Le Antigen Research Articles

Correlation between the number of apoptotic cells and expression of the apoptosis-related antigens Fas, Le(y) and bcl-2 protein in non-Hodgkin's lymphomas.

The relationship between the number of apoptotic cells and the expression of apoptosis-related antigens was examined in 56 cases of non-Hodgkin's lymphomas and in 10 cases of reactive hyperplastic lymph nodes (RHL). Apoptosis was visually quantified by the in situ end-labeling (ISEL) method, and the expression of Fas, Le(y) antigens and bcl-2 protein was examined by immunohistochemistry. The expression of Le(y) antigen was observed in germinal centers of RHL and 45% of non-Hodgkin's lymphomas. The apoptotic cell count (AC) in follicular lymphomas was significantly less than that in diffuse lymphomas. The distribution pattern of apoptotic cells in follicular lymphomas was inverse to that in RHL. In follicular lymphomas, AC was lower in follicles than in interfollicular areas. In contrast, AC was higher in follicles than in interfollicular areas in RHL. Le(y) antigen-positive lymphomas showed a significantly higher AC than the negative cases. The Fas antigen-positive lymphomas showed a higher AC than the negative cases. However, AC in bcl-2 protein-positive and negative cases was not significantly different. These results suggest that Ley and Fas antigens appear to be involved in the apoptotic tendency of tumor cells in non-Hodgkin's lymphomas, whereas bcl-2 does not necessarily.

Infants and young children with cystic fibrosis have high levels of serum sialyl Lewis(a) antigen.

Previous studies suggest that serum levels of the mucin-associated sialyl Lewis(a) (Le(a)) antigen (NeuAC alpha 2-3 Gal beta 1-3 [Fuc alpha 1-4]GlcNAc beta 1...) correlate with the pulmonary status of cystic fibrosis (CF) patients who have Le(a) or Lewis(b) blood types and can form the antigen. However, there is little information on serum sialyl Le(a) antigen levels in CF patients or normal children younger than 9 y. We measured serum antigen levels using the MAb 19-9 in normal term neonates, CF infants and young children, and infants and children who had bronchopulmonary dysplasia or asthma. The mean serum sialyl Le(a) antigen level of the CF patients was 46.7 U/mL, significantly above (p < 0.01) the mean levels of the three other groups. The mean serum sialyl Le(a) levels of the three non-CF groups were not significantly different from one another or from published normal values. We conclude that serum sialyl Le(a) antigen levels are elevated early in CF, but are normal in asthma and bronchopulmonary dysplasia patients.

Lewis X Structure Increases Cell Substratum Adhesion in L Cells

cDNAs of alpha-1,3-fucosyltransferase as well as alpha-1,3/4-fucosyltransferase were placed under the control of a beta-actin promoter and cytomegalovirus enhancer and were introduced into L cells. The transfected cells expressing Le(x) antigen showed increased cell substratum adhesion as compared to the antigen-negative cells, when they were cultured for 2 to 4 h in Dulbecco-modified minimum essential medium containing 0.05% bovine serum albumin. The increased cell substratum adhesion was completely inhibited by cycloheximide and anti-integrin antiserum, and partly by an RGD peptide and EGTA. These findings indicate that Le(x) structure promotes cell adhesion to substratum-bound material secreted by cells, and that the increased adhesion is mediated by integrin. Western blotting experiments have revealed an 85 kDa protein and a 50-60 kDa protein as carriers of Le(x) antigen in transfected cells. The latter is likely to be basigin, which is a member of the immunoglobulin superfamily and is considered to be an integrin-associated protein. We hypothesize that fucosylation of basigin enhances integrin-mediated cell substratum adhesion.

Evaluation of blood-group-related carbohydrate antigens in thyroid neoplasms

An immunohistochemical study using a panel of monoclonal antibodies (mAbs) against blood group A, B, H and Lewis antigens Le(a), Le(b), Le(x) and Le(y) and lectins, such as PNA, VVA-B4 and UEA -1, was carried out to investigate the aberrant expression of carbohydrate antigens in formalin-fixed, paraffin-embedded tissue sections of thyroid neoplasms. The tissues examined consisted of 26 papillary carcinomas, seven follicular adenomas, seven follicular carcinomas, one anaplastic carcinoma and one medullary carcinoma. MAbs against A, B and H antigens reacted strongly with papillary carcinomas from most of the individuals studied (22 of 26 individuals) and their reactivity corresponded well to the blood groups of individuals. In follicular adenomas and carcinomas, these mAbs reacted weakly with a small number of neoplastic cells from two of seven, and four of seven individuals, respectively. Positive staining with mAbs against Le(a) and Le(b) antigens was found in almost all papillary carcinomas. On the contrary, these mAbs rarely reacted with cells from follicular adenomas and carcinomas. T and Tn antigens which were recognized by PNA and VVA-B4, respectively, were only weakly expressed in a limited number of cells from the thyroid neoplasms. Normal cells adjacent to malignant cells were not stained by any of the stains used in this study. These results are discussed in light of our recent findings that polylactosamine structures produced in papillary carcinomas are quite different from those expressed in other types of neoplasms.

SIALYL-LEWIS-X ANTIGEN IMMUNOREACTION OF COLORECTAL-CANCER AND ITS RELATIONSHIP TO HEMATOGENOUS METASTASIS

The adhesion of cancer cells to endothelial cells of the target organ is one of the most important steps of hematogenous metastasis. Especially, sialyl Le(X) plays an important role in defining the metastasis. The expression of sialyl Le(X) antigen in colorectal cancer and its usefulness not only as an indicator of metastatic potential but also as a prognostic factor was studied immunohistochemically. Fifty-five (32.4%) sialyl Le(X) antigen-positive tumors were found in 170 colorectal cancers. There was a significant correlation between the expression of sialyl Le(X) antigen and the histological tumor type, venous invasion, lymph node metastasis, as well as liver metastasis. Hematogenous metastases were significantly more frequent in patients with sialyl Le(X)-positive tumor than in those with sialyl Le(X)-negative tumor; and prognosis was significantly poorer in the former. The results suggest that sialyl Le(X) antigen plays a role in hematogenous metastasis of colorectal cancer, and that the expression of sialyl Le(X) is associated with poor prognosis.

Type-1 chain histo-blood group antigens (Le(a), monosialosyl-Le(a), disialosyl-Le(a), Le(b), and H) in normal and malignant human endometrium.

Type-1 chain histo-blood group antigens such as the Lewis (Le)a, monosialosyl-Le(a), Le(b) and H antigens show an increased expression in endometrial carcinomas. However, the possibility that these antigens are expressed under genetic or hormonal influence in endometrial carcinomas has not been considered. In the present study, the expression of type-1 chain carbohydrate antigens in normal and malignant endometrium was evaluated by immunohistochemistry and related to both genetic and hormonal factors. The glands of normal, non-secretory endometria expressed, in contrast with surface epithelial cells, Le(a), Le(b), disialosyl-Le(a), and H determinants infrequently. Adenomatous hyperplasias and endometrial carcinomas showed an increased expression of type-1 chain carbohydrates that was qualitatively influenced by the erythrocyte Lewis phenotype and the secretor status. Whereas Le(a+b-) non-secretors mainly accumulated Le(a) antigen, and only limited amounts of Le(b) antigen, Le(a-b+) secretors expressed H, Le(b) and Le(a) antigens. The expression of type-1 chain antigens showed no association with the serum-oestrogen level or to the hormone-receptor status. Thus the Lewis secretor status has a qualitative influence on the increased expression of type-1 chain antigens, which, however, seem to be unrelated to hormonal factors. Our findings suggest an increased activity of the Se-gene-defined or a closely related fucosyl-transferase in neoplastic endometrial epithelial cells.

An immunohistochemical study of the distribution of blood group substances and related antigens in primary colorectal carcinomas and metastatic lymph node and liver lesions, using monoclonal antibodies against A, B, H type 2, Le(a), and Le(x) antigens.

Using indirect immunoperoxidase staining, we examined the distribution of blood group substances and related antigens, including Le(a), A, B, H type 2, and Le(x), in 87 carcinomas and 42 normal mucosal specimens of colon and rectum, as well as in metastatic lesions of 32 lymph nodes and 9 liver specimens. The compatible expression rate of A/B/H type 2 antigens was 33.3% (3/9) in proximal normal colon, but only 6.1% (2/33) in distal normal colon. Compatible expression was observed in 14 of 25 carcinomas in the proximal colon (56.0%), but these antigens in distal colon cancers reappeared with a high positive rate, 62.9% (39/62). The rate of H type 2 accumulation with the deletion of A and/or B antigens was 6.9% (6/87). Incompatible expression was not observed in colorectal cancer. Le(a) and Le(x) antigens were expressed in normal mucosa and primary carcinoma throughout the colon. Le(a) and Le(x) in primary carcinoma that showed A/B deletion with/without H type 2 accumulation was expressed more than in carcinoma with A/B/H type 2 compatible expression (88.2% vs 71.7% for Le(a); 94.1% vs 88.7% for Le(x), respectively). In 16 of 32 patients (50.0%), A/B/H type 2 antigen expression in metastatic lesions had disappeared or was decreased compared with that in primary carcinoma, followed by metastasis to regional lymph nodes. These results suggested that: (a) A/B/H type 2 blood group substances in the distal colon behave as tumor-associated antigens in colorectal carcinoma. (b) Most frequently, A/B/H type 2 antigens expressed in primary carcinoma were weakened or had disappeared in metastatic lymph nodes. Further investigation of the biological function of carbohydrate chains, such as those of blood group substances and related antigens on cancer cell surfaces may lead to a solution of the problem of the metastatic behavior of tumor cells.

Expression of type-2 histo-blood group carbohydrate antigens (Le(x), Le(y), and H) in normal and malignant human endometrium.

Changes in expression of histo-blood group ABH and Lewis antigens are common alterations in carcinomas. Using immunohistochemistry, we have evaluated the expression of type-2 histo-blood group antigens in normal and malignant endometrial tissues in relation to genetic and hormonal factors. The Le(x), sialosyl-Le(x), and Le(y) antigens were inconstantly expressed in the normal endometrium. The expression was uninfluenced by the secretor status but was related to the ABO blood group status in Oestradiol (E2) stimulated endometria. Le(y) was expressed most frequently in proliferating endometria from blood group 0 individuals. Le(x) and Le(y) were maximally expressed in atrophic endometria, and Le(x) and Le(y) staining scores correlated inversely with serum levels of E2 in normal, non-secretory endometria. No correlation was found in adenomatous hyperplasias and endometrial carcinomas, which when compared with atrophic endometria, showed a loss of Le(x) and Le(y) and an increased H-carbohydrate expression at apical membranes. Carcinomas from non-secretors showed lower expression of Le(y) and H-antigens than carcinomas from secretors. Our findings suggest that the genetic and hormonal influence on glycosylation based on type-2 chain carbohydrates differ between normal and malignant endometrium. This difference is probably related to specific tumour-associated qualitative and quantitative changes in the fucosyltransferases.

Different sialyltransferase activities in human colorectal carcinoma cells from surgical specimens detected by specific glycoprotein and glycolipid acceptors.

The amount and type of sialylation of tumor cell membranes depends on the activity of a number of different sialyltransferase enzymes. For the detection of specific activities in human colorectal carcinoma tissue several glycoprotein and glycolipid acceptors were used: desialylated fetuin, alpha 1-acid glycoprotein, beta 2-glycoprotein I, ovine submaxillaris mucin, and the gangliosides GM1, GM2, GM3 and GD1a. Because of their possible relevance for metastasis, precursors of Le(a) and Le(x) antigens, too, were employed, namely neoglycolipids produced by coupling LcOse4 or NeoLcOse4 oligosaccharides to L-alpha-phosphatidyl-ethanol-amine-dipalmitoyl. Our data indicate that human colorectal tumor tissue contains two highly active sialyltransferase enzymes, which are only weakly expressed in normal mucosa. These are a N-glycan-specific alpha 2,6-sialyltransferase, which was significantly increased in metastasizing tumors, and a Gal beta 1,3Gal-NAc-specific sialyltransferase, which was increased in tumors of early stages. A shift to enhanced alpha 2,6-sialylation of membrane glycoproteins during carcinogenesis was demonstrated by lectin ELISA analysis of magneto-bead separated tumor cells. Quantitative determination of specific sialyltransferase activities may be a sensitive tool for detection and monitoring of colon carcinoma.

An immunohistochemical employer monoclonal antibodies against Le(a), sialyl Le(a), Le(x), and sialyl Le(x) antigens in primary colorectal, carcinomas and lymph node and hepatic lesions.

The immunohistochemical expression of sialylated and non-sialylated forms of both Le(x) and Le(a) were studied in 87 carcinomas and 42 normal mucosal specimens of colon and rectum, as well as in 32 metastatic lymph nodes and 9 hepatic lesions, using an indirect immunoperoxidase staining. Their antigens were expressed in normal mucosa with the following frequencies: Le(a), 95.2% (40/42); sialyl Le(a), 88.1% (37/42); Le(x), 95.2% (40/42); and sialyl Le(x), 17.0% (7/42), whereas in carcinomas, the respective rate of frequency were: 78.2% (68/87); 78.2% (68/87); 90.8% (79/87); and 93.1% (81/87). Sialyl Le(x) antigen showed the highest tumor specificity compared to other antigens. In three normal mucosal specimens and four carcinomas with Le(a-b-) phenotype, the expression of type 1 antigens (Le(a) and sialyl Le(a)) was not consistent, whereas type 2 antigens (Le(x) and sialyl Le(x)) were consistently observed in carcinomas. The staining of type 1 antigens and Le(x) was decreased in metastatic lesions compared with primary carcinomas, whereas sialyl Le(x) antigen had the same positive-staining rate in both. Metastatic carcinoma expressed the sialylated form more predominantly than the non-sialylated form in type 2 antigens whereas the opposite result was observed in type 1 antigens. These results suggested that: (a) sialyl Le(x), defined by monoclonal antibody CSLEX1, may be useful as a tumor-associated antigen in colorectal carcinoma, and (b) the alteration of Lewis-related carbohydrate antigens in cancer cell membranes, including sialylation and/or aberrant glycosylation, may be related to metastatic behavior.

Native and sialic acid masked Lewis(a) antigen reactivity in medullary thyroid carcinoma. Distinct tumour-associated and prognostic relevant antigens.

Forty-six medullary thyroid carcinomas (MTC) were subjected to a qualitative and quantitative characterization of native and sialic acid masked Lewis(a) (Le(a)) antigens. Immunohistochemical investigations included monoclonal antibodies (MABs) directed against alpha(2,3)-sialyl-Le(a), i.e. CA19-9 (MAB 19-9), native Le(a) (MAB anti Le(a)) and alpha(2,3)sialyl type 1 structure, i.e. CA 50 (MAB C50). To detect sialic acid masked Le(a) reactivity, MAB anti-Le(a) was also applied to native and enzymatically desialylated tissue sections with and without masking of sialic acid residues by sialic acid and sequence specific lectins. Only 7 MTC (15%) displayed a weak expression of CA19-9, while 16 (33%) showed moderate positive staining for native Le(a). Twenty-seven tumours exhibited a strong staining by the N'ase MAB anti Le(a) staining sequence. The latter could most effectively be inhibited by the simultaneous masking of alpha(2,3)-and alpha-(2,6)-linked sialic acid residues due to the competitive binding of sialic acid and sequence specific lectins: Maackia amurensis agglutinin (specific alpha(2,3)-linked sialic acid) and Sambucus nigra agglutinin (specific alpha(2,6)-linked sialic acid). Thus, in MTC the major portion of sialic acid masked Le(a) antigen reactivity is different from that detected by the MAB 19-9. The antigen reactivity is probably due to Le(a) structures containing both alpha(2,3) and alpha(2,6)-linked sialic acid residues. A highly significant correlation between the expression of CA50 and that detected by the N'ase MAB anti-Le(a) staining sequence indicates that the alpha(2,3)-sialyl type 1 chain represents a common intermediate structure within the pathway of the biosynthesis of sialylated Le(a) antigens, excluding the formation of CA19-9 via the formation of the disialyl type 1 structure. This is subsequently fucosylated to the corresponding sialic acid masked Le(a). Preliminary clinicopathological studies indicate that the sialic acid masked Le(a) antigens detected by the N'ase MAB anti-Le(a) staining sequence are related to biologically aggressive MTC.

Detection of a novel lung adenocarcinoma — Associated serum antigen defined by two monoclonal antibodies, KM 432 and KM 227 : Shitara K, Hanai N, Yoshida H. Division of Immunology, Tokyo Research Laboratories, Kyowa Hakko Kogyo Co Ltd, Tokyo 194. Anticancer Res 1993;13:579–86

Two monoclonal antibodies, KM227 (IgM) and KM432 (IgG1), were established by immunizing mice with the crude membrane fraction of lung squamous cell carcinoma and the high-molecular-weight antigen in pleural perfusion from patients with lung adenocarcinoma, respectively. They were found to recognize the carbohydrate epitope and the peptide epitope on the antigens, respectively. Both KM227 and KM432 bound to the serum antigens associated with lung adenocarcinoma and lung benign diseases. A double determinant assay in which KM432 was used as 1st antibody and biotinylated KM227 was used as 2nd antibody selectively detected the antigen, designated KA432/227, elevated in sera from patients with lung adenocarcinoma. The molecular weight of KA432/227 in pleural perfusion from patients with lung adenocarcinoma was estimated to be more than 900 kD by the gel filtration chromatography. Serum with a high KA432/227 level was most frequently found in lung adenocarcinoma (47.8%), and occasionally found in lung squamous cell carcinoma (23.1%), lung large cell carcinoma (22.2%), pancreatic carcinoma (20.0%) and carcinoma of the bile-duct and the gall bladder (25.0%). In contrast, positive percentages in benign diseases and healthy adults were very low (0 to 7.7%). No significant correlation was found between the serum level of KA432/227 and other lung adenocarcinoma associated antigens, such as sialyl Le(x). Combined detection of KA432/227 and sialyl Le(x) antigen elevated the positive percentages in lung adenocarcinoma (67.2%).

Elevated Levels of Serum Mucin-associated Antigen in Adult Patients with Cystic Fibrosis

Previous studies using the CA 19-9 antibody have demonstrated that serum mucin levels in patients with cystic fibrosis (CF) are elevated and that the degree of elevation relates to the age of the patient and possibly to his or her clinical status. However, CA 19-9 only recognizes the mucin-associated blood group sialyl Le(a+) antigen, so mucin levels cannot be measured in patients without Lewis antigens. The present study used the 17B1 monoclonal antibody to measure serum mucin levels in normal subjects, and in patients with CF, patients with chronic obstructive pulmonary disease (COPD), and patients with lung transplants. Serum mucin levels were 25 ng/ml (+/- 1 SEM, n = 8) in normal subjects, 13,853 ng/ml (+/- 1,281, n = 25) in patients with CF, and 25.5 ng/ml (+/- 1.9, n = 17) in patients with COPD. Patients with CF who were sialyl Le(a-b-) also had elevated serum mucin levels (715 +/- 152, n = 2). Serum mucin levels of six lung transplant recipients with CF were elevated compared with those in normal subjects (4,621 +/- 765 ng/ml), but they were not different from serum mucin levels in six lung transplant recipients without CF (5,307 +/- 1.677 ng/ml). Preliminary characterization of the serum mucin antigen showed that: (1) in CF sera, the antigen is polydisperse and smaller than the antigen in normal sera; (2) the mucin antigen is distinct from ABO blood group antigens. Serum mucin levels may be a useful marker to follow a specific patient's response to therapy.

Enhancement of retroviral infectionin vitroby anti-LeyIgG: reversal by humanization of monoclonal mouse antibody

Monoclonal mouse IgG3 antibody (ABL 364) against the carbohydrate Le(y) antigen enhanced infection in vitro with HTLV-1 and with HIV-1 when propagated in both transformed and normal lymphocytes. Enhancement was independent of complement, occurred with both lymphocytes and monocytes as target cells, and did not use either L(ey) epitopes on target cells for cross-linkage of virus to the cell or the Fc part of the antibody as a ligand for any cellular receptor. For enhancement to occur, binding of anti-Le(y) antibody to virus was required to take place before virus binding to its specific receptor with no indication of any alternative pathway of infection, as evidenced by abrogation of enhancement by anti-CD4 MAb or soluble recombinant CD4, and also the inability of anti-Le(y) MAb to mediate HIV infection of HSB-2 cells in which HTLV-1/HIV pseudovirus infection was enhanced. While F(ab)2 fragments of ABL 364 also enhanced infection, a human/mouse chimeric antibody and a fully humanized antibody had no enhancing effect on free virus infection. We suggest that binding of anti-Le(y) ABL 364 or its F(ab)2 fragment induced a conformational change in the gp120 oligomers facilitating the process of infection, and that this function was abrogated by the IgG1 Fc of the chimeric and the humanized antibodies. The observations indicate that the non-paratope domains of antiviral antibodies can influence their function as neutralizing or enhancing for infection.

Synergetic effect of interleukin-2 and cellular cytotoxicity against a novel tumor-associated carbohydrate antigen Le(a)/Le(a) (dimeric Le(a)) mediated by monoclonal antibody NCC-ST-421 in adoptive immunization using SCID mice.

The murine IgG3 monoclonal antibody NCC-ST-421, raised against a human gastric cancer, shows strong reactivity with dimeric Le(a) (Le(a)/Le(a); V4FucIII4FucLc6Cer) expressed on gastrointestinal cancer cells. ST-421 reacted minimally with non-dimeric or simple Le(a) expressed on normal tissues. ST-421 is capable of mediating both antibody-dependent cellular cytotoxicity (ADCC) with human peripheral blood lymphocytes, and complement-dependent cytotoxicity with human complement. Interleukin-2 (IL-2) modulates the function of immunocytes, in particular inducing lymphokine-activated killer (LAK) cell activity and enhancing ADCC. We therefore employed combination immunotherapy with IL-2, LAK, and ST-421-induced ADCC in vitro and in mice with severe combined immunodeficiency (SCID), using target tumor cells expressing Le(a)/Le(a) antigen. ADCC against human colon cancer cell lines in vitro was enhanced three to four times after preincubation with IL-2. Addition of IL-2 reduced the amount of ST-421 required for efficient ADCC 10- to 100-fold. ADCC was activated by IL-2 earlier (1 day) than the generation of LAK cells (3-4 days), and at lower concentration of IL-2. These effects were specific for ST-421, as demonstrated by experiments with irrelevant antibody or irrelevant target cells. An anti-(Fc receptor) antibody blocked the ADCC but not the LAK activity in vitro. The enhancement of ADCC by IL-2 may be caused by activation of effector cells expressing Fc receptors. In vivo experiments using SCID mice inoculated with human colon cancer showed a significant tumor-growth-suppressive effect after combined therapy using human peripheral blood lymphocytes, LAK, IL-2, and ST-421. In summary, adoptive immunization with human lymphocytes activated by IL-2 and ST-421 effectively suppressed growth of gastrointestinal cancer cells expressing Le(a)/Le(a).

Histochemical analysis of blood group antigens in human sublingual glands and pancreas. An application of high-performance liquid chromatography to estimate the quantity of galactose liberated from tissue sections by alpha-galactosidase digestion.

Using immunostaining with monoclonal antibodies (mAbs) from three different sources as well as anti-B lectin, GSAI-B4 staining and alpha-galactosidase digestion, blood group B antigens were localized and analysed in tissue sections of sublingual glands from blood group B and AB individuals. Quantitative analysis of galactose was simultaneously carried out on the supernatant enzyme solution used for treating tissue sections by high-performance liquid chromatography (HPLC). In addition, galactose liberated from the pancreas tissues of blood group B and AB individuals was also estimated by HPLC analysis in order to compare the content of antigens. mAb-B(H079) and GSAI-B4 reacted uniformly with the mucous cells from blood group B and AB secretors. On the other hand, other mAbs-B(B006 and A582) recognized the antigen in a limited number of cells or was even negative in some cases of blood group AB individuals. Only mAb-B(H079) recognized the B antigens in mucous cells from non-secretors. Digestion with alpha-galactosidase resulted in the consistent appearance of H and Le(b) antigens in the mucous cells of all the secretors examined, although the reduction of staining intensity with anti-B reagents was not so marked. Le(y) antigens also appeared in some cases after the enzyme digestion. In non-secretors, Le(b) and Le(y) antigens, but not H antigens, appeared in some mucous cells following enzyme digestion. HPLC analysis of galactose revealed that alpha-galactosidase can specifically liberate the terminal galactose residues of B antigens, and no marked difference was present in the content of liberated galactose from mucous cells of sublingual glands among the individuals investigated (8.5-11.7 nmoles cm-2). No galactose was detected in samples from the sublingual glands of non-secretors, and only a trace amount of galactose was detected in the samples from pancreas tissues. These results suggest that the observed difference in the reactivity of different reagents with each tissue site can be ascribed to both quantitative and qualitative heterogeneity of B antigens.

Human Lewis alpha 1-->3/4fucosyltransferase: specificity of fucose transfer to GlcNAc beta 1-->3Gal beta 1-->4Glc beta 1-->1Cer (LcOse3Cer).

Biosynthesis of the Lex series of carbohydrate antigens proceeds by fucose transfer in alpha 1-->3-linkage to the pen-ultimate GlcNAc residue of a neolacto-series oligosaccharide acceptor, a reaction catalysed by multiple enzymes expressed in human tissues. Particularly broad acceptor specificity, including the ability to catalyse fucose transfer to both lacto- and neolacto-series acceptors as well as the precursor Lc3 structure (where Lc3, lactotriaosylceramide, is GlcNAc beta 1-->3Gal beta 1-->4Glc beta 1-->1Cer), exists for one human fucosyltransferase form, the Lewis alpha 1-->3/4fucosyltransferase (FucT-III). To determine if fucose transfer to Lc3 may represent an alternate early step in Le(x) or Le(a) antigen biosynthesis with this enzyme, the chemical structure of the fucosylated Lc3 reaction product formed by the Lewis alpha 1-->3/4fucosyltransferase from Colo 205 cells has been defined. Transfer of [14C]fucose to Lc3 yielded a labelled product migrating as a tetrasaccharide on thin layer chromatography plates. This product remained an acceptor for both beta 1-->3- and beta 1-->4-galactosyl transfer on the terminal GlcNAc residue. The product was degraded to a fucosylated trisaccharide derivative by bovine kidney beta-N-acetylglucosaminidase. Fast atom bombardment mass spectrometry and methylation analysis confirmed that the product was composed exclusively of the following structure containing a fucose linked to the 3-position of the internal Glc residue: [formula; see text] Such a structure does not represent an intermediate in Le(x) or Le(a) antigen biosynthesis. Thus, the evidence suggests that Le(x) or Le(a) antigen synthesis results exclusively from fucosylation of complete core chains.

The distribution of type 1 chain ABH and related histo-blood group antigens in normal cycling human endometrium.

Cycling endometrial tissue was examined immunohistochemically for blood group antigens using a panel of monoclonal antibodies with specificity to type 1 chain carbohydrates. Staining was evaluated against the genetic background (ABO, Lewis, and saliva-secretor status) and the subphase of the menstrual cycle. Expression of type 1 chain antigens in most cases correlated with the genetic background; however, Le(a) and Le(b) antigens were in a few cases demonstrated in endometria with the erythrocyte Le(a-b-) phenotype and Le(b) antigen in erythrocyte Le(a + b-) endometria also. In addition, Le(b) antigen was preferentially expressed in endometria from blood group O individuals. Type 1 chain antigens were, in general, maximally demonstrated in the surface epithelium. Chain 1 A, H, ALe(b), Le(a), and disialosyl-Le(a) (dsLe(a)) determinants were demonstrated only sporadically in functionalis and basalis glands of cycling endometria. Staining for most type 1 chain antigens showed variations that were related to the histomorphology (layer and menstrual phase), and monosialosyl-Le(a) (msLe(a)) seemed to be a marker of secretory differentiation in the endometrium. Our results support the view that synthesis and expression of type 1 chain antigens in human endometrium is influenced by the genetic background and is modulated by the hormonal environment.

Immunohistochemical detection of sialyl Le(x) antigen on mucosal Langerhans cells of human oral mucosa following neuraminidase pretreatment.

Histochemical assessment of selected carbohydrate sequences on Langerhans cells of human oral mucosa was made by combined use of enzyme digestion and immunostaining with monoclonal antibodies against specific carbohydrate structures. In both frozen sections and epithelial sheets without the enzyme pretreatment, mucosal Langerhans cells, identified by positive staining with anti-CD1a and HLA-DR antibodies, did not express any carbohydrate antigens on their surface. In contrast, following neuraminidase pretreatment of both types of material, the fucosylated type 2 chain (Le(x)) became detectable on Langerhans cells, indicating that sialic acid is the terminal residue of this sequence. Other enzymes were ineffective in this apparent unmasking, and the staining patterns of the other related carbohydrate sequences (Le(y)+, Le(a), Le(b)) remained unaffected by pretreatment with any of the enzymes used. These findings suggest that the mucosal Langerhans cells possess a unique carbohydrate chain, the sialyl fucosylated type 2 sequence (sialyl Le(x) antigen).

Le(y) antigen expression is correlated with apoptosis (programmed cell death).

Apoptosis (programmed cell death) is a basic physiological process which determines specific patterns of tissue size and shape, and balance of cell number, during morphogenesis, and seems to play an integral role in oncogenic progression. Since dramatic changes of cellular glycosylation pattern are well known to be closely correlated with differentiation, development and oncogenesis, it is likely that similar specific changes are associated with apoptosis. However, this possibility has not been systematically investigated. We therefore carried out histological studies of many tumours and normal tissues for which a high incidence of apoptosis is believed to occur. Sections were stained with monoclonal antibodies (MoAbs) directed to carbohydrate antigens Le(y) and Le(x), proliferating cellular nuclear antigen (PCNA) and Fas (previously claimed to be an apoptosis-inducing antigen). Antibody staining patterns were compared with morphological cell characteristics as revealed by haematoxylin/eosin staining, and DNA fragmentation patterns (a marker of apoptosis) as revealed by 3'-OH nick-end labelling technique. We found that expression of Le(y) (defined by MoAb BM1) is closely correlated with the process of apoptosis, but not with cell proliferation or necrosis. Within Le(y)-positive areas of tissue sections, typical apoptotic morphological changes and DNA fragmentation (as revealed by positive nick-end labelling) were frequently observed in certain loci, although not all Le(y)-positive cells showed such signs of apoptosis. Le(y)-positive areas showed consistent negative staining by MoAb directed to PCNA and negative or weak staining by MoAb directed to Fas antigen, regardless of tissue source. No such trends were observed for Le(x) glycosylation. We conclude that Le(y) expression is a useful phenotypic marker predictive of apoptosis, i.e. some (although not all) Le(y)-positive cells subsequently become apoptotic.