Sialyl Lewis(x)-mediated, PSGL-1-independent rolling adhesion on P-selectin.
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Selectin-mediated cell adhesion is an essential component of the inflammatory response. In an attempt to unambiguously identify molecular features of ligands that are necessary to support rolling adhesion on P-selectin, we have used a reconstituted ("cell-free") system in which ligand-coated beads are perfused over soluble P-selectin surfaces. We find that beads coated with the saccharides sialyl Lewis(x) (sLe(x)), sialyl Lewis(a) (sLe(a)), and sulfated Lewis(x) (HSO(3)Le(x) support rolling adhesion on P-selectin surfaces. Although it has been suggested that glycosylation and sulfation of P-selectin glycoprotein ligand-1 (PSGL-1) is required for high-affinity binding and rolling on P-selectin, our findings indicate that sulfation of N-terminal tyrosine residues is not required for binding or rolling. However, beads coated with a tyrosine-sulfated, sLe(x)-modified, PSGL-1-Fc chimera support slower rolling on P-selectin than beads coated with sLe(x) alone, suggesting that sulfation improves rolling adhesion by modulating binding to P-selectin. In addition, we find it is not necessary that P-selectin carbohydrate ligands be multivalent for robust rolling to occur. Our results demonstrate that beads coated with monovalent sLe(x), exhibiting a more sparse distribution of carbohydrate than a similar amount of the multivalent form, are sufficient to yield rolling adhesion. The relative abilities of various ligands to support rolling on P-selectin are quantitatively examined among themselves and in comparison to human neutrophils. Using stop-time distributions, rolling dynamics at video frame rate resolution, and the average and variance of the rolling velocity, we find that P-selectin ligands display the following quantitative trend, in order of decreasing ability to support rolling adhesion on P-selectin: PSGL-1-Fc > sLe(a) approximately sLe(x) > HSO(3)Le(x). (+info)
Detection and isolation of MUC1 mucin from larynx squamous cell carcinoma.
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The progression from uncontrolled cell proliferation to invasion and metastasis of epithelial tumors is partially understood. Alteration of epithelial mucin expression have been described in different malignant localizations but only few attempts have been made to identify mucin expression in malignant laryngeal tumors. In the present report, results are shown of studies on the expression of mucins and carbohydrate related antigens in laryngeal cancer and on the isolation of MUC1 mucin from this tumor tissue. Malignant laryngeal specimens were processed for immunohistochemical analysis and for extranuclear membrane fractions (ENM) which were obtained by ultracentrifugation. Subsequently, ENM samples were centrifuged in density-gradient; the analysis of fractions was performed by means of SDS-PAGE and Western-blotting. The panel of monoclonal antibodies (MAbs) included anti MUC1 mucin, anti Lewis x, anti sialyl Lewis x, anti Lewis y, anti MUC-5B, anti oral mucin (gp230), anti Tn hapten, anti p53 and anti cytokeratins. By immunohistochemistry, it was possible to detect MUC1 mucin, Lewis x and Lewis y showing strong reactions while sialyl-Lewis x and Tn antigen only reacted weakly in a few cells; cytokeratins were detected in all samples. In ENM derived fractions obtained by CsCl centrifugation, MUC1 was demonstrated by Western blotting. CONCLUSIONS: (1) laryngeal cancer antigenic expression comprises mostly MUC1 mucin, Lewis x, Lewis y as well as Tn antigen and (2) the methodology here employed is useful to isolate MUC1 from tumor samples. (+info)
Role of fucosyltransferases in the association between apomucin and Lewis antigen expression in normal and malignant gastric epithelium.
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BACKGROUND: In normal gastric epithelium, MUC5AC is detected in superficial epithelium associated with Lewis type 1 antigens and MUC6 is detected in antral glands with Lewis type 2. Therefore, the stomach constitutes an excellent model to examine the role of glycosyltransferases in determining the specificity of apomucin glycosylation. AIMS: To determine the molecular basis of this association and to examine changes in expression of gastric and intestinal apomucins and their association with Lewis antigens during the gastric carcinogenesis process. METHODS: Fucosyltransferase (FUT1, FUT2, FUT3) and mucin (MUC5AC, MUC6) transcripts were detected using reverse transcription-polymerase chain reaction. Apomucin (MUC2, MUC4, MUC5AC, MUC6) and Lewis antigen (types 1 and 2) expression were analysed using single and double immunohistochemistry and in situ hybridisation. RESULTS: In the normal stomach, FUT1 is exclusively detected associated with MUC6; FUT2 is only detected when MUC5AC is present. This co-regulation is lost in gastric tumours, as is differential expression of MUC5AC and MUC6 in normal gastric epithelial cells. In gastric tumours, especially those with the intestinal phenotype, MUC2 and MUC4 genes are upregulated, and gastric-type and intestinal-type mucins are coexpressed. These changes are early events in the gastric carcinogenesis process, as they are detected in intestinal metaplasia. CONCLUSIONS: The glycosylation pattern found in normal gastric epithelium is dictated by the specific set of fucosyltranferases expressed by the cells rather than by the apomucin sequence. The development of intestinal metaplasia and gastric cancer is associated with the appearance of cellular phenotypes that are absent from normal epithelium. (+info)
European Task Force on Lymphoma project on lymphocyte predominance Hodgkin disease: histologic and immunohistologic analysis of submitted cases reveals 2 types of Hodgkin disease with a nodular growth pattern and abundant lymphocytes.
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Paraffin blocks and clinical data from 521 patients with lymphocyte predominance Hodgkin disease (LPHD) diagnosed between 1970 and 1994 were collected from 16 European and United States oncological centers to establish the pathologic and clinical characteristics of a large patient cohort, to determine how frequent T-cell-rich large B-cell lymphoma (TCRLBCL) is among LPHD, and to find differential diagnostic criteria distinguishing between the 2 lymphoma categories. For this purpose, conventionally and immunohistologically stained sections were reviewed by a panel of hematopathologists. The diagnosis of LPHD was confirmed in only 219 of the 388 assessable cases (56.5%). This low confirmation rate was due mainly to the presence of a new variant of classical Hodgkin disease (CHD), which resembled, in terms of nodular growth and lymphocyte-richness, nodular LPHD and, in terms of the immunophenotype of the tumor cells, CHD and was designated nodular lymphocyte-rich CHD (NLRCHD). The nodules of LRCHD consisted-as in nodular LPHD-predominantly of B cells but differed from those present in LPHD in that they represented expanded mantle zones with atrophic germinal centers. Clinically, patients with LPHD and NLRCHD showed similar disease characteristics at presentation but differed in the frequency of multiple relapses and prognosis after relapse. Patients with LPHD and NLRCHD clearly differed from patients with CHD with nodular sclerosis or mixed cellularity, as they presented with an earlier disease stage and infrequent mediastinal involvement. As 97% of the LPHD cases showed a complete or partial nodular growth pattern, their differentiation from TCRLBCL was a rare problem in the present series. (Blood. 2000;96:1889-1899) (+info)
Colonization and immune responses in mice to Helicobacter pylori expressing different Lewis antigens.
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BACKGROUND AND AIMS: A mouse model was established to compare colonization by the H. pylori Sydney strain SS1 with several clinical isolates expressing different Lewis antigens on their surface. In addition, both humoral and cell mediated immune responses were determined for different H. pylori strains. METHODS: Mice were inoculated intragastrically separately with the Sydney strain as well as with five clinical isolates of H. pylori expressing different Lewis (Le) antigen phenotypes. Colonization of the mouse stomach by the bacteria was monitored from two to fourteen weeks post inoculation by four independent methods namely, urease, PCR (using CagA primers), bacterial culture and histology. Antibody titers and cellular immune responses were monitored by ELISA and antigen stimulation test respectively. RESULTS: Different degrees of colonization were observed in C57, CD1 and Balb/c mice inoculated with H. pylori strain SS1 (Le(x), Le(y)) and clinical isolates UA948 (Le(a), Le(x)), UA861 (alpha-glucosyl polyLacNAc), UA1258 (Le(y)), UA802 (Le(y)) and UA1264 (no Le antigen) starting from week two post inoculation. All three mice strains mounted high immune responses against different H. pylori antigens. Treatment of mice with vancomycin prior to inoculation has no effect either on colonization of the stomach or the immune response of the mice. Histological evaluation established colonization after 10 weeks post inoculation but not gastritis. CONCLUSIONS: Stomach of mice can be colonized consistently, with H. pylori strain SS1, and colonization was also achieved with all clinical isolates that were not mouse adapted. These strains could be detected more consistently by PCR in the early stages, then by culture only after 8 - 10 weeks. In our study, Lewis(x) expressing bacterial strain (UA948) failed to colonize Balb/c mice, whereas the Le(y) expressing strain (UA1258) did not colonize C57/BL6 mice. (+info)
Soluble isoforms of CEACAM1 containing the A2 domain: increased serum levels in patients with obstructive jaundice and differences in 3-fucosyl-N-acetyl-lactosamine moiety.
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CEACAM1 (biliary glycoprotein or CD66a) is a member of the carcinoembryonic antigen (CEA) subgroup of the CEA family. Eleven RNA isoforms derived from the splicing of a single CEACAM1 gene have been described. Some of the CEACAM1 isoforms have been recognized by the CD66 antibodies in T and B lymphocytes, natural killer cells, granulocytes and epithelial cells in several human tissues. Although it is also present in soluble form in bile and serum, and elevated levels have been found in the serum of patients with liver diseases, it is not known which isoforms are primarily involved. In order to learn more about the distribution and properties of particular CEACAM1 isoforms, we have prepared a monoclonal antibody specific for the A2 domain of CEACAM1, designated TEC-11. This antibody does not cross-react with other members of the CEA family. Immunoblotting analysis revealed that the TEC-11 epitope was present in all cell types expressing CEACAM1 containing the A2 domain [CEACAM1(A2)], including granulocytes (160 000 MW isoform) and sperm cells (140 000 MW isoform). A 115 000 MW isoform of CEACAM1(A2) was present in human serum, bile, saliva and seminal fluid. Human bile, saliva and seminal fluid also contained the 160 000 MW CEACAM1(A2) isoform. Significantly higher serum levels of the 115 000 MW CEACAM1(A2) isoform were detected in patients with obstructive jaundice. The 160 000 MW isoform of CEACAM1(A2) in bile, but not a 115 000 MW isoform in serum and bile, carried the 3-fucosyl-N-acetyl-lactosamine moiety. The combined data indicate that various isoforms of CEACAM1(A2) are present in different body fluids where they could take part in different CEACAM1-mediated functions. (+info)
Altered CD15 glycolipid expression in the developing rat cerebellum following treatment with antithyroid drug, propylthiouracil.
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Thyroid hormone plays an important role during central nervous system (CNS) development. Experimentally-induced perinatal hypothyroid rats show abnormal cerebellar cytoarchitecture, but the underlying mechanism is not fully understood. Altered cell-cell interactions may contribute to such abnormalities, since the expression of NCAM is increased in hypothyroid animals. In the present study, we examined the expression of carbohydrate epitope 3-fucosyl-N-acetyl-lactosamine (CD15 antigen), that can be localized on both astrocytes and neurons in the developing brain, and is considered to play an important role in glial-neuronal interaction and cell migration. Newborn rats were treated with an antithyroid drug, propylthiouracil (PTU) and the CD15 glycolipid levels in the cerebellum were examined by enzyme-linked immunosolvent assay (ELISA) using 7A monoclonal antibody raised against rat forebrain antigen. A transient elevation of CD15 level was observed on postnatal day 10 in PTU-treated animals. Analysis of neutral glycolipids on high performance thin layer chromatography (HPTLC), revealed two distinct immunoreactive bands, corresponding to Fuc-nLc6 and Fuc-nLc4. The Fuc-nLc4 is preferentially increased in the PTU-treated group. These results suggest that a transient increase in CD15 glycoconjugates with isoform-specific manner induced by PTU may contribute to morphological abnormalities in hypothyroid rat cerebellum affecting granule cell migration. (+info)
Spontaneous apoptosis in neutrophils is associated with downregulation of HLA Class I and is prevented by ligation of Class I.
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In many types of cells, ligation of human leukocyte antigens (HLA) Class I molecules with specific mAbs results in the transduction of signals that trigger different cell functions. We have investigated the effects of Class I ligation in human neutrophils. After several hours in culture, neutrophils split spontaneously into two subpopulations, one with normal and the other with reduced levels of Class I. The latter subpopulation displayed high binding capacity for Annexin V, showed a hypodiploid peak, electrophoretic DNA fragmentation, and morphological features of apoptotic cells. The addition of drugs known to delay apoptosis (GM-CSF or cAMP) resulted in a reduction of Class I modulation. Furthermore, ligation of surface Class I with F(ab')2 fragments of the anti-Class I mAb W6/32 resulted in a delay in the progression of apoptosis. These data indicate that this surface Class I molecule is a marker of age-related apoptosis, and the ligation of these molecules results in the transduction of a signal that inhibits apoptosis. Thus, the downregulation of HLA Class I molecules in aging neutrophils prevents their halting the apoptotic process. (+info)