Tissue distribution of 2-3 and 2-6 sialyl Lewis A antigens and significance of the ratio of two antigens for the differential diagnosis of malignant and benign disorders of the digestive tract

The authors investigated the tissue distribution of two kinds of sialylated derivatives of Lewis A (Le(a)) antigen in patients with cancers of the digestive system using specific monoclonal antibodies, and evaluated the significance of determining the 2-3 and 2-6 sialylated Le(a) antigen levels for the diagnosis of cancer. In most specimens from patients with cancers of the pancreas, biliary tract, stomach, and colon, the 2-3 sialylated Le(a) antigen was strongly expressed in cancer cells. However, 2-6 sialylated Le(a) antigen was less frequently expressed in cancer cells. The former is therefore more specific to cancer than the latter. Also, the serum level of the 2-3 sialylated Le(a) antigen was significantly higher than that of the 2-6 counterpart in patients with cancers of pancreas, biliary tract, stomach, and colon. The resulting ratio of serum 2-3/2-6 sialylated Le(a) antigens was frequently high in patients with malignancy and was low in patients with benign disorders of these digestive organs. Therefore, the 2-3/2-6 sialylated Le(a) antigen ratio is a useful for the differential diagnosis of malignant disorders in these organs. However, liver disorders were found to be exceptional in that both antigens were mostly absent in hepatocellular carcinoma (HCC) cells in immunohistologic examination, as well as in nonmalignant parenchymal liver cells. Only the epithelial cells of the intrahepatic bile ducts expressed the 2-6 sialylated Le(a) antigen strongly, and expressed the 2-3 sialylated Le(a) antigen moderately. The levels of both antigens were sometimes high in patients with liver disorders, but the ratio always remained low in patients with HCC as well as benign liver disorders such as cirrhosis or chronic hepatitis. The sialylated Le(a) antigens, which sometimes accumulate in the sera of patients with HCC, were concluded to originate from the epithelial cells of the proliferating small bile ducts, and those serum antigens cannot be considered as evidence for the presence of liver cancer cells.     

Hypoxic culture induces expression of sialin, a sialic acid transporter, and cancer-associated gangliosides containing non-human sialic acid on human cancer cells

Tumor hypoxia figures heavily in malignant progression by altering the intracellular glucose metabolism and inducing angiogenic factor production, thus, selecting and expanding more aggressive cancer cell clones. Little is known, however, regarding hypoxia-induced antigenic changes in cancers. We investigated the expression of N-glycolyl sialic acid (NeuGc)-G(M2), a cancer-associated ganglioside containing non-human sialic acid, NeuGc, in human cancers. Cancer tissues prepared from patients with colon cancers frequently expressed NeuGc-G(M2), whereas it was virtually absent in nonmalignant colonic epithelia. Studies on cultured cancer cells indicated that the non-human sialic acid was incorporated from culture medium. Hypoxic culture markedly induced mRNA for a sialic acid transporter, sialin, and this accompanied enhanced incorporation of NeuGc as well as N-acetyl sialic acid. Transfection of cells with sialin gene conferred accelerated sialic acid transport and induced cell surface expression of NeuGc-G(M2). We propose that the preferential expression of NeuGc-G(M2) in cancers is closely associated with tumor hypoxia. Hypoxic culture of tumor cells induces expression of the sialic acid transporter, and enhances the incorporation of non-human sialic acid from the external milieu. A consequence of this is the acquisition of cancer-associated cell surface gangliosides, typically G(M2), containing non-human sialic acid (NeuGc), which is not endogenously synthesized through CMP-N-acetyl sialic acid hydroxylase because humans lack the gene for the synthetic enzyme. As hypoxia is associated with diminished response to radiotherapy and chemotherapy, NeuGc-G(M2) is a potential therapeutic target for hypoxic cancer cells.     

6-Sulfo LacNAc,a novel carbohydrate modification of PSGL-1, defines an inflammatory type of human dendritic cells

The monoclonal antibody M-DC8 defines a major subset of human blood dendritic cells (DCs). Here we identify the M-DC8 structure as 6-sulfo LacNAc, a novel carbohydrate modification of the P selectin glycoprotein ligand 1 (PSGL-1). In contrast to previously described blood DCs, M-DC8+ DCs lack the cutaneous lymphocyte antigen (CLA) on PSGL-1 and fail to bind P and E selectin. Yet they express anaphylatoxin receptors (C5aR and C3aR) and the Fcgamma receptor III (CD16), which recruit cells to inflammatory sites. While sharing with DC1 the expression of myeloid markers and a potent capacity to prime T cells in vitro, M-DC8+ DCs produce far more TNF-alpha in response to the bacterial endotoxin lipopolysaccharide (LPS). Thus, 6-sulfo LacNAc-expressing DCs appear as a novel proinflammatory DC subset.     

Rejuvenation of antioxidant system in central nervous system of aged rats by grape seed extract

Oxidative stress is considered as a major risk factor that contributes to age-related increase in lipid peroxidation and declined antioxidants in the central nervous system during aging. Grape seed extract, one of the bioflavonoid, is widely used for its medicinal properties. In the present study, we evaluated the role of grape seed extract on lipid peroxidation and antioxidant status in discrete regions of the central nervous system of young and aged rats. Male albino rats of Wistar strain were divided into four groups: Group I-control young rats, Group II-young rats treated with grape seed extract (100 mg/kg body weight) for 30 days, Group III-aged control rats and Group IV-aged rats supplemented with grape seed extract (100 mg/kg body weight) for 30 days. Age-associated increase in lipid peroxidation was observed in the spinal cord, cerebral cortex, striatum and the hippocampus regions of aged rats (Group III). Activities of antioxidant enzymes like superoxide dismutase, catalase, glutathione peroxidase and levels of non-enzymic antioxidants like reduced glutathione, Vitamin C and Vitamin E were found to be significantly decreased in all the brain regions studied in aged rats when compared to young rats. However, normalized lipid peroxidation and antioxidant defenses were reported in the grape seed extract-supplemented aged rats. These findings demonstrated that grape seed extract enhanced the antioxidant status and decreased the incidence of free radical-induced lipid peroxidation in the central nervous system of aged rats.     

A role for SHPS-1/SIRPalpha in Concanavalin A-dependent production of MMP-9

SHPS-1/SIRPalpha1 is a transmembrane glycoprotein that belongs to the immunoglobulin (Ig) super family. In the present study, we show that SHPS-1 strongly associates with Concanavalin A (Con A), a plant lectin obtained from jack beans. Further studies with SHPS-1 mutants reveal that the extracellular domain of SHPS-1 containing the Ig sequence is responsible for its association with Con A. Con A treatment induces cross-linking and multimerization of the SHPS-1 protein in the plasma membrane, accompanied by its tyrosine phosphorylation and recruitment of SHP-2. In contrast, Ricinus communis agglutinin (RCA), another lectin obtained from castor bean, does not bind or activate tyrosine phosphorylation of SHPS-1. Moreover, Con A activates Akt in a SHP-2-dependent manner. Treatment of mouse embryonic fibroblasts (MEFs) with Con A induces secretion of matrix metalloproteinase (MMP)-9, a phenomenon that is inhibited in cells expressing YF mutant of SHPS-1, a dominant negative form of Akt or in cells pre-treated with an Akt inhibitor, LY294002 or extracellular-signal regulated kinase (Erk) inhibitor, U0126. In addition, expression of the YF mutant of SHPS-1 inhibits Con A-dependent activation of Akt and Erk kinases. Taken together, our results suggest that SHPS-1 is a receptor for Con A that mediates Con A-dependent MMP-9 secretion through SHP-2-promoted activation of both Akt and Erk pathways.     

Carbohydrate antigen profiles of human erythroleukemia cell lines HEL and K562

The expression of major carbohydrate antigens carried by polylactosaminyl chains in human erythroleukemia cell lines, K562 and HEL, was investigated by applying monoclonal antibodies recognizing specific carbohydrate determinants. The two cell lines showed common differences in their glycolipid compositions: (1) the presence of significant amounts of ganglio-series glycolipids, which are absent in normal erythrocytes; and (2) a remarkable reduction in the amount of globo-series glycolipids, which are the major glycolipids in normal human erythrocytes. A variety of differences were also detected in the carbohydrate antigens carried by lacto-series glycolipids and glycoproteins having related carbohydrate chains. K562 cells were i+H- X+, with a minor population of I+ cells. HEL cells were I-i+H+X-. The presence of the I+ population in K562 cells is particularly noteworthy, since I-antigen is characteristic of adult mature erythrocytes and is absent in most human leukemic cell lines. Several clones showing I+, I+/-i+/-, or I-i+ specificities were isolated from K562 cells by cloning in either methylcellulose media or limiting dilution, and I+ and I- cells were sorted by FACS fluorometer. HEL cells and these K562 clones provide a useful experimental model for studying the biologic significance and enzymatic control in expression of cell surface polylactosamines.     

Generation of two murine monoclonal antibodies that can discriminate N-glycolyl and N-acetyl neuraminic acid residues of GM2 gangliosides

Since the preliminary analyses of the glycolipids of small cell carcinomas of the lung showed an increase of GM2 ganglioside, we generated new murine monoclonal antibodies directed to GM2 to identify the molecular species of the glycolipid. The monoclonal antibodies MK2-34 and MK1-16 (both IgM), which specifically detect N-glycolyl GM2 and N-acetyl GM2, respectively, were generated by immunizing mice with liposomes containing monophosphoryl lipid A, trehalose dimycolate, and the antigenic ganglioside. Among the glycolipid preparations extracted from the cancer tissues of 39 patients with lung cancer, a significant amount of N-acetyl GM2 was detected with MK1-16 antibody in 70% of the squamous cell carcinoma cases, 50% of the lung adenocarcinoma cases, 33% of the large cell carcinoma cases, and 100% of the cases of small cell carcinoma of the lung. On the other hand, N-glycolyl GM2 which was defined by the monoclonal MK2-34 was not found in any of the glycolipid fractions prepared from the lung cancer tissues examined in this study. Immunohistochemical studies of the lung cancer tissues with the MK1-16 antibody showed that the N-acetyl GM2 was present not only in small cell carcinoma tissues as one of the antigens related to tumors of neuroectodermal origin, but also in the squamous cell carcinoma and adenocarcinoma of the lung with a comparable frequency. The appearance of the N-acetyl GM2 antigen correlated well with the degree of differentiation of the cancer cells in patients with squamous cell carcinoma and adenocarcinoma of the lung.