Biomedical subjects

Gerlinde R Van de Walle

Publications and source records attributed to Gerlinde R Van de Walle.

8 recordsLinked to original sources

PubMed2006

Activation of alphaIIbbeta3 is a sufficient but also an imperative prerequisite for activation of alpha2beta1 on platelets.

Platelet integrins alpha2beta1 and alphaIIbbeta3 play critical roles in platelet adhesion and thrombus formation after vascular injury. On resting platelets, both integrins are in a low-affinity state. However, agonist stimulation results in conformational changes that enable ligand binding that can be detected with conformation dependent monoclonal antibodies (mAbs). By using such conformation-dependent mAbs, we could demonstrate that activation of integrin alphaIIbbeta3 is not only sufficient, but also a prerequisite for alpha2beta1 activation. Compared with platelets in plasma, stimulation of washed platelets resulted in only a minor activation of alpha2beta1, as detected with the activation-sensitive mAb IAC-1. Addition of fibrinogen to stimulated washed platelets greatly potentiated activation of this integrin. Also, treatment of alphaIIbbeta3 with the ligand-mimetic peptide RGDS, resulting in outside-in signaling, led to a powerful alpha2beta1 activation, even in the absence of overall platelet activation, involving tyrosine kinase activity but no protein kinase C activation. The absolute necessity of alphaIIbbeta3 for proper alpha2beta1 activation on platelets was demonstrated by using the alphaIIbbeta3 antagonist aggrastat, which was able to completely abolish alpha2beta1 activation, both under static and flow conditions. In addition, analogous experiments with Glanzmann platelets lacking alphaIIbbeta3 confirmed the indispensability of alphaIIbbeta3 for alpha2beta1 activation.

Animals↗

PubMed2005

Herpesvirus interference with virus-specific antibodies: bridging antibodies, internalizing antibodies, and hiding from antibodies.

Herpesviruses have developed different tools to thwart efficient antibody-dependent neutralisation and lysis of virions and elimination of infected cells. This overview will briefly summarize different of these tools, including (i) viral Fc receptors and the resulting process of antibody bridging, (ii) internalization of individual viral proteins and clustered antibody-antigen complexes from the plasma membrane of infected cells, and (iii) directed egress of virus particles to sites of intimate cell-cell contact that are difficult to access for antibodies.

Animals↗

PubMed2005

Two functional active conformations of the integrin {alpha}2{beta}1, depending on activation condition and cell type.

For several integrins, the existence of multiple conformational states has been studied intensively. For the integrin alpha2beta1, a major collagen receptor on platelets and other cell types, however, no such experimental data were available thus far. Recently, our group has developed a monoclonal antibody IAC-1 sensitive to the molecular conformation of alpha2beta1 because it only binds to the activated state of alpha2beta1 on platelets, induced upon inside-out signaling. By investigating IAC-1 binding in combination with collagen binding after inside-out stimulation and outside manipulation, we demonstrated the existence of three different conformations of alpha2beta1 on platelets and Chinese hamster ovary cells as follows: (i) a nonactivated, resting state with no collagen nor IAC-1 binding; (ii) an intermediate state, induced by outside manipulation, with collagen but no IAC-1 binding; and (iii) a fully activated state, induced after inside-out stimulation, with both collagen and IAC-1 binding. Moreover, these different conformational states of alpha2beta1 are dependent on the cell type where alpha2beta1 is expressed, as IAC-1 binding to peripheral blood mononuclear cells and Jurkat cells could also be induced by outside manipulation, in contrast to platelets and alpha2beta1-expressing Chinese hamster ovary cells. Finally, we revealed a functional relevance for these different conformational states because the conformation of alpha2beta1, induced after outside manipulation, resulted in significantly more cell spreading on coated collagen compared with nonactivated or inside-out stimulated cells.

Actins↗

PubMed2003

Antibody-induced internalization of viral glycoproteins and gE-gI Fc receptor activity protect pseudorabies virus-infected monocytes from efficient complement-mediated lysis.

Pseudorabies virus (PRV)-infected blood monocytes are able to transport virus throughout the body of vaccination-immune pigs. PRV-infected monocytes express viral glycoproteins in their plasma membrane that can be recognized by virus-specific antibodies. Recently, it has been shown that addition of PRV-specific polyclonal immunoglobulins to PRV-infected monocytes at 37 degrees C induces internalization of the majority of plasma membrane-expressed viral glycoproteins. This study investigated whether this process may interfere with efficient antibody-dependent complement-mediated lysis (ADCML) of infected monocytes. Therefore, an ADCML assay was set up in vitro. A significant decrease in the percentage of cells lysed by ADCML was observed when antibody-induced internalization of PRV glycoproteins occurred (P<0.005). Furthermore, it is shown (i) that the PRV gE-gI complex, which, like certain other alpha herpesvirus orthologues, possesses IgG-binding capacity, aids in avoiding efficient ADCML of PRV-infected monocytes and (ii) that the efficiency of PRV gE-gI-mediated evasion of ADCML can be decreased by the presence of gE-gI-specific antibodies.

Animals↗

PubMed2003

Virus complement evasion strategies.

The immune system has a variety of tools at its disposal to combat virus infections. These can be subdivided roughly into two categories: 'first line defence', consisting of the non-specific, innate immune system, and 'adaptive immune response', acquired over time following virus infection or vaccination. During evolution, viruses have developed numerous, and often very ingenious, strategies to counteract efficient recognition of virions or virus-infected cells by both innate and adaptive immunity. This review will focus on the different strategies that viruses use to avoid recognition by one of the components of the immune system: the complement system. Complement evasion is of particular importance for viruses, since complement activation is a crucial component of innate immunity (alternative and mannan-binding lectin activation pathway) as well as of adaptive immunity (classical, antibody-dependent complement activation).

Animals↗

PubMed2003

Transmission of pseudorabies virus from immune-masked blood monocytes to endothelial cells.

Pseudorabies virus (PRV) may cause abortion, even in the presence of vaccination-induced immunity. Blood monocytes are essential to transport the virus in these immune animals, including transport to the pregnant uterus. Infected monocytes express viral proteins on their cell surface. Specific antibodies recognize these proteins and should activate antibody-dependent cell lysis. Previous work showed that addition of PRV-specific polyclonal antibodies to PRV-infected monocytes induced internalization of viral cell surface proteins, protecting the cells from efficient antibody-dependent lysis in vitro (immune-masked monocytes). As a first step to reach the pregnant uterus, PRV has to cross the endothelial cell barrier of the maternal blood vessels. The current aim was to investigate in vitro whether immune-masked PRV-infected monocytes can transmit PRV in the presence of virus-neutralizing antibodies via adhesion and fusion of these monocytes with endothelial cells. Porcine blood monocytes, infected with a lacZ-carrying PRV strain, were incubated with PRV-specific antibodies to induce internalization. Then, cells were co-cultivated with endothelial cells for different periods of time. Only PRV-infected monocytes with internalized viral cell surface proteins adhered efficiently to endothelial cells. LacZ transmission to endothelial cells, as a measure for monocyte-endothelial cell fusion, could be detected after co-cultivation from 30 min onwards. Virus transmission was confirmed by the appearance of plaques. Adhesion of immune-masked PRV-infected monocytes to endothelial cells was mediated by cellular adhesion complex CD11b-CD18 and subsequent fusion was mediated by the virus. In conclusion, immune-masked PRV-infected monocytes can adhere and subsequently transmit virus to endothelial cells in the presence of PRV-neutralizing antibodies.

Animals↗

PubMed2003

Pseudorabies virus (PRV)-specific antibodies suppress intracellular viral protein levels in PRV-infected monocytes.

Blood monocytes infected with pseudorabies virus (PRV), a swine alphaherpesvirus, are not eliminated efficiently by antibody-dependent immunity and may occasionally transport PRV to the pregnant uterus of vaccinated animals. This study examines in vitro the long-term fate of PRV-infected monocytes cultivated in the presence of porcine PRV-specific antibodies. All monocytes were infected and expressed viral late proteins, and 30 % of PRV-infected monocytes cultivated with PRV-specific antibodies survived up to 194 h post-infection (p.i.), the end of the experiment (compared to 0 % for cells cultivated with PRV-negative antibodies). Of these surviving cells, +/-75 % no longer expressed microscopically detectable viral late proteins from 144 h p.i. onwards. Remarkably, monocytes infected with a PRV gB-null virus did not survive in the presence of PRV-specific antibodies. These data suggest that PRV-specific antibodies suppress viral protein levels in infected monocytes, perhaps helping the virus to persist and reach internal organs in vaccinated animals.

Animals↗

PubMed2002

Antibody-induced internalization of viral glycoproteins in pseudorabies virus-infected monocytes and role of the cytoskeleton: a confocal study.

Addition of pseudorabies virus (PrV)-specific polyclonal immunoglobulins to PrV-infected monocytes induces internalization of plasma membrane anchored viral glycoproteins. This process may interfere with antibody-dependent cell lysis and resembles the well-studied physiological endocytosis process. A confocal study was designed to investigate whether the major cellular components, involved in physiological endocytosis (clathrin, actin, dynein and microtubules), play a role in this virological internalization process. In order to visualize the interaction of endosomes, which contain the internalized viral glycoproteins, with clathrin, actin, dynein and microtubules, a double labeling of viral glycoproteins and different cellular proteins was performed. Porcine monocytes were inoculated with the PrV-strain 89V87 at a multiplicity of infection of 50 for 13h. After the addition of FITC-labeled porcine polyclonal PrV-specific antibodies, cells were fixed with para-formaldehyde at different time points and afterwards permeabilized. The different cellular components were visualized with monoclonal antibodies and a Texas Red-conjugate, with the exception of actin, which was stained with phalloidin-Texas Red. The cells were analyzed by confocal microscopy. A clear co-localization was observed between the viral glycoproteins and clathrin and dynein during the internalization process. The microtubules were in close contact with the internalized vesicles. For actin no co-localization could be observed. It can be stated that clathrin, dynein and microtubules, important components during physiological endocytosis, are also of importance during the antibody-induced internalization of viral glycoproteins.

Actins↗