Ilse Sienaert | KU Leuven (original) (raw)
Papers by Ilse Sienaert
Multiphoton Microscopy in the Biomedical Sciences, 2001
Multi-photon imaging has generated intense interest from investigators using fluorescent imaging ... more Multi-photon imaging has generated intense interest from investigators using fluorescent imaging assays in live cells. It can be argued that this technique is rapidly overtaking confocal microscopy as the method of choice for three- dimensional fluorescent imaging of in vivo preparations. Because of the cost involved in purchasing a commercial multi- photon imaging microscope, many investigators have elected to build
Integrative Aspects of Calcium Signalling, 1998
The intracellular Ca2+-release channels consist of two gene families each represented by three di... more The intracellular Ca2+-release channels consist of two gene families each represented by three different isoforms with similar properties: the inositol 1,4,5-trisphosphate receptor (InsP3R) family (Furuichi et al., 1994; Joseph, 1996; Missiaen et al., 1996c; Mikoshiba, 1997) and the ryanodine receptor (RyR) family (Sorrentino & Volpe, 1993; McPherson & Campbell, 1993; Meissner, 1994; Sorrentino, 1995; Sutko & Airey, 1996; Franzini-Armstrong & Protasi, 1997). InsP3Rs and RyRs are located on intracellular membranes which form the Ca2+ stores. These Ca2+ stores represent a part of or the whole of the endo- (ER) or sarcoplasmic reticulum (SR), depending on the cell type (reviewed by Pozzan et al., 1994). The general tetrameric structure of the intracellular Ca2+ channels and their regulatory properties are very well conserved, not only within each family but also between both the families (Furuichi et al., 1994). A striking feature of these intracellular Ca2+ channels is the presence of very large cytoplasmic regions, which contain roughly 80% of the protein structure, including the N-terminal end. The channel domain is located at the C-terminal end of the protein. As both families have an even number of transmembrane domains, the outermost C-terminal part is also located in the cytoplasm. The general structure of InsP3Rs and RyRs with their large cytoplasmic domains is compatible with a role of these Ca2+ channels as integrators of a large number of cellular mediators.
Journal of Interferon and Cytokine Research, 2006
Elevated production of tumor necrosis factor (TNF) plays a central role in the pathogenesis of ma... more Elevated production of tumor necrosis factor (TNF) plays a central role in the pathogenesis of many inflammatory diseases, such as rheumatoid arthritis and Crohn's disease. Naturally occurring pteridine analogs have been reported to have potent immunomodulatory activity, especially on TNF production. The aim of this study is to identify small molecule TNF inhibitiors derived from pteridine and to prove their in vivo efficacy in an inflammatory model. A focused chemical library based on the pteridine scaffold was screened in vitro on lipopolysaccharide (LPS)-induced TNF production in peripheral blood mononuclear cells (PBMC). One synthetic pteridine analog (4AZA2096), shown to have strong inhibitory activity, was selected and tested for its efficacy to treat trinitrobenzenesulfonate (TNBS)-induced colitis in mice, a model of Crohn's disease. Colitis was induced by rectal administration of 1 mg TNBS in 50% ethanol after presensitization via the skin. The synthetic pteridine analog 4AZA2096 was shown to potently inhibit LPS-induced TNF production in vitro. Colitic mice treated with 4AZA2096 orally (20 mg/kg/day) recovered more rapidly and, histologically, had a reduction of inflammatory lesions, less edema, a reduction of goblet cell loss, and reduced wall thickness. Cell infiltration in the colon, especially infiltration of neutrophils, as shown by myeloperoxidase (MPO) activity, was reduced in 4AZA2096-treated animals. Intralesional TNF production was lower in mice of the treated groups, whereas interleukin-18 (IL-18) and interferon-gamma (IFN-gamma) mRNA were not affected. Treatment had no effect on anti-TNBS antibody production, arguing against generalized immunosuppression. In conclusion, we identified a pteridine derivative, 4AZA2096, with strong inhibitory activity on TNF production and a remission- inducing effect in TNBS colitis, supporting further preclinical and clinical development of this novel TNF inhibitor for treatment of inflammatory diseases.
Clinical Immunology, 2007
Bioorganic & Medicinal Chemistry Letters, 2011
Screening of a pteridine-based compound library led to the identification of compounds exhibiting... more Screening of a pteridine-based compound library led to the identification of compounds exhibiting immunosuppressive as well as anti-inflammatory activity. Optimization afforded a series of 2-amino-4-N-piperazinyl-6-(3,4-dimethoxyphenyl)pteridine analogues. The most potent congeners in this series displayed low nM IC(50) values in the Mixed Lymphocyte Reaction (MLR) assay. In addition, these compounds also have potent anti-inflammatory activity as measured in the Tumor Necrosis Factor (TNF) assay.
Multi-photon imaging has generated intense interest from investigators using fluorescent imaging ... more Multi-photon imaging has generated intense interest from investigators using fluorescent imaging assays in live cells. It can be argued that this technique is rapidly overtaking confocal microscopy as the method of choice for three- dimensional fluorescent imaging of in vivo preparations. Because of the cost involved in purchasing a commercial multi- photon imaging microscope, many investigators have elected to build their own system by adapting in-house confocal laser scanning microscopes. One of the components used for this adaptation involves the construction of an external pulse compressor. Pulse compressors are used to add negative dispersion to pulsed radiation, which undergoes group velocity dispersion when traversing optical elements. In this chapter, we describe a practical guide to building an external pulse compressor.
Journal of Biological Chemistry, 1997
Pflugers Archiv-european Journal of Physiology, 1996
Intracellular Ca2+ signals in response to inositol 1,4,5-trisphosphate-producing agents often pre... more Intracellular Ca2+ signals in response to inositol 1,4,5-trisphosphate-producing agents often present themselves as Ca2+ oscillations and propagating Ca2+ waves originating at discrete initiation sites. We studied the spatial organization of the Ca2+ signal in single CPAE endothelial cells stimulated with adenosine triphosphate. The long, thin processes presented a higher agonist sensitivity and, for the same agonist concentration, a faster rise
Biochimica Et Biophysica Acta-proteins and Proteomics, 2002
Biochemical Journal, 2000
Biochemical Journal, 2002
Pflugers Archiv-european Journal of Physiology, 2003
In almost all cells, cytosolic Ca2+ is a crucial intracellular messenger, regulating many cellula... more In almost all cells, cytosolic Ca2+ is a crucial intracellular messenger, regulating many cellular processes. In non-excitable as well as in some excitable cells, Ca2+ release from the intracellular stores into the cytoplasm is primarily initiated by the second messenger inositol 1,4,5-trisphosphate (IP3), which interacts with the IP3 receptor (IP3R), a tetrameric intracellular Ca2+-release channel. This review focuses on the pharmacological modulation of the various functionally important sub-domains of the IP3R, including the IP3-binding domain, calmodulin-binding sites, adenine nucleotide-binding sites and the sites for interaction for FK506-binding proteins and other regulators. We will particularly focus on the pharmacological tools that interfere with these domains and discuss their relative specificity for the IP3R, thereby indicating their potential usefulness for unraveling the complex functional regulation of the IP3R.
Pflugers Archiv-european Journal of Physiology, 1998
Ca2+-dependent Cl– secretion in the respiratory tract occurs physiologically or under pathophysi... more Ca2+-dependent Cl– secretion in the respiratory tract occurs physiologically or under pathophysiological conditions when inflammatory mediators are released. The mechanism of intracellular Ca2+ release was investigated in the immortalized bronchial epithelial cell line 16HBE14o-. Experiments on both intact and permeabilized cells revealed that only inositol 1,4,5-trisphosphate (InsP 3) receptors and not ryanodine receptors are involved in intracellular Ca2+ release. The expression pattern of the three InsP 3 receptor isoforms was assessed both at the mRNA and at the protein level. The level of expression at the mRNA level was type 3 (92.5%) >> type 2 (5.4%) > type 1 (2.1%) and this rank order was also observed at the protein level. The ATP-induced Ca2+ signals in the intact cell, consisting of abortive Ca2+ spikes or fully developed [Ca2+] rises and intracellular Ca2+ waves, were indicative of positive feedback of Ca2+ on the InsP 3 receptors. Low Ca2+ concentrations stimulated and high Ca2+ concentrations inhibited InsP 3-induced Ca2+ release in permeabilized 16HBE14o- cells. We localized a cytosolic Ca2+-binding site between amino acid residues 2077 and 2101 in the type-2 InsP 3 receptor and between amino acids 2030 and 2050 in the type-3 InsP 3 receptor by expressing the respective parts of these receptors as glutathione S-transferase fusion proteins in bacteria. We conclude that the InsP 3 receptor isoforms expressed in 16HBE14o- cells (mainly type-3 and type-2) are stimulated by Ca2+ and that this phenomenon contributes to the ATP-induced Ca2+ signals in intact 16HBE14o- cells.
Pflugers Archiv-european Journal of Physiology, 1996
Intracellular Ca2+ signals in response to inositol 1,4,5-trisphosphate-producing agents often pre... more Intracellular Ca2+ signals in response to inositol 1,4,5-trisphosphate-producing agents often present themselves as Ca2+ oscillations and propagating Ca2+ waves originating at discrete initiation sites. We studied the spatial organization of the Ca2+ signal in single CPAE endothelial cells stimulated with adenosine triphosphate. The long, thin processes presented a higher agonist sensitivity and, for the same agonist concentration, a faster rise in cytoplasmic Ca2+ concentration and rate of wave propagation than the cell body. Ca2+ waves originated preferentially in one of these processes and then invaded the cell body. Removal of external Ca2+ induced a progressive inhibition up to blockade of the response in the process but not in the cell body. These findings suggest that CPAE cells contain many individual store units, each of which has the inherent ability to set the stage for Ca2+ release. A diffusing messenger originating from the initiation zone then coordinates the events leading to Ca2+ release in the individual store units to produce a Ca2+ wave.
Biochemical and Biophysical Research Communications, 1995
Multiphoton Microscopy in the Biomedical Sciences, 2001
Multi-photon imaging has generated intense interest from investigators using fluorescent imaging ... more Multi-photon imaging has generated intense interest from investigators using fluorescent imaging assays in live cells. It can be argued that this technique is rapidly overtaking confocal microscopy as the method of choice for three- dimensional fluorescent imaging of in vivo preparations. Because of the cost involved in purchasing a commercial multi- photon imaging microscope, many investigators have elected to build
Integrative Aspects of Calcium Signalling, 1998
The intracellular Ca2+-release channels consist of two gene families each represented by three di... more The intracellular Ca2+-release channels consist of two gene families each represented by three different isoforms with similar properties: the inositol 1,4,5-trisphosphate receptor (InsP3R) family (Furuichi et al., 1994; Joseph, 1996; Missiaen et al., 1996c; Mikoshiba, 1997) and the ryanodine receptor (RyR) family (Sorrentino & Volpe, 1993; McPherson & Campbell, 1993; Meissner, 1994; Sorrentino, 1995; Sutko & Airey, 1996; Franzini-Armstrong & Protasi, 1997). InsP3Rs and RyRs are located on intracellular membranes which form the Ca2+ stores. These Ca2+ stores represent a part of or the whole of the endo- (ER) or sarcoplasmic reticulum (SR), depending on the cell type (reviewed by Pozzan et al., 1994). The general tetrameric structure of the intracellular Ca2+ channels and their regulatory properties are very well conserved, not only within each family but also between both the families (Furuichi et al., 1994). A striking feature of these intracellular Ca2+ channels is the presence of very large cytoplasmic regions, which contain roughly 80% of the protein structure, including the N-terminal end. The channel domain is located at the C-terminal end of the protein. As both families have an even number of transmembrane domains, the outermost C-terminal part is also located in the cytoplasm. The general structure of InsP3Rs and RyRs with their large cytoplasmic domains is compatible with a role of these Ca2+ channels as integrators of a large number of cellular mediators.
Journal of Interferon and Cytokine Research, 2006
Elevated production of tumor necrosis factor (TNF) plays a central role in the pathogenesis of ma... more Elevated production of tumor necrosis factor (TNF) plays a central role in the pathogenesis of many inflammatory diseases, such as rheumatoid arthritis and Crohn's disease. Naturally occurring pteridine analogs have been reported to have potent immunomodulatory activity, especially on TNF production. The aim of this study is to identify small molecule TNF inhibitiors derived from pteridine and to prove their in vivo efficacy in an inflammatory model. A focused chemical library based on the pteridine scaffold was screened in vitro on lipopolysaccharide (LPS)-induced TNF production in peripheral blood mononuclear cells (PBMC). One synthetic pteridine analog (4AZA2096), shown to have strong inhibitory activity, was selected and tested for its efficacy to treat trinitrobenzenesulfonate (TNBS)-induced colitis in mice, a model of Crohn's disease. Colitis was induced by rectal administration of 1 mg TNBS in 50% ethanol after presensitization via the skin. The synthetic pteridine analog 4AZA2096 was shown to potently inhibit LPS-induced TNF production in vitro. Colitic mice treated with 4AZA2096 orally (20 mg/kg/day) recovered more rapidly and, histologically, had a reduction of inflammatory lesions, less edema, a reduction of goblet cell loss, and reduced wall thickness. Cell infiltration in the colon, especially infiltration of neutrophils, as shown by myeloperoxidase (MPO) activity, was reduced in 4AZA2096-treated animals. Intralesional TNF production was lower in mice of the treated groups, whereas interleukin-18 (IL-18) and interferon-gamma (IFN-gamma) mRNA were not affected. Treatment had no effect on anti-TNBS antibody production, arguing against generalized immunosuppression. In conclusion, we identified a pteridine derivative, 4AZA2096, with strong inhibitory activity on TNF production and a remission- inducing effect in TNBS colitis, supporting further preclinical and clinical development of this novel TNF inhibitor for treatment of inflammatory diseases.
Clinical Immunology, 2007
Bioorganic & Medicinal Chemistry Letters, 2011
Screening of a pteridine-based compound library led to the identification of compounds exhibiting... more Screening of a pteridine-based compound library led to the identification of compounds exhibiting immunosuppressive as well as anti-inflammatory activity. Optimization afforded a series of 2-amino-4-N-piperazinyl-6-(3,4-dimethoxyphenyl)pteridine analogues. The most potent congeners in this series displayed low nM IC(50) values in the Mixed Lymphocyte Reaction (MLR) assay. In addition, these compounds also have potent anti-inflammatory activity as measured in the Tumor Necrosis Factor (TNF) assay.
Multi-photon imaging has generated intense interest from investigators using fluorescent imaging ... more Multi-photon imaging has generated intense interest from investigators using fluorescent imaging assays in live cells. It can be argued that this technique is rapidly overtaking confocal microscopy as the method of choice for three- dimensional fluorescent imaging of in vivo preparations. Because of the cost involved in purchasing a commercial multi- photon imaging microscope, many investigators have elected to build their own system by adapting in-house confocal laser scanning microscopes. One of the components used for this adaptation involves the construction of an external pulse compressor. Pulse compressors are used to add negative dispersion to pulsed radiation, which undergoes group velocity dispersion when traversing optical elements. In this chapter, we describe a practical guide to building an external pulse compressor.
Journal of Biological Chemistry, 1997
Pflugers Archiv-european Journal of Physiology, 1996
Intracellular Ca2+ signals in response to inositol 1,4,5-trisphosphate-producing agents often pre... more Intracellular Ca2+ signals in response to inositol 1,4,5-trisphosphate-producing agents often present themselves as Ca2+ oscillations and propagating Ca2+ waves originating at discrete initiation sites. We studied the spatial organization of the Ca2+ signal in single CPAE endothelial cells stimulated with adenosine triphosphate. The long, thin processes presented a higher agonist sensitivity and, for the same agonist concentration, a faster rise
Biochimica Et Biophysica Acta-proteins and Proteomics, 2002
Biochemical Journal, 2000
Biochemical Journal, 2002
Pflugers Archiv-european Journal of Physiology, 2003
In almost all cells, cytosolic Ca2+ is a crucial intracellular messenger, regulating many cellula... more In almost all cells, cytosolic Ca2+ is a crucial intracellular messenger, regulating many cellular processes. In non-excitable as well as in some excitable cells, Ca2+ release from the intracellular stores into the cytoplasm is primarily initiated by the second messenger inositol 1,4,5-trisphosphate (IP3), which interacts with the IP3 receptor (IP3R), a tetrameric intracellular Ca2+-release channel. This review focuses on the pharmacological modulation of the various functionally important sub-domains of the IP3R, including the IP3-binding domain, calmodulin-binding sites, adenine nucleotide-binding sites and the sites for interaction for FK506-binding proteins and other regulators. We will particularly focus on the pharmacological tools that interfere with these domains and discuss their relative specificity for the IP3R, thereby indicating their potential usefulness for unraveling the complex functional regulation of the IP3R.
Pflugers Archiv-european Journal of Physiology, 1998
Ca2+-dependent Cl– secretion in the respiratory tract occurs physiologically or under pathophysi... more Ca2+-dependent Cl– secretion in the respiratory tract occurs physiologically or under pathophysiological conditions when inflammatory mediators are released. The mechanism of intracellular Ca2+ release was investigated in the immortalized bronchial epithelial cell line 16HBE14o-. Experiments on both intact and permeabilized cells revealed that only inositol 1,4,5-trisphosphate (InsP 3) receptors and not ryanodine receptors are involved in intracellular Ca2+ release. The expression pattern of the three InsP 3 receptor isoforms was assessed both at the mRNA and at the protein level. The level of expression at the mRNA level was type 3 (92.5%) >> type 2 (5.4%) > type 1 (2.1%) and this rank order was also observed at the protein level. The ATP-induced Ca2+ signals in the intact cell, consisting of abortive Ca2+ spikes or fully developed [Ca2+] rises and intracellular Ca2+ waves, were indicative of positive feedback of Ca2+ on the InsP 3 receptors. Low Ca2+ concentrations stimulated and high Ca2+ concentrations inhibited InsP 3-induced Ca2+ release in permeabilized 16HBE14o- cells. We localized a cytosolic Ca2+-binding site between amino acid residues 2077 and 2101 in the type-2 InsP 3 receptor and between amino acids 2030 and 2050 in the type-3 InsP 3 receptor by expressing the respective parts of these receptors as glutathione S-transferase fusion proteins in bacteria. We conclude that the InsP 3 receptor isoforms expressed in 16HBE14o- cells (mainly type-3 and type-2) are stimulated by Ca2+ and that this phenomenon contributes to the ATP-induced Ca2+ signals in intact 16HBE14o- cells.
Pflugers Archiv-european Journal of Physiology, 1996
Intracellular Ca2+ signals in response to inositol 1,4,5-trisphosphate-producing agents often pre... more Intracellular Ca2+ signals in response to inositol 1,4,5-trisphosphate-producing agents often present themselves as Ca2+ oscillations and propagating Ca2+ waves originating at discrete initiation sites. We studied the spatial organization of the Ca2+ signal in single CPAE endothelial cells stimulated with adenosine triphosphate. The long, thin processes presented a higher agonist sensitivity and, for the same agonist concentration, a faster rise in cytoplasmic Ca2+ concentration and rate of wave propagation than the cell body. Ca2+ waves originated preferentially in one of these processes and then invaded the cell body. Removal of external Ca2+ induced a progressive inhibition up to blockade of the response in the process but not in the cell body. These findings suggest that CPAE cells contain many individual store units, each of which has the inherent ability to set the stage for Ca2+ release. A diffusing messenger originating from the initiation zone then coordinates the events leading to Ca2+ release in the individual store units to produce a Ca2+ wave.
Biochemical and Biophysical Research Communications, 1995