice. Using this model, tumor cells with different liver-metastasizing potential were selected and shown to possess several properties known to be associated with other metastatic cell lines. The ability of tumor cells to invade a reconstituted basement membrane and to secrete type IV collagenase was directly proportional to their metastatic ability. In addition, liver-metastasizing cells preferentially migrated toward liver extracts in a Boyden chamber assay, as compared to extracts of brain or lung, and adhered rapidly to highly purified hepatic sinusoidal endothelial cells versus hepatic parenchymal cells in vitro. This model may thus be useful for studying many aspects of the pathogenesis of colon cancer metastasis.

Lewisx- and sialylated Lewisx-related antigen expression in human malignant and nonmalignant colonic tissues.

Biochemical studies have revealed that some normal cells express the LeX trisaccharide Gal beta 1----4(Fuc alpha 1----3)GlcNAc either on short-chain fucolipids or as a single immunodeterminant on glycolipid oligosaccharide side chains. Cancer cells, including those from colonic adenocarcinomas, express this antigen on longer type 2 blood group side chains as difucosylated or trifucosylated fucolipids. Moreover, sialylated forms of difucosylated LeX also accumulate in colon cancer but not in normal colonic mucosa. In the present study, six monoclonal antibodies which selectively recognize the various LeX-related antigens were used for immunohistochemical examination of these antigens in serial sections of human colonic tissue. All of these antigens were oncodevelopmental in human colon. Monoclonal antibodies anti-SSEA-1 and AH8-183, directed against short-chain, monofucosylated LeX, were unable to discriminate well between normal and malignant colonic tissue. However, the other four antibodies were much better at distinguishing cancer from normal tissue. FH6 was the most specific in that no normal tissues bound this antibody. However, FH6 failed to stain poorly differentiated cancers and some colloid-type carcinomas. FH4, which was also highly specific, stained almost all cancers, regardless of the degree of differentiation. FH4 primarily stained cancer cell cytoplasm, whereas the sialylated antigen defined by FH6 predominantly stained cell membranes. Differences were noted between the expression of LeX-related antigens in autopsied normal mucosa compared to mucosa of benign colonic diseases. Monoclonal antibodies recognizing long-chain polyfucosylated and sialylated LeX-related antigens appear to be useful tools for detection of colon cancer.

Comparison of T-antigen expression in normal, premalignant, and malignant human colonic tissue using lectin and antibody immunohistochemistry.

The Thomsen-Friedenreich antigen has been implicated as a cancer-associated antigen in some human organs including the colon. Most previous studies of Thomsen-Friedenreich antigen expression in the colon used peanut agglutinin (PNA) to identify the immunodeterminant in tissues. However, evidence from other organs suggests that anti-T antibodies have specificities which differ from those of peanut lectin. To elucidate the nature of the T-immunodeterminant in colonic mucosa, we compared staining by PNA to that of a polyclonal (PAb) and monoclonal (MAb) anti-T antibody. PNA demonstrated the best sensitivity (91%) in cancer tissues but the lowest specificity (68%) in normal mucosa. Staining with MAb was only 76% sensitive but 100% specific. Sensitivity and specificity of PAb were intermediate between PNA and MAb. MAb stained fewer adenomatous polyps than either PNA or PAb, but staining appeared to correlate with premalignant features of the polyps. PNA-binding sites were more prevalent than either PAb or MAb in hyperplastic polyps. Cell cytoplasm was stained by both antibodies more often than by PNA. The majority of fetal colonic specimens stained with all three reagents suggesting that Thomsen-Friedenreich antigen may be an oncodevelopmental antigen in human colon. Differences in staining patterns in some tissues may be due to different antigenic specificities among PNA, PAb, and MAb.

Expression of Tn, sialosyl-Tn, and T antigens in human colon cancer.

Mucin glycoproteins are major secretory products of the colon and contain O-linked oligosaccharides synthesized on a polypeptide backbone. The initial step in the synthesis of O-linked oligosaccharides is the addition of N-acetylgalactosamine to serine or threonine residues forming the Tn antigen. This substance can then receive additional carbohydrate residues such as sialic acid to form sialosyl-Tn antigen, or galactose to form T antigen. In the colon, the T antigen is an oncodevelopmental cancer-associated antigen but little is known about Tn and sialosyl-Tn expression. The present comparative immunohistochemical study was performed to analyze the expression of these antigens in fetal, normal adult, and malignant colorectal tissues with an aim toward elucidating whether Tn and sialosyl-Tn are also oncodevelopmental colon cancer-associated antigens and to gain insight into the earliest steps of mucin glycosylation in colonocytes. We used three reagents to detect Tn antigen (two monoclonal antibodies ETn1.01 and CU-1, and one lectin Vicia villosa), two reagents to detect sialosyl-Tn (monoclonal antibodies TKH2 and B72.3) and one to detect T antigen (monoclonal antibody AH9-16). Except for occasional reactivity with VVA and CU-1, cells of normal colonic mucosa did not express Tn, sialosyl-Tn, or T antigens. However, in the transitional mucosa immediately adjacent to cancer, all three antigens were expressed (ranging from 35 to 67% of cases depending upon the reagent). In colon cancers, the percentage of cases expressing each antigen were as follows: Tn 72-81%, sialosyl-Tn 93-96%, and T 71%. Unlike T antigen, which was preferentially expressed by moderately well- and well-differentiated adenocarcinomas, both Tn and sialosyl-Tn antigens were expressed by most histological subsets of colon cancers, including poorly differentiated adenocarcinomas and mucinous (colloid and signet ring cell type) carcinomas. The majority of cancers expressed both Tn and sialosyl-Tn, usually in association with T antigen. Only one cancer lacked all three antigens. Fetal colonic mucosal cells expressed all three antigens, particularly in goblet cell mucin. These results indicate that like T antigen, Tn and sialosyl-Tn are oncodevelopmental cancer-associated antigens in the colon. Moreover, Tn and sialosyl-Tn antigens appear to be useful markers of poorly differentiated adenocarcinomas and mucinous carcinomas: two histological subsets that often fail to express other cancer-associated antigens and that are often associated with a poor clinical outcome.(ABSTRACT TRUNCATED AT 400 WORDS)

Immune sera and monoclonal antibodies define two configurations for the sialyl Tn tumor antigen.

Sialyl Tn (sTn) is a mucin-associated carbohydrate antigen expressed in most types of human adenocarcinoma. Defining the configuration of tumor cell surface sTn recognized by antibodies is important for understanding the basis for the cancer cell specificity of sTn-reactive mAbs, for the development of more effective mAbs, and for designing cancer vaccines against sTn. In this study, we compared the immunogenicity of synthetic single sTn disaccharide epitopes and clusters [sTn(C)] of 3 sTn epitopes covalently linked via serine to keyhole limpet hemocyanin [KLH; sTn-KLH and sTn(C)-KLH, respectively]. The cell surface sTn configurations were analyzed with the use of sera from mice immunized with these neoglycoproteins and a panel of sTn-reactive mAb. Sera from mice immunized with sTn-KLH reacted in direct and inhibition assays with sTn-human serum albumin (HSA) but only weakly with sTn(C)-HSA, whereas sera from mice immunized with sTn(C)-KLH reacted with sTn(C)-HSA but not with sTn-HSA. Both anti-sTn and anti-sTn(C) sera reacted with ovine submaxillary mucin (a natural source of sTn) and with sTn-positive human tumor cell line LS-C but not with sTn-negative LS-B cells. With regard to the sTn-reactive mAbs, B72.3 reacted exclusively with clustered sTn, whereas mAb B195.3R11 reacted preferentially with unclustered sTn. Results with mAbs TKH2, B239.1, and CC49 were less clear, although all reacted more strongly with clustered sTn than with unclustered sTn. These results suggest that sTn is recognized at the tumor cell surface in at least two quite distinct configurations, as clustered and nonclustered arrays.

Resolution of factor X deficiency in primary amyloidosis following splenectomy.

A 57-year-old man with primary amyloidosis was initially seen with hematuria, cutaneous bleeding, and hepatosplenomegaly. Factor X was determined to be 10% to 16% of normal plasma values. Administration of vitamin K-dependent factor concentrate transiently improved in vitro clotting tests but did not alter the clinical course. Following a splenectomy, bleeding ceased and factor X levels returned to normal, remaining so despite discontinuation of factor concentrate infusion. Amyloid fibrils extracted from the patient's spleen were determined to be derived from lambda V1 light chains. The importance of splenectomy as an effective therapeutic modality is discussed.

Monoclonal antibody COL-1 reacts with restricted epitopes on carcinoembryonic antigen: an immunohistochemical study.

We used a monoclonal antibody, MAb COL-1, which recognized a restricted epitope on the carcinoembryonic antigen (CEA) molecule, to stain a wide variety of human normal and cancerous tissues. None of the 35 different types of normal tissue stained with COL-1. Of 59 types of benign and malignant tissues, COL-1 reacted with neoplasms of epithelial origin, especially the gastrointestinal tract, breast, lung, and bladder. In benign adenomatous colon polyps, villous adenomas were more frequently stained than tubular adenomas. Normal colon tissue from individuals without colon disease was unreactive, but very weak reactivity was noted in normal-appearing mucosa several centimeters remote from colon cancers. In contrast, another anti-CEA antibody with a less restricted epitope reacted frequently with both normal and remote colon mucosa. These results indicate that MAb COL-1 recognizes a restricted CEA epitope expressed only on pre-malignant or malignant cells and therefore may be a useful reagent for immunopathology.