Characterization of anticoagulant heparinoids by immunoprofiling

Heparinoids are used in the clinic as anticoagulants. A specific pentasaccharide in heparinoids activates antithrombin III, resulting in inactivation of factor Xa and–when additional saccharides are present–inactivation of factor IIa. Structural and functional analysis of the heterogeneous heparinoids generally requires advanced equipment, is time consuming, and needs (extensive) sample preparation. In this study, a novel and fast method for the characterization of heparinoids is introduced based on reactivity with nine unique anti-heparin antibodies. Eight heparinoids were biochemically analyzed by electrophoresis and their reactivity with domain-specific anti-heparin antibodies was established by ELISA. Each heparinoid displayed a distinct immunoprofile matching its structural characteristics. The immunoprofile could also be linked to biological characteristics, such as the anti-Xa/anti-IIa ratio, which was reflected by reactivity of the heparinoids with antibodies HS4C3 (indicative for 3-O-sulfates) and HS4E4 (indicative for domains allowing anti-factor IIa activity). In addition, the immunoprofile could be indicative for heparinoid-induced side-effects, such as heparin-induced thrombocytopenia, as illustrated by reactivity with antibody NS4F5, which defines a very high sulfated domain. In conclusion, immunoprofiling provides a novel, fast, and simple methodology for the characterization of heparinoids, and allows high-throughput screening of (new) heparinoids for defined structural and biological characteristics

Cell Surface GPI-anchoring of CD45 isoforms

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Alternative splicing of CD45 pre-mRNA is uniquely obedient to conditions in lymphoid cells

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School en pedagogische functie

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Pedagogische waarden in het basisonderwijs

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Differential expression of heparan sulfate domains in rat spleen.

The microarchitecture of the spleen is composed of a meshwork of reticulum cells and their matrix. Heparan sulfates (HS) are important components of this meshwork and are involved in processes such as cell adhesion, cell migration, and cytokine/growth factor binding. The expression of HS epitopes was analyzed using anti-HS antibodies. Four different staining patterns were observed, as exemplified by antibodies RB4EA12, HS4E4, AO4B08, and HS4C3. These antibodies recognize different chemical modifications in HS. In adult spleen, RB4EA12 stained only the reticular meshwork and blood vessels in the red pulp and marginal zone. HS4E4 stained blood vessel-associated basal lamina. AO4B08 and HS4C3 stained the reticular meshwork and blood vessels throughout the spleen, but only AO4B08 strongly stained smooth muscle cells and ring fibers. Interleukin-2 localized in the red pulp and marginal zone and was bound to HS. AO4B08, HS4C3, and RB4EA12 but not HS4E4 co-localized with interleukin-2. In 10-day-old spleen, HS4E4 recognized reticular fibers, which were not stained in the adult stage. Immunoelectron microscopy revealed that HS was restricted to basal laminae and reticular fibers. Taken together, data show that HS epitopes are differentially expressed in the spleen and that this may create specific extracellular environments for immunological processes

Differential expression of heparan sulfate domains in rat spleen.

Item does not contain fulltextThe microarchitecture of the spleen is composed of a meshwork of reticulum cells and their matrix. Heparan sulfates (HS) are important components of this meshwork and are involved in processes such as cell adhesion, cell migration, and cytokine/growth factor binding. The expression of HS epitopes was analyzed using anti-HS antibodies. Four different staining patterns were observed, as exemplified by antibodies RB4EA12, HS4E4, AO4B08, and HS4C3. These antibodies recognize different chemical modifications in HS. In adult spleen, RB4EA12 stained only the reticular meshwork and blood vessels in the red pulp and marginal zone. HS4E4 stained blood vessel-associated basal lamina. AO4B08 and HS4C3 stained the reticular meshwork and blood vessels throughout the spleen, but only AO4B08 strongly stained smooth muscle cells and ring fibers. Interleukin-2 localized in the red pulp and marginal zone and was bound to HS. AO4B08, HS4C3, and RB4EA12 but not HS4E4 co-localized with interleukin-2. In 10-day-old spleen, HS4E4 recognized reticular fibers, which were not stained in the adult stage. Immunoelectron microscopy revealed that HS was restricted to basal laminae and reticular fibers. Taken together, data show that HS epitopes are differentially expressed in the spleen and that this may create specific extracellular environments for immunological processes

Highly sulfated chondroitin sulfates, a novel class of prognostic biomarkers in ovarian cancer tissue.

OBJECTIVE: Clinical decision making in ovarian cancer needs new (prognostic) biomarkers. Although the search for biomarkers has traditionally focused on tumor cells, their surrounding contains important information as well. Glycosaminoglycans, heterogeneous polysaccharides which are abundantly present in the stromal compartment, are indicated in several pathological processes including cancer. In this study we investigated a specific glycosaminoglycan motif (4,6-disulfated chondroitin sulfate) for its potential as a prognostic biomarker in ovarian cancer. METHODS: 4,6-Disulfated chondroitin sulfate presence was studied immunohistochemically using the single chain antibody GD3G7 on 148 ovarian tumors including benign and malignant tumors, and tumors with low malignant potential. For comparative purposes p53 and Ki-67 were evaluated. X(2) tests, univariate and multivariate Cox proportional hazards analyses were applied for statistical analysis. RESULTS: The stroma of malignant tumors showed significantly increased expression of 4,6-disulfated chondroitin sulfate (GD3G7 epitope) compared with benign tumors and tumors with LMP (p-values<0.000 and 0.002, respectively). Expression of GD3G7 in malignant tumors was significantly correlated with serous subtype, high tumor grade, advanced FIGO-stage and high CA-125 levels. In patients with advanced FIGO stage GD3G7 expression was significantly correlated with incomplete debulking and good response to platinum-based chemotherapy. GD3G7 surpassed both p53 and Ki-67 in statistical analysis. Multivariate survival analysis revealed GD3G7 expression as an independent predictor for progression free survival. CONCLUSION: Glycosaminoglycan motifs may form a new class of biomarkers for (ovarian) cancer, as indicated here for the GD3G7 epitope. Expression of GD3G7 may contribute in therapeutic decision making and constitutes a potential biomarker for poor prognosis