Biomedical subjects

Kurt Vermeire

Publications and source records attributed to Kurt Vermeire.

23 records · Page 2Linked to original sources

PubMed2003

Evaluation of SDF-1/CXCR4-induced Ca2+ signaling by fluorometric imaging plate reader (FLIPR) and flow cytometry.

BACKGROUND: The chemokine receptors CXCR4 and CCR5 are the main coreceptors for human immunodeficiency virus (HIV) 1 to enter its target cells. The antiviral activity of their natural ligands (stromal cell-derived factor 1 [SDF-1], regulated on activation normal T-cell expressed and secreted, (RANTES) and macrophage inflammatory proteins 1alpha and 1beta, MIP-1alpha and MIP-1beta) and the finding that individuals deficient in CCR5 are relatively resistant to HIV infection led to the concept that chemokine receptor antagonists can play an important role in anti-HIV therapy. AMD3100, the prototype compound of the bicyclams, is one of the most potent and selective CXCR4 antagonists described to date. The search for new chemokine receptor antagonists involves the evaluation of compounds for their ability to block the specific chemokine-induced transient intracellular Ca(2+) flux. We evaluated two cell-based-fluorescent methods with the use of the Fluorometric Imaging Plate Reader (FLIPR) system and a flow cytometric assay to measure the SDF-1-induced intracellular Ca(2+) mobilization via CXCR4. Both assay systems were compared for their sensitivity, advantages, and system-dependent limitations. METHODS: CXCR4(+) lymphocytic and monocytic cell lines commonly used in laboratories studying acquired immunodeficiency syndrome and freshly isolated peripheral blood mononuclear cells (PBMCs) were loaded with the Ca(2+) indicator Fluo-3. After washing, the cells were preincubated with the CXCR4 antagonist AMD3100. Then the increase in intracellular Ca(2+) concentration after stimulation with SDF-1 was examined by the FLIPR and the flow cytometer, which monitored the change in green fluorescence intensity of Ca(2+)-bound Fluo-3. Surface CXCR4 expression was also determined flow cytometrically. RESULTS: In all five CXCR4(+) cell lines, SDF-1 elicited a transient increase in intracellular Ca(2+) concentration. For each cell line, the magnitude of response was related to the level of CXCR4 expression: the cells with the highest CXCR4 level showed the strongest Ca(2+) response. AMD3100 effected a dose-dependent inhibition of the SDF-1-induced Ca(2+) flux in all cell lines examined. The FLIPR was more sensitive than the flow cytometer in detecting minor Ca(2+) responses. In freshly isolated PBMCs, the Ca(2+) response was due solely to the stimulation of monocytes and granulocytes, whereas the lymphocyte population, although expressing CXCR4, did not respond at all. This phenomenon could be observed with the flow cytometer and not with the FLIPR. CONCLUSIONS: The FLIPR system is a most adequate to monitor intracellular Ca(2+) mobilization and to evaluate chemokine receptor antagonists. However, flow cytometry and its multiparameter analysis allow additional characterization of the cells involved in the chemokine receptor-mediated signal transduction.

Aniline Compounds↗

PubMed2003

The antiviral activity of the CXCR4 antagonist AMD3100 is independent of the cytokine-induced CXCR4/HIV coreceptor expression level.

The chemokine receptor CXCR4 is the main coreceptor used by T-tropic X4 HIV-1 strains to infect its target T cells. It has been proven that the CXCR4 expression level in T cells is strongly up-regulated by interleukin (IL)-4, a Th2-type cytokine that is secreted preferentially in HIV-infected patients in a later stage of disease. This results in an enhancement of HIV-1 replication in CD4+ T-lymphocytes. We have now evaluated the potency of the CXCR4 antagonist AMD3100 in phytohemagglutinin (PHA)/IL-2- versus PHA/IL-4-activated T cells in order to determine whether the compound has comparable CXCR4-antagonistic and anti-HIV-1 effects under these different cytokine treatments. We analyzed the CXCR4 expression level and the dose-dependent inhibition of CXCR4 expression by AMD3100, by monitoring the binding of an anti-CXCR4 monoclonal antibody (clone 12G5). We also determined stromal cell-derived factor (SDF)-1-induced intracellular calcium signaling and HIV-1 replication in these cells in the absence and presence of AMD3100. The CXCR4 expression level in PHA/IL-4-stimulated cells was much higher than in PHA/IL-2-stimulated cells. However, the potency of the bicyclam AMD3100 to block anti-CXCR4 mAb binding, SDF-1-induced intracellular calcium signaling, and HIV-1 replication of the X4 NL4.3 strain and three primary isolates remained unchanged. Our data indicate that CXCR4 antagonists such as AMD3100 act independently of the HIV-1 coreceptor expression level. These compounds should therefore be useful in suppressing HIV-1 infection in all stages of the disease.

Anti-HIV Agents↗

PubMed2003

The Anti-HIV potency of cyclotriazadisulfonamide analogs is directly correlated with their ability to down-modulate the CD4 receptor.

9-Benzyl-3-methylene-1,5-di-p-toluenesulfonyl-1,5,9-triazacyclododecane (CADA) has been identified as a novel antiviral lead compound with significant anti-human immunodeficiency virus and anti-human herpesvirus 7 activity. Surprisingly, this compound selectively decreased the expression of the CD4 glycoprotein, the primary receptor needed for the entry of both viruses. Herein, we describe the CD4 down-modulating and antiviral potencies of more than 25 CADA derivatives. Flow cytometric evaluation of cellular CD4 receptor expression in T cells demonstrated the specific CD4 down-modulating capacity of the CADA derivatives, with IC(50) values similar to those obtained in the antiviral assays. The close correlation observed between the CD4 down-regulating and anti-HIV potencies of the CADA derivatives further points to CD4 receptor down-modulation as the primary mode of antiviral action for this group of compounds.

Anti-HIV Agents↗

PubMed2002

CADA inhibits human immunodeficiency virus and human herpesvirus 7 replication by down-modulation of the cellular CD4 receptor.

The novel antiviral agent cyclotriazadisulfonamide (CADA) inhibited human immunodeficiency virus (HIV) (IC50, 0.3-3.2 microM) and human herpesvirus 7 (HHV-7) infection (IC50, 0.3-1.5 microM) in T-cell lines and PBMCs. When T-cells were pretreated with CADA for 24 h, they became markedly protected from viral infection. Flow cytometric analysis revealed a significant decrease in the expression of the CD4 glycoprotein, the primary receptor needed for entry of both viruses. Moreover, the antiviral activity of CADA correlated with its ability to down-modulate the CD4 receptor. CADA did not alter the expression of any other cellular receptor (or HIV coreceptor) examined. Time course experiments showed that CD4 down-modulation by CADA differs in mechanism from the effects of aurintricarboxylic acid, which binds directly to CD4, and phorbol myristate acetate, which activates protein kinase C. Further analysis of CD4 mRNA levels suggested that CADA was not involved in the regulation of CD4 expression at a transcriptional level, but very likely at (post) translational levels. This unique mechanism of action makes CADA an important lead in developing new drugs for treatment of AIDS, autoimmune diseases, and inflammatory disorders.

Anti-Infective Agents↗

PubMed1998

Immunomodulatory properties of interferon-gamma. An update.

During the early aspecific phase of host defense, production of interferon (IFN)-gamma by natural killer cells plays an important role in bringing about acute inflammation, mainly because of the activating effects of IFN-gamma on adhesive properties of endothelial cells and on mediator production by mononuclear phagocytes (MPCs). In the subsequent antigen-specific phase of the immune response, IFN-gamma acts as a regulator of antigen presentation and of proliferation and differentiation of lymphocyte populations. Immunosuppressive as well as immunostimulatory effects may result from these actions. High-level production of IFN-gamma during this phase of host defense is now classically seen as a hallmark of a T-helper 1 (TH1)-type reaction, characterized by activation of antimicrobial activity of macrophages and by inflammatory reactions with a DTH character. Development of TH1-type lymphocyte populations producing IFN-gamma is regulated by other cytokines including interleukin (IL)-12. In many systems IL-12 and IFN-gamma act in a similar fashion, and a current subject of debate is the question of whether all activities of IL-12 are mediated by IFN-gamma. Another question is whether IFN-gamma, by its ability to potentiate MPCs' ability to produce IL-12, plays a role in bringing about or stabilizing TH1 type responses. In two model systems of autoimmune disease, experimental autoimmune encephalomyelitis and collagen-induced arthritis, IL-12 and IFN-gamma were found to act independently.

Animals↗