Danuta Dus

Human NK Cells Lyse Organ-Specific Endothelial Cells: Analysis of Adhesion and Cytotoxic Mechanisms

Human organ-specific microvascular endothelial cells (ECs) were established and used in the present study to investigate their susceptibility to natural killer cell line (NKL)-induced lysis. Our data indicate that although IL-2-stimulated NKL (NKL2) cells adhered to the human peripheral (HPLNEC.B3), mesenteric lymph node (HMLNEC), brain (HBrMEC), and lung (HLMEC) and skin (HSkMEC.2) ECs, they significantly killed these cells quite differently. A more pronounced lysis of OSECs was also observed when IL-2-stimulated, purified peripheral blood NK cells were used as effector cells. In line with the correlation observed between adhesion pattern and the susceptibility to NKL2-mediated killing, we demonstrated using different chelators that the necessary adhesion step was governed by an Mg2+-dependent, but Ca2+-independent, mechanism as opposed to the subsequent Ca2+-dependent killing. To identify the cytotoxic pathway used by NKL2 cells, the involvement of the classical and alternate pathways was examined. Blocking of the Ca2+-dependent cytotoxicity pathway by EGTA/MgCl2 significantly inhibited endothelial target cell killing, suggesting a predominant role for the perforin/granzyme pathway. Furthermore, using confocal microscopy, we demonstrated that the interaction between NKL2 effectors and ECs induced cytochrome c release and Bid translocation in target cells, indicating an involvement of the mitochondrial pathway in NKL2-induced EC death. In addition, although all tested cells were sensitive to the cytotoxic action of TNF, no susceptibility to TRAIL or anti-Fas mAb was observed. The present studies emphasize that human NK cell cytotoxicity toward ECs may be a potential target to block vascular injury.

CD133 positive progenitor endothelial cell lines from human cord blood

Endothelial progenitor cells (EPCs) modulate postnatal vascularization and contribute to vessel regeneration in adults. Stem cells and progenitor cells were found in umbilical cord blood, bone marrow, and mobilized peripheral blood cells, from where they were isolated and cultured. However, the yield of progenitor cells is usually not sufficient for clinical application and the quality of progenitor cells varies. The aim of the study was the immortalization of early progenitor cells with high proliferative potential, capable to differentiate to EPCs and, further, toward endothelial cells. Two cell lines, namely HEPC‐CB.1 and HEPC‐CB.2 (human endothelial progenitor cells—cord blood) were isolated. As assessed by specific antibody labeling and flow cytometric analysis, they express a panel of stem cell markers: CD133, CD13, CD271, CD90 and also endothelial cell markers: CD202b, CD309 (VEGFR2), CD146, CD105, and CD143 but they do not present markers of finally differentiated endothelial cells: CD31, vWf, nor CD45 which is a specific hematopoietic cell marker. Using the multiplex Cytometric Bead Assay, the simultaneous production of proangiogenic cytokines IL8, angiogenin, and VEGF was demonstrated in normoxia and was shown to be increased by hypoxia. Both cell lines, similarly as mature endothelial cells, underwent in vitro pre‐angiogenic process, formed pseudovessel structures and present an accelerated angiogenesis in hypoxic conditions. To date, these are the first CD133 positive established cell lines from human cord blood cells.

The effect of calcium ions on subcellular localization of aldolase-FBPase complex in skeletal muscle

In skeletal muscles, FBPase–aldolase complex is located on α‐actinin of the Z‐line. In the present paper, we show evidence that stability of the complex is regulated by calcium ions. Real time interaction analysis, confocal microscopy and the protein exchange method have revealed that elevated calcium concentration decreases association constant of FBPase–aldolase and FBPase‐α‐actinin complex, causes fast dissociation of FBPase from the Z‐line and slow accumulation of aldolase within the I‐band and M‐line. Therefore, the release of Ca2+ during muscle contraction might result, simultaneously, in the inhibition of glyconeogenesis and in the acceleration of glycolysis.

Selective human endothelial cell activation by chemokines as a guide to cell homing

An original model of organo‐specific, immortalized and stabilized endothelial cell lines was used to delineate the part played by some chemokines (CCL21, CX3CL1, CCL5 and CXCL12) and their receptors in endothelium organo‐specificity. Chemokine receptor expression and chemokine presentation were investigated on organo‐specific human endothelial cell lines. Although the chemokines showed distinct binding patterns for the various endothelial cell lines, these were not correlated with the expression of the corresponding receptors (CX3CR1, CXCR4, CCR5 and CCR7). Experiments with CCL21 on peripheral lymph node endothelial cells demonstrated that the chemokine did not co‐localize with its receptor but was associated with extracellular matrix components. The specific activity of chemokines was clearly shown to be related to the endothelial cell origin. Indeed, CX3CL1 and CCL21 promoted lymphocyte recruitment by endothelial cells from the appendix and peripheral lymph nodes, respectively, while CX3CL1 pro‐angiogenic activity was restricted to endothelial cells from the appendix and skin. The high specificity of the chemokine/endothelium interaction allowed the design of a direct in vitro endothelial cell targeting assay. This unique cellular model demonstrated a fundamental role for chemokines in conferring on the endothelium its organo‐specificity and its potential for tissue targeting through the selective binding, presentation and activation properties of chemokines.

Lectins from the Red Marine Algal Species Bryothamnion seaforthii and Bryothamnion triquetrum as Tools to Differentiate Human Colon Carcinoma Cells

The carbohydrate-binding activity of the algal lectins from the closely related red marine algal species Bryothamnion triquetrum (BTL) and Bryothamnion seaforthii (BSL) was used to differentiate human colon carcinoma cell variants with respect to their cell membrane glyco-receptors. These lectins interacted with the cells tested in a dose-dependent manner. Moreover, the fluorescence spectra of both lectins clearly differentiated the cells used as shown by FACS profiles. Furthermore, as observed by confocal microscopy, BTL and BSL bound to cell surface glycoproteins underwent intense internalization, which makes them possible tools in targeting strategies.

Structures of the alpha(1-3)-galactose-containing asparagine-linked glycans of a Lewis lung carcinoma cell subline resistant to Aleuria aurantia agglutinin: elucidation by 1H-NMR spectroscopy.

Four bi-antennary glycan fractions of theN-acetyllactosamine-type, derived from a Lewis lung carcinoma (LL2) cell subline resistant to theAleuria aurantia agglutinin were studied by 400 MHz1H-NMR spectroscopy. By this method, their antennae were found to be terminated either by α(2-3 or 6)-linkedN-acetylneuraminic acid or α(1-3)-linked galactose residues. The primary structure of glycans of these four glycopeptide or derived oligosaccharide-alditols has been determined in full detail.

Metastatic potential and saccharide antigens expression of human colon cancer cells xenotransplanted into athymic nude mice.

Aberrant glycosylation is a common phenomenon observed in colon carcinoma cells. As a consequence, the increased expression of Lewis blood group family antigens, particularly Le x, sialyl Le x, and sialyl Le a has been reported. Recently, it has been demonstrated that these antigenic epitopes may play an important role in cell-cell adhesive interactions during progressive growth and metastasis (Hakomori 1996). The aim of this work was to study the relationship between Table I. Decrease m expresston of saccharide antigens in CX-1.1 AS5

Glycosphingolipids of human urothelial cell lines with different grades of transformation

Neutral glycolipids and gangliosides from seven human urothelial cell lines, differing in grades of transformation (TGr), were characterized by fast atom bombardment mass spectrometry, exoglycosidase treatment and an immunostaining procedure. The major neutral glycolipids identified in all cell lines studied included CMH, CDH, CTH, globoside and paragloboside, the gangliosides were GM3, GM2, sialosylparagloboside and GD1a. The following observations were made: 1. GM2 was the major ganglioside in the TGrll cell lines (non-tumorigenic, non-invasive), but a minor component in the TGrIII cell lines (tumorigenic, invasive). 2. All components showed C16:0 and C24:0 as major fatty acids, but in the TGrIII cell lines the fatty acid composition of CMH and some of the gangliosides were more complex showing unsaturated and hydroxy-fatty, acids as well.

Expression of c-myc oncoprotein in laryngeal squamous cell carcinoma.

Objective c-myc seems to play a pivotal role in normal growth and development as well in cellular transformation and carcinogenesis. Overexpression of the c-myc oncogene has been observed in many hematopoetic and solid tumors. The role of c-myc protein in squamous cell carcinoma of the head and neck in general and laryngeal squamous cell carcinomas (LSCCs) in particular is far from clear. The aim of this study was to evaluate the relations between the level of c-myc protein in LSCCs and the clinicopathological data of patients, DNA ploidy and the SG2M phase index (PI). Material and Methods The c-myc protein level was evaluated immunohistochemically in tumor specimens from 50 patients with LSCC. The DNA index and SG2M PI were determined by means of flow cytometry. Results We found c-myc protein in 34 (68%) tumors. Expression of c-myc protein was demonstrated to be frequent in non-metastatic cases (p=0.016). There was no association between c-myc protein level and age, primary tumor size, histological grading or type of cancer. In 13 (26%) cases we observed DNA aneuploid tumors. The mean value of the SG2M PI was 22.5%. Expression of c-myc protein was not related to SG2M PI or DNA ploidy. Conclusions We have shown that c-myc oncoprotein may be involved in the genesis of LSCC. Our findings suggest that the detectability of c-myc protein is associated with a lower metastatic potential. The c-myc oncogene is probably not as important in laryngeal cancers compared to other cancers. Further investigations must be performed to establish the value of predicting nodal metastases in LSCC.

The anti-angiogenic activity of IL-12 is increased in iNOS−/− mice and involves NK cells

We have previously reported that the in vivo transfer of murine interleukin-12 (IL-12) gene using a Semliki Forest virus vector induced tumor regression through inhibition of tumor blood vessel formation. To examine whether IL-12 anti-angiogenic activity interferes with the NO pathway, we used inducible nitric oxide synthase-deficient mice (iNOS−/−) and demonstrated that the anti-tumor effect of IL-12 is more pronounced in these mice. In addition, despite the increased level of intratumoral VEGF in iNOS−/− mice, IL-12 induced a stronger inhibition of blood vessel formation. Histological analysis of SFV-IL-12-treated tumors showed an increase in natural killer (NK) perivascular infiltration in iNOS−/− as compared to control mice. In vitro IL-12-stimulated murine splenic NK cells displayed significant killing activity towards established murine endothelial cells used as targets. These studies indicate that the anti-angiogenic activity of IL-12 interferes with iNOS pathway and involves NK cell recruitment.