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Biomedical subjects

M A Verdaasdonk

Publications and source records attributed to M A Verdaasdonk.

12 recordsLinked to original sources
PubMed1997

The actin binding domain of the epidermal growth factor receptor is required for EGF-stimulated tissue invasion.

NIH-3T3 fibroblasts expressing epidermal growth factor receptors (EGFRs) lacking the actin binding domain (ABD) were analyzed for their EGF-induced capacity to invade a bone marrow stromal cell (BMSC) monolayer. The fibroblasts display a reduction in the percentage of cytoskeleton-associated EGFRs. Furthermore, EGF-induced tyrosine kinase activity is unaffected by the mutation. Cells expressing the mutant EGFRs hardly invade a BMSC monolayer upon EGF stimulation in contrast to cells expressing wild-type EGFRs. Using the same cells no difference was observed in PDGF-induced invasion, which ligand was as potent in both cell types as EGF was in wild-type cells. Inhibition of both the phosphatidyl inositol-3-kinase (PI-3-K) and lipoxygenase pathways in wild-type cells mimicked the effect of the ABD deletion. Our results point to an important role for the ABD of the EGFR in EGF-induced tissue invasion.

3T3 Cells↗
PubMed1997

Fibronectin distribution in human bone marrow stroma: matrix assembly and tumor cell adhesion via alpha5 beta1 integrin.

Tumor cell interactions with fibronectin (FN) are important for the development of secondary tumors inside the bone marrow stroma. We studied and compared the in situ distribution of FN in paraffin-embedded human bone marrow sections and investigated the in vitro regulation of FN assemblage by bone marrow stromal cells (BMSC). Finally, the role of FN in the interaction of BMSC with tumor cells was studied. Fine elongated FN-positive cell extensions, probably of stromal cell origin, were observed as well as a limited amount of extracellular FN deposits in connective tissues around capillaries and sinusoids. In vitro studies, using the confocal laser scanning microscope, showed that BMSC produced a high amount of FN with a characteristic extracellular matrix formation in an extensive network. FN matrix formation was predominantly detected at contact sites between cultured BMSC. In in vitro cultures with low cell concentrations and in vivo with a limited number of stromal cell contacts only limited matrix was found. From previous studies it is known that the alpha5 beta1 integrin is involved in the regulation of FN assembly. Here the role of the alpha5-subunit of this integrin was investigated. By using two different monoclonal antibodies (mAb) against the alpha5-subunit (2H6 and mAb16) the assembly of endogenous FN was completely blocked, indicating that these antibodies are directed against the active epitope. Another mAb (mAb11) against the alpha5-subunit did not affect the FN assemblage. Codistribution analysis of alpha5-subunits, alpha v-subunits, actin, and FN demonstrated that the alpha5 beta1 integrin is associated with FN and not with intracellular actin. Integrins alpha(v) beta1, alpha(v) beta3, and alpha(v) beta5, also ligands of FN, did not colocalize with FN. Codistribution of alpha v with the terminal ends of actin and not with FN indicates that alpha(v)-subunits are mainly directed to vitronectin rather than to FN. The dominant role of alpha5 beta1 in FN interaction is underlined by effective blocking of tumor cell adhesion with BMSC using anti-alpha5, anti-beta1, and anti-FN antibodies. These results emphasize the important role of alpha5 integrin subunit in FN matrix assembly in human BMSC and an exclusive role of alpha5 beta1 in the anchorage and regulation of FN-mediated adhesion processes in the bone marrow.

Actins↗
PubMed1996

beta1-Integrins dominate cell traffic of leukemic cells in human bone-marrow stroma.

Migration patterns of leukemic cells in bone marrow are largely regulated by cell contacts between leukemic cells and stromal cells or extra-cellular matrix. The mechanism of this interaction with bone-marrow stromal cells was studied in a human in vitro model. Migration behavior of erythroleukemia cell line K562, derived from a patient with chronic myeloid leukemia, was compared with that of the erythroleukemia cell line HEL92.1.7 and the promyelocytic leukemia cell line HL60 from acute leukemias. Interaction varied between low binding affinity (K562) to intensive cell interaction (HEL92.1.7) followed by invasion into the stromal cell monolayer. Some of the HL60 cells adhered to stromal cells, while the remainder migrated into the stromal cell monolayer. The role of adhesion molecules in these cell interactions was determined. Distinct expression of beta1-integrins ICAM-1, CD44 and VCAM-1 was detected on the different cell lines. Inhibition studies pointed to a dominant role of VLA-4- and VLA-5-mediated interactions. K562 lacked VLA-4 and a low binding affinity of the VLA-5 on these cells resulted in an absence of binding to the bone-marrow stroma. These results indicate the VLA-5/fibronectin, VLA-4/fibronectin and the VLA-4/VCAM-1 interaction pathways between leukemic cells and bone-marrow stroma.

Bone Marrow↗
PubMed1994

Interaction of tumour cells with cultured stromal cells from human bone marrow.

Adhesion of tumour cells to cultured bone marrow stromal cells has been studied in an in vitro model system. Stromal cells were isolated from bone marrow aspirates. Immunohistochemical and electron microscopical analysis revealed a uniform cell monolayer of myofibroblastic cells, expressing fibroblast antigens and smooth muscle actin. Cell interactions with tumour cells lines showed different patterns. The K562 cells bound in low numbers to stromal cells. HEL-DR- and HL60 cells adhered to stromal cells showing an enlarged cell contact area (spreading) attenuated by distinct contact sites and they invaded the monolayer. Adhesion molecules, important for cell contacts, were detected on tumor cells. Different VLA antigens were detected on tumour cells, but on stromal cells only VLA-5 and CD29 were found. In vitro inhibition studies with mAbs against adhesion molecules indicated two major pathways for binding of tumour cells to stromal cells: VCAM-1/VLA-4 and fibronectin/VLA-5. Variation in inhibition of mAbs to VLA-4 and VCAM-1 indicated the existence of critical epitopes in the adhesion of tumour cells.

Antibodies, Monoclonal↗
PubMed1992

An improved and rapid method for the isolation of rat lymph node or spleen T lymphocytes for T cell proliferation assays.

To detect and compare the capacity of antigen presenting cells to present antigen in a T cell proliferation assay, it is necessary to obtain a pure population of antigen-primed T cells that gives low background proliferative responses. Therefore in this paper we present a newly developed isolation method of antigen-primed T lymphocytes from rat spleen or lymph nodes. This method uses a nylon wool column to deplete most of the adherent cells and B cells, followed by an indirect elimination method with magnetic beads to remove contaminating Ia-positive cells. We compared this method with two commonly used isolation methods, namely a 1.5 h adherence step, followed by a nylon wool column and a Sephadex G-10 column and a 1.5 h adherence step followed by a passage through two consecutive columns of Sephadex G-10. The best T cell enrichment (98% OX-19/52-positive cells) was achieved with the newly developed method, in which the contamination of Ia-positive cells, predominantly B cells and dendritic cells (DC), was diminished to less than 2%. The background response of this population was low and differed significantly with the common methods. Antigen-specific T cell responses induced by splenic DC, expressed as stimulation index, gave very specific responses and showed a steep rise with increasing DC concentrations compared to the common methods. Therefore we conclude that we developed an improved, rapid and reproducible method for the isolation of rat spleen or lymph node T lymphocytes suitable for T cell proliferation assays.

Animals↗
PubMed1992

Induction of an increased number of dendritic cells in the peritoneal cavity of rats by intraperitoneal administration of Bacillus Calmette-Guérin.

Recently we described the presence of a small number of DC among the peritoneal cells of steady state rats. These DC had the same morphological characteristics and a similar antigen-presenting capacity as DC isolated from the spleen. This study shows that in the peritoneal cavity, which is a non-lymphoid microenvironment, the number of DC increases after i.p. administration of BCG. Next to this relatively small influx of DC, the approximately three-fold increase of the total number of cells is predominantly caused by an enormous influx of neutrophilic granulocytes, and to a lesser extent by an influx of macrophages. The phenotype and the antigen-presenting capacity of peritoneal DC has not changed, while the number of Ia-positive M phi has increased. Nevertheless, due to a suppressive effect of the peritoneal M phi, the total peritoneal cell suspension is no longer capable of presenting antigen.

Animals↗
PubMed1991

Morphological and functional characteristics of rat steady state peritoneal dendritic cells.

Dendritic cells (DC) are present in lymphoid organs and also in many non-lymphoid tissues. In this study, DC in the steady state peritoneal cavity of rats were identified morphologically and functionally. Approximately 1% of the peritoneal cells are DC. On cytocentrifuge preparations these cells had the same characteristics as lymph node and spleen DC: they had an irregular outline, all were strongly MHC class II positive and had acid phosphatase activity in a spot in a juxtanuclear position. Also ultrastructurally, peritoneal DC were similar to DC isolated from lymph node and spleen. Enrichment of peritoneal DC, using overnight culture and a Nycodenz gradient, resulted in a highly purified DC fraction. Functionally, peritoneal DC appeared to be very potent antigen-presenting cells, far more potent than peritoneal macrophages, which had an inhibitory rather than an accessory function.

Acid Phosphatase↗