Katrien Van Beneden

Publications of Katrien Van Beneden

• Galactose-modified iNKT cell agonists stabilized by an induced fit of CD1d prevent tumour metastasis.

  Authors: Sandrine Aspeslagh, Yali Li, Esther Dawen Yu, Nora Pauwels, Matthias Trappeniers, Enrico Girardi, Tine Decruy, Katrien Van Beneden, Koen Venken, Michael Drennan, Luc Leybaert, Jing Wang, Richard W Franck, Serge Van Calenbergh, Dirk M Zajonc, Dirk Elewaut

  The EMBO journal. 06/2011; 30(11):2294-305.

  Invariant natural killer T (iNKT) cells are known to have marked immunomodulatory capacity due to their ability to produce copious amounts of effector cytokines. Here, we report the structure andInvariant natural killer T (iNKT) cells are known to have marked immunomodulatory capacity due to their ability to produce copious amounts of effector cytokines. Here, we report the structure and function of a novel class of aromatic α-galactosylceramide structurally related glycolipids with marked Th1 bias in both mice and men, leading to superior tumour protection in vivo. The strength of the Th1 response correlates well with enhanced lipid binding to CD1d as a result of an induced fit mechanism that binds the aromatic substitution as a third anchor, in addition to the two lipid chains. This induced fit is in contrast to another Th1-biasing glycolipid, α-C-GalCer, whose CD1d binding follows a conventional key-lock principle. These findings highlight the previously unexploited flexibility of CD1d in accommodating galactose-modified glycolipids and broaden the range of glycolipids that can stimulate iNKT cells. We speculate that glycolipids can be designed that induce a similar fit, thereby leading to superior and more sustained iNKT cell responses in vivo.

• An anti-inflammatory selective glucocorticoid receptor modulator preserves osteoblast differentiation.

  Authors: Alexander Rauch, Valerie Gossye, Debby Bracke, Elien Gevaert, Peggy Jacques, Katrien Van Beneden, Bernard Vandooren, Martina Rauner, Lorenz C Hofbauer, Guy Haegeman, Dirk Elewaut, Jan P Tuckermann, Karolien De Bosscher

  FASEB journal : official publication of the Federation of American Societies for Experimental Biology. 01/2011; 25(4):1323-32.

  Glucocorticoids (GCs) are in widespread use to treat inflammatory bone diseases, such as rheumatoid arthritis (RA). Their anti-inflammatory efficacy, however, is accompanied by deleterious effects onGlucocorticoids (GCs) are in widespread use to treat inflammatory bone diseases, such as rheumatoid arthritis (RA). Their anti-inflammatory efficacy, however, is accompanied by deleterious effects on bone, leading to GC-induced osteoporosis (GIO). These effects include up-regulation of the receptor activator of NF-κB ligand/osteoprotegerin (RANKL/OPG) ratio to promote bone-resorbing osteoclasts and include inhibition of bone-forming osteoblasts. We previously identified suppression of osteoblast differentiation by the monomer glucocorticoid receptor (GR) via the inhibition of Il11 expression as a crucial mechanism for GIO. Here we show that the GR-modulating substance compound A (CpdA), which does not induce GR dimerization, still suppresses proinflammatory cytokines in fibroblast-like synovial cells from patients with RA and in osteoblasts. In contrast to the full GR agonist dexamethasone, it does not unfavorably alter the RANKL/OPG ratio and does not affect Il11 expression and subsequent STAT3 phosphorylation in these cells. Notably, while dexamethasone inhibits osteoblast differentiation, CpdA does not affect osteoblast differentiation in vitro and in vivo. We describe here for the first time that selective GR modulators can act against inflammation, while not impairing osteoblast differentiation.

• Invariant natural killer T cells are natural regulators of murine spondylarthritis.

  Authors: Peggy Jacques, Koen Venken, Katrien Van Beneden, Hamida Hammad, Sylvie Seeuws, Michael B Drennan, Dieter Deforce, Gust Verbruggen, Maria Apostolaki, George Kollias, Bart N Lambrecht, Martine De Vos, Dirk Elewaut

  Arthritis and rheumatism. 04/2010; 62(4):988-99.

  To investigate the role of invariant natural killer T (iNKT) cells in TNF(DeltaARE/+) mice, an animal model of spondylarthritis (SpA) with both gut and joint inflammation. The frequency andTo investigate the role of invariant natural killer T (iNKT) cells in TNF(DeltaARE/+) mice, an animal model of spondylarthritis (SpA) with both gut and joint inflammation. The frequency and activation of iNKT cells were analyzed on mononuclear cells from the lymph nodes and livers of mice, using flow cytometry with alpha-galactosylceramide/CD1d tetramers and quantitative polymerase chain reaction for the invariant V(alpha)14-J(alpha)18 rearrangement. Bone marrow-derived dendritic cells (DCs) were obtained by expansion of primary cells with granulocyte-macrophage colony-stimulating factor followed by coculture with iNKT cell hybridomas, and interleukin-2 release into the cocultures was then measured by enzyme-linked immunosorbent assay (ELISA). Cytokine levels were determined by ELISA or cytometric bead array analyses of freshly isolated DCs and iNKT cells in mixed cocultures. TNF(DeltaARE/+) mice were backcrossed onto J(alpha)18(-/-) and CD1d(-/-) mice, and disease onset was evaluated by clinical scoring, positron emission tomography, and histology. CD1d levels were analyzed on mononuclear cells in paired blood and synovial fluid samples from patients with SpA compared with healthy control subjects. In the absence of iNKT cells, symptoms of gut and joint inflammation in TNF(DeltaARE/+)mice were aggravated. Invariant NKT cells were activated during the course of the disease. This was linked to an enrichment of inflammatory DCs, characterized by high levels of CD1d, particularly at draining sites of inflammation. A similar increase in CD1d levels was observed on DCs from patients with SpA. Inflammatory DCs from TNF(DeltaARE/+) mice stimulated iNKT cells to produce immunomodulatory cytokines, in the absence of exogenous stimulation. Prolonged, continuous exposure, but not short-term exposure, to tumor necrosis factor (TNF) was found to be responsible for the enhanced DC-NKT cell crosstalk. This mode of iNKT cell activation represents a natural counterregulatory mechanism for the dampening of TNF-driven inflammation.

• Cutting edge: The chemokine receptor CXCR3 retains invariant NK T cells in the thymus.

  Authors: Michael B Drennan, Ann Sophie Franki, Pieter Dewint, Katrien Van Beneden, Sylvie Seeuws, Serge A van de Pavert, Emma C Reilly, Gust Verbruggen, Thomas E Lane, Reina E Mebius, Dieter Deforce, Dirk Elewaut

  Journal of immunology (Baltimore, Md. : 1950). 09/2009; 183(4):2213-6.

  The current model used to define T cell export from the thymus suggests that emigrating lymphocytes seed the peripheral organs as functionally mature cells. This model holds true for the majority ofThe current model used to define T cell export from the thymus suggests that emigrating lymphocytes seed the peripheral organs as functionally mature cells. This model holds true for the majority of T cells exported from the thymus with the exception of invariant NK T (iNKT) cells. iNKT cells undergo lineage expansion after positive selection and acquire NK receptor expression once fully mature; yet, the majority of mature iNKT cells are retained in the thymus by an as of yet unidentified mechanism. In this study we demonstrate that mature iNKT cells are retained in the thymus by the chemokine receptor CXCR3. We propose that the expression of CXCR3 ligands in the thymic medullary epithelium promotes the chemotactic retention of mature iNKT thymocytes and prevents leakage of iNKT cells into the peripheral circulation.

• Application of a new anti-zearalenone monoclonal antibody in different immunoassay formats.

  Authors: Natalia Burmistrova, Irina Goryacheva, Evgenia Basova, Ann Sophie Franki, Dirk Elewaut, Katrien Van Beneden, Dieter Deforce, Carlos Van Peteghem, Sarah De Saeger

  Analytical and bioanalytical chemistry. 08/2009;

  Monoclonal antibodies against zearalenone (ZEA) were raised in mice according to the hybridoma technology and applied in different immunochemical techniques. More specifically, three formats based onMonoclonal antibodies against zearalenone (ZEA) were raised in mice according to the hybridoma technology and applied in different immunochemical techniques. More specifically, three formats based on the competitive direct enzyme immunoassay principle were developed: an enzyme-linked immunosorbent assay (ELISA), a flow-through gel-based immunoassay column and a flow-through membrane-based immunoassay. In ELISA, the 50% inhibitory concentration (IC50) was 0.8 ng/mL, and the limit of detection for ZEA standard solutions was 0.1 ng/mL. The antibodies showed a high ZEA (100%) and alpha-zearalenol (alpha-ZOL) (69%) recognition, while cross-reactivities with alpha-zearalanol, zearalanone, beta-zearalenol and beta- zearalanol were 42%, 22%, <1% and <1%, respectively. For standard solutions, a cut-off level at 10 ng/mL could be established for the gel- and membrane-based enzyme immunoassays. Assay time of both non-instrumental tests was 25 min for 10 samples. By including a simple sample extraction procedure, the methods were applied to wheat with IC50s in ELISA of 80 and 120 mug/kg (dilution up to 5% and 15% (v/v) of wheat matrix, respectively). The cut-off level of the gel- and membrane-based immunoassays was established at 100 microg/kg. Potentials and limitations of the developed methods were compared. The possible application for multi-mycotoxin analysis of the ELISA method based on a single monoclonal antibody was investigated. Therefore, principal component analysis and partial least squares regression data modelling were used to separate the immunoassay responses of two cross-reactants (ZEA and alpha-ZOL).

• A plant-derived glucocorticoid receptor modulator attenuates inflammation without provoking ligand-induced resistance.

  Authors: Valerie Gossye, Dirk Elewaut, Katrien Van Beneden, Pieter Dewint, Guy Haegeman, Karolien De Bosscher

  Annals of the rheumatic diseases. 02/2009;

  BACKGROUND: Acquired resistance to glucocorticoids (GCs) constitutes a major clinical challenge, often overlooked in the search for improved alternatives to classical steroids. We sought to unravelBACKGROUND: Acquired resistance to glucocorticoids (GCs) constitutes a major clinical challenge, often overlooked in the search for improved alternatives to classical steroids. We sought to unravel how two glucocorticoid receptor (GR) activating compounds, Dexamethasone (DEX) and CompoundA (CpdA), influence GR levels and how this can be correlated to their gene regulatory potential. METHODS: CpdA and DEX were applied in a short-term and in a long-term treatment protocol. Via Q-PCR analysis in fibroblast-like synoviocytes (FLS) the gene regulatory potential of both compounds in the two experimental conditions was analysed. A parallel Western blot assay revealed the GR protein levels in both conditions (ex vivo). Additionally, we examined the effect of systemic administration of DEX and CpdA, in concentrations effective to inhibit collagen-induced arthritis (CIA), in DBA/1 mice on GR levels (in vivo). RESULTS: CpdA does not induce a homologous down-regulation of GR in vivo and ex vivo, thereby retaining its anti-inflammatory effects after prolonged treatment in FLS. This is in sharp contrast to DEX, showing a direct link between prolonged DEX treatment, decreasing GR levels, and the abolishment of inflammatory gene repression in FLS. We additionally observed that the acquired low receptor levels after prolonged DEX treatment are still sufficient to sustain the transactivation of endogenous GRE-driven genes in FLS, a mechanism partially held accountable for the metabolic side effects. CONCLUSION: /B> CpdA is less likely to evoke therapy resistance, as it does not lead to homologous GR down-regulation, which is in contrast to classical GCs.

• The Synthesis and in vivo Evaluation of 2',2'-Difluoro KRN7000.

  Authors: Leo Leung, Cyrille Tomassi, Katrien Van Beneden, Tine Decruy, Matthias Trappeniers, Dirk Elewaut, Yifang Gao, Tim Elliott, Aymen Al-Shamkhani, Christian Ottensmeier, Jörn M Werner, Anthony Williams, Serge Van Calenbergh, Bruno Linclau

  ChemMedChem. 02/2009;

• 6'-derivatised alpha-GalCer analogues capable of inducing strong CD1d-mediated Th1-biased NKT cell responses in mice.

  Authors: Matthias Trappeniers, Katrien Van Beneden, Tine Decruy, Ulrik Hillaert, Bruno Linclau, Dirk Elewaut, Serge Van Calenbergh

  Journal of the American Chemical Society. 01/2009; 130(49):16468-9.

• Reversible changes in serum immunoglobulin galactosylation during the immune response and treatment of inflammatory auto-immune arthritis.

  Authors: Katrien Van Beneden, Ken Coppieters, Wouter Laroy, Filip De Keyser, Ilse E Hoffman, Filip Van den Bosch, Bert Vander Cruyssen, Michael Drennan, Peggy Jacques, Pieter Rottiers, Gust Verbruggen, Roland Contreras, Nico Callewaert, Dirk Elewaut

  Annals of the rheumatic diseases. 10/2008;

  OBJECTIVES: In this study we used the improved DNA-sequencer aided FACE (DSA-FACE) technology to monitor the changes in the galactosylation status of serum immunoglobulins during the immune responseOBJECTIVES: In this study we used the improved DNA-sequencer aided FACE (DSA-FACE) technology to monitor the changes in the galactosylation status of serum immunoglobulins during the immune response and therapy of auto-immune arthritis. METHODS: Collagen-induced arthritis (CIA) was induced in susceptible DBA/1 mice and the undergalactosylation status (UGS) of serum immunoglobulins was determined using the improved DSA-FACE technology. Prophylactic intravenous tolerization with type-II collagen as well as semi-therapeutic treatment with Dexamethasone (DEX) were performed, and UGS was analyzed. Next, we studied the serum immunoglobulin glycosylation profile in both rheumatoid arthritis (RA) and spondyloarthropathy (SpA) patients, and followed changes in the UGS scores during anti-TNFalpha therapy. RESULTS: In the longitudinal CIA study, the undergalactosylation state of immunoglobulins was found to be significantly correlated with the clinical arthritis scores. Upon collagen-specific tolerization as well as glucocorticoid semi-therapeutic treatment, improvement of the clinical arthritis scores correlated with decreased levels of UGS. We also demonstrated that withdrawal of DEX was associated with an increased UGS score. Interestingly, reversibility in the UGS was also shown during treatment of both RA and SpA patients with anti-TNFalpha. CONCLUSIONS: These findings demonstrate that the UGS of serum immunoglobulins changes during the disease course of CIA and that this UGS is inhibited by antigen-specific and antigen-independent treatment procedures. The observation that Ig galactosylation is a reversible process is also documented during treatment of both RA and SpA patients with anti-TNFalpha.

• Synthesis and In Vivo Evaluation of 4-Deoxy-4,4-difluoro-KRN7000.

  Authors: Leo Leung, Cyrille Tomassi, Katrien Van Beneden, Tine Decruy, Dirk Elewaut, Tim Elliott, Aymen Al-Shamkhani, Christian Ottensmeier, Serge Van Calenbergh, Joern Werner, Tony Williams, Bruno Linclau

  Organic letters. 10/2008;

  The synthesis of 4-deoxy-4,4-difluoro-KRN7000 starting from phytosphingosine is described. Key steps include a regioselective benzylation of azidophytosphingosine and a deoxofluor-mediatedThe synthesis of 4-deoxy-4,4-difluoro-KRN7000 starting from phytosphingosine is described. Key steps include a regioselective benzylation of azidophytosphingosine and a deoxofluor-mediated fluorination of the corresponding 4-ketone. This fluorination failed completely when the adjacent 3-OH was protected as benzyl ether but proceeded well when a benzoyl group was used. The biological evaluation reveals a bias toward Th1 cytokine induction upon Natural Killer T cell activation.

• Synthesis and in vitro evaluation of alpha-GalCer epimers.

  Authors: Matthias Trappeniers, Stijn Goormans, Katrien Van Beneden, Tine Decruy, Bruno Linclau, Aymen Al-Shamkhani, Tim Elliott, Christian Ottensmeier, Joern M Werner, Dirk Elewaut, Serge Van Calenbergh

  ChemMedChem. 08/2008; 3(7):1061-70.

  alpha-GalCer (also known as KRN7000) is an immunomodulatory glycolipid that is known to potently activate invariant natural killer T (NKT) cells upon CD1d-mediated stimulation. Because Th1 and Th2alpha-GalCer (also known as KRN7000) is an immunomodulatory glycolipid that is known to potently activate invariant natural killer T (NKT) cells upon CD1d-mediated stimulation. Because Th1 and Th2 cytokines, which are released after alpha-GalCer presentation, antagonize each other's effects, alpha-GalCer analogues that induce a biased Th1/Th2 response are highly awaited. In this context, we report the synthesis and in vitro evaluation of alpha-Gal-D-xylo-Cer and two alpha-Gal-L-lyxo-Cer analogues, one with the natural acyl chain, the other with a truncated chain.

• A plant-derived ligand favoring monomeric glucocorticoid receptor conformation with impaired transactivation potential attenuates collagen-induced arthritis.

  Authors: Pieter Dewint, Valerie Gossye, Karolien De Bosscher, Wim Vanden Berghe, Katrien Van Beneden, Dieter Deforce, Serge Van Calenbergh, Ulf Müller-Ladner, Bert Vander Cruyssen, Gust Verbruggen, Guy Haegeman, Dirk Elewaut

  Journal of immunology (Baltimore, Md. : 1950). 03/2008; 180(4):2608-15.

  The glucocorticoid receptor (GR) is a transcription factor regulating its target genes either positively, through direct binding to the promoter of target genes, or negatively by the interferenceThe glucocorticoid receptor (GR) is a transcription factor regulating its target genes either positively, through direct binding to the promoter of target genes, or negatively by the interference with the activity of transcription factors involved in proinflammatory gene expression. The well-known adverse effects of glucocorticoids are believed to be mainly caused by their GR-mediated gene-activating properties. Although dimerization of GR is thought to be essential for gene-activating properties, no compound has yet been described which selectively imposes GR monomer formation and interference with other transcription factors. In the present study, we report on a GR-binding, plant-derived compound with marked dissociative properties in rheumatoid arthritis fibroblast-like synoviocytes, which are important effector cells in inflammation and matrix degradation in rheumatoid arthritis. In addition, these findings could be extended in vivo in murine collagen-induced arthritis, in which joint inflammation was markedly inhibited without inducing hyperinsulinemia. Therefore, we conclude that GR monomers are sufficient for inhibition of inflammation in vivo.

• A single early activation of invariant NK T cells confers long-term protection against collagen-induced arthritis in a ligand-specific manner.

  Authors: Ken Coppieters, Katrien Van Beneden, Peggy Jacques, Pieter Dewint, Ann Vervloet, Bert Vander Cruyssen, Serge Van Calenbergh, Guangwu Chen, Richard W Franck, Gust Verbruggen, Dieter Deforce, Patrick Matthys, Moriya Tsuji, Pieter Rottiers, Dirk Elewaut

  Journal of immunology (Baltimore, Md. : 1950). 09/2007; 179(4):2300-9.

  The glycosphingolipid alpha-galactosylceramide (alpha-GalCer) has been shown to be a potent activator of invariant NKT (iNKT) cells, rapidly inducing large amounts of both Th1 and Th2 cytokines uponThe glycosphingolipid alpha-galactosylceramide (alpha-GalCer) has been shown to be a potent activator of invariant NKT (iNKT) cells, rapidly inducing large amounts of both Th1 and Th2 cytokines upon injection in mice. The C-glycoside analog of alpha-GalCer (alpha-C-GalCer), by contrast, results in an enhanced Th1-type response upon activation of iNKT cells. We administered a single dose of these Ags to DBA/1 mice during the early induction phase of collagen-induced arthritis and demonstrated therapeutic efficacy of alpha-GalCer when administered early rather than late during the disease. Surprisingly, the Th1-polarizing analog alpha-C-GalCer also conferred protection. Furthermore, a biphasic role of IFN-gamma in the effect of iNKT cell stimulation was observed. Whereas in vivo neutralization of IFN-gamma release induced by either alpha-GalCer or alpha-C-GalCer early during the course of disease resulted in partial improvement of clinical arthritis symptoms, blockade of IFN-gamma release later on resulted in a more rapid onset of arthritis. Although no phenotypic changes in conventional T cells, macrophages, or APCs could be detected, important functional differences in T cell cytokine production in serum were observed upon polyclonal T cell activation, 2 wk after onset of arthritis. Whereas alpha-GalCer-treated mice produced significantly higher amounts of IL-10 upon systemic anti-CD3 stimulation compared with PBS controls, T cells from alpha-C-GalCer-treated mice, by contrast, produced substantially lower levels of cytokines, suggesting the involvement of different protective mechanisms. In conclusion, these findings suggest long-term, ligand-specific, time-dependent, and partially IFN-gamma-dependent immunomodulatory effects of iNKT cells in collagen-induced arthritis.

• A unique lymphotoxin {alpha}beta-dependent pathway regulates thymic emigration of V{alpha}14 invariant natural killer T cells.

  Authors: Ann Sophie Franki, Katrien Van Beneden, Pieter Dewint, Kirsten J L Hammond, Stijn Lambrecht, Georges Leclercq, Mitchell Kronenberg, Dieter Deforce, Dirk Elewaut

  Proceedings of the National Academy of Sciences of the United States of America. 07/2006; 103(24):9160-5.

  Natural killer (NK) T cells using an invariant Valpha14 (Valpha14i) T cell receptor rearrangement form a distinct immunoregulatory T cell lineage. Several studies indicated that a NK1.1(-) Valpha14iNatural killer (NK) T cells using an invariant Valpha14 (Valpha14i) T cell receptor rearrangement form a distinct immunoregulatory T cell lineage. Several studies indicated that a NK1.1(-) Valpha14i NKT precursor cell differentiates and expands within the thymus before export to the peripheral tissues occurs. However, little is known about the signals that cause the emigration of Valpha14i NKT cells from the thymus to the periphery. Here we show that signaling of lymphotoxin (LT) alphabeta through the LTbeta receptor (LTbetaR) is indispensable for regulating peripheral but not thymic Valpha14i NKT cell numbers. Homing to and homeostatic proliferation of thymic Valpha14i NKT cells in peripheral organs, however, was not dependent on LTbetaR. Instead, our data indicate that a LTbetaR-expressing thymic stromal cell regulates the thymic emigration of Valpha14i NKT cells but not conventional T cell receptor alphabeta cells.

• Ly49 and CD94/NKG2 receptor acquisition by NK cells does not require lymphotoxin-beta receptor expression.

  Authors: Frederik Stevenaert, Katrien Van Beneden, Veerle De Colvenaer, Ann Sophie Franki, Veronique Debacker, Tom Boterberg, Dieter Deforce, Klaus Pfeffer, Jean Plum, Dirk Elewaut, Georges Leclercq

  Blood. 09/2005; 106(3):956-62.

  A crucial step in murine natural killer (NK) cell development, mediated by bone marrow stromal cells, is the induction of Ly49 and CD94/NKG2 receptor expression. The signals that regulate Ly49A crucial step in murine natural killer (NK) cell development, mediated by bone marrow stromal cells, is the induction of Ly49 and CD94/NKG2 receptor expression. The signals that regulate Ly49 receptor expression are still largely undetermined. It has been shown that interaction between lymphotoxin alpha1beta2 (LTalpha1beta2) and LTbeta receptor (LTbetaR), expressed on lymphoid progenitor cells and nonlymphoid bone marrow stromal cells, respectively, is important for both quantitative and functional NK cell development. Therefore, we have investigated the role of LT-LTbetaR-mediated signaling in Ly49 and CD94/NKG2 receptor acquisition. We show that the NK receptor repertoire of LTbetaR-/- mice can only be partially analyzed because of the residual 129/Ola mouse genetic background, due to a physical linkage of the LTbetaR locus and the loci encoding the Ly49 and CD94/NKG2 receptors. Therefore, we transferred wild-type B6 lymphoid-committed progenitor cells into LTbetaR-/- mice, which differentiated into NK cells with a normal NK cell receptor repertoire. Also, administration of LTbetaR-immunoglobulin (Ig), which acts as a soluble receptor for LTalpha1beta2, resulted in reduced NK cell percentages but did not influence the Ly49 and CD94/NKG2 receptor acquisition on remaining NK cells. These results indicate that LTbetaR-mediated signals are not required for Ly49 and CD94/NKG2 receptor acquisition.

• Chronically inflamed synovium from spondyloarthropathy and rheumatoid arthritis investigated by protein expression profiling followed by tandem mass spectrometry.

  Authors: Kelly Tilleman, Katrien Van Beneden, Aline Dhondt, Ilse Hoffman, Filip De Keyser, Eric Veys, Dirk Elewaut, Dieter Deforce

  Proteomics. 05/2005; 5(8):2247-57.

  We investigated the cytosolic proteome of inflamed synovial tissue by hierarchical clustering analysis and validated the feasibility of this proteome analysis by identifying proteins that wereWe investigated the cytosolic proteome of inflamed synovial tissue by hierarchical clustering analysis and validated the feasibility of this proteome analysis by identifying proteins that were differentially expressed between rheumatoid arthritis (RA), spondyloarthropathy (SpA), and osteoarthritis (OA). Synovial biopsy samples were obtained from 18 patients undergoing needle arthroscopy for knee synovitis associated with RA (n = 6) and SpA (n = 6), and for joint effusion of the knee associated with OA (n = 6). Cytosolic proteins were extracted from the tissue and subjected to two-dimensional gel electrophoresis. Protein expression patterns were statistically analyzed and used for hierarchical cluster analysis. Proteins of interest were independently identified by matrix-assisted laser desorption/ionization- and electrospray ionization-mass spectrometry. Hierarchical cluster analysis of the complete match set, containing 640 spots, remarkably segregated SpA from RA and OA. Next, we used a subset of spots that was statistically, differentially expressed (P < 0.01), between RA and SpA, SpA and OA, or RA and OA, in both Student's t-test and Mann-Whitney U-test. The dendrograms revealed distinct clustering of RA versus SpA and RA versus OA. Spots that were differentially expressed between the groups were identified by tandem mass spectrometry. Fructose bisphosphate aldolase A and alpha-enolase showed higher expression levels in SpA than in OA (P < 0.01). Calgranulin A myeloid related protein-8 (MRP-8) was markedly up-regulated in RA and SpA patients in comparison to OA patients where this spot was below detection limit. The analysis of the cytosolic proteome of synovial tissue is a useful approach to identify disease-associated proteins in chronic inflammatory arthritis.

• Lymphotoxin alpha 1 beta 2: a critical mediator in V alpha 14i NKT cell differentiation.

  Authors: Ann Sophie Franki, Katrien Van Beneden, Pieter Dewint, Ivan Meeus, Eric Veys, Dieter Deforce, Dirk Elewaut

  Molecular immunology. 03/2005; 42(4):413-7.

  Lymphotoxin (LT) alpha 1 beta 2, a tumour necrosis factor (TNF) cytokine critically involved in lymphoid organogenesis, is indispensable for the differentiation of V alpha 14i natural killer T (NKT)Lymphotoxin (LT) alpha 1 beta 2, a tumour necrosis factor (TNF) cytokine critically involved in lymphoid organogenesis, is indispensable for the differentiation of V alpha 14i natural killer T (NKT) cells, a lymphocyte subset with important immunoregulatory properties. However, it is not required for the development of conventional T-cells. LT alpha 1 beta 2 signals through the LT beta receptor, which is expressed on non-lymphoid cells. Triggering of this receptor induces a unique signalling cascade leading to the activation of the transcription factor RelB through activation of NF-kappa B inducing kinase. This pathway is required for V alpha 14i NKT cell differentiation as appears from studies in gene-deficient animals. By reciprocal bone marrow chimeras, it was shown that RelB is required in a radiation-resistant host cell or stromal cell for normal V alpha 14i NKT cell development, presumably in the thymic stroma. These stromal cells are not required for the positive selection of these cells but rather play a prominent role in their terminal differentiation. Altogether, these observations underscore the unique developmental requirements of this particular lymphocyte subset.

• Mapping of the leptin binding sites and design of a leptin antagonist.

  Authors: Frank Peelman, Katrien Van Beneden, Lennart Zabeau, Hannes Iserentant, Peter Ulrichts, Delphine Defeau, Annick Verhee, Dominiek Catteeuw, Dirk Elewaut, Jan Tavernier

  The Journal of biological chemistry. 10/2004; 279(39):41038-46.

  The leptin/leptin receptor system shows strong similarities to the long-chain cytokine interleukin-6 (IL-6) and granulocyte colony-stimulating factor cytokine/receptor systems. The IL-6 familyThe leptin/leptin receptor system shows strong similarities to the long-chain cytokine interleukin-6 (IL-6) and granulocyte colony-stimulating factor cytokine/receptor systems. The IL-6 family cytokines interact with their receptors through three different binding sites I-III. The leptin structure was superposed on the crystal structures of several long-chain cytokines, and a series of leptin mutants was generated focusing on binding sites I-III. The effect of the mutations on leptin receptor (LR) signaling and on binding to the membrane proximal cytokine receptor homology domain (CRH2) of the LR was determined. Mutations in binding site I at the C terminus of helix D show a modest effect on signaling and do not affect binding to CRH2. Binding site II is composed of residues at the surface of helices A and C. Mutations in this site impair binding to CRH2 but have only limited effect on signaling. Site III mutations around the N terminus of helix D impair receptor activation without affecting binding to CRH2. We identified an S120A/T121A mutant in binding site III, which lacks any signaling capacity, but which still binds to CRH2 with wild type affinity. This leptin mutant behaves as a potent leptin antagonist both in vitro and in vivo.

• Ly49E expression points toward overlapping, but distinct, natural killer (NK) cell differentiation kinetics and potential of fetal versus adult lymphoid progenitors.

  Authors: Frederik Stevenaert, Katrien Van Beneden, An De Creus, Veronique Debacker, Jean Plum, Georges Leclercq

  Journal of leukocyte biology. 07/2003; 73(6):731-8.

  Using a new antibody, we found previously that contrary to adult natural killer (NK) cells, fetal NK cells have a unique phenotype, as they exclusively express Ly49E. This can be explained by anUsing a new antibody, we found previously that contrary to adult natural killer (NK) cells, fetal NK cells have a unique phenotype, as they exclusively express Ly49E. This can be explained by an intrinsic different NK differentiation potential of fetal versus adult lymphoid progenitors, by immaturity of fetal NK cells or by instability of Ly49E expression. Here, we show that adult progenitor cells were still capable of differentiating into Ly49E-expressing NK cells but at a much lower frequency. Surprisingly, Ly49E expression in vitro did not require stromal cells. Kinetic analysis in vivo showed that Ly49E was expressed early, together with CD94/NKG2 and Ly49G2, followed by Ly49C, and finally Ly49D. Transfer of sorted Ly49E-positive fetal NK cells showed stable Ly49E expression, and later, part of these cells up-regulated other Ly49 members. These data indicate that although there are intrinsic differences, there is no strict fetal and adult wave of NK cell differentiation.

• Developmental and functional defects of thymic and epidermal V gamma 3 cells in IL-15-deficient and IFN regulatory factor-1-deficient mice.

  Authors: An De Creus, Katrien Van Beneden, Frederik Stevenaert, Veronique Debacker, Jean Plum, Georges Leclercq

  Journal of immunology (Baltimore, Md. : 1950). 07/2002; 168(12):6486-93.

  In this study, the role of IL-15 and its regulation by the transcription factor IFN regulatory factor-1 (IRF-1) in murine V gamma 3 T cell development and activity is assessed. Compared withIn this study, the role of IL-15 and its regulation by the transcription factor IFN regulatory factor-1 (IRF-1) in murine V gamma 3 T cell development and activity is assessed. Compared with wild-type (WT) mice, reduced numbers of mature V gamma 3 cells were found in the fetal thymus of IL-15(-/-) mice, while IRF-1(-/-) mice displayed normal frequencies. V gamma 3(+) dendritic epidermal T cells (DETCs) were absent in IL-15(-/-) mice but present in IRF-1(-/-) mice. DETCs from IRF-1(-/-) mice displayed morphologically a less mature phenotype and showed different emergence kinetics during ontogeny. This corresponded with lower IL-15 mRNA levels in the skin epidermis. Comparable levels of IL-7 were found in the skin of WT and IL-15(-/-) mice. Adoptive transfer experiments of WT fetal thymocytes into IL-15(-/-) mice did not result in the development of V gamma 3(+) DETCs, confirming the nonredundant role of IL-15 in the skin during DETC development. In vitro, cytolytic activity of IL-15(-/-) V gamma 3 cells was normal after stimulation with IL-15 and was further enhanced by addition of IL-12. In contrast, cytolytic activity of IRF-1(-/-) V gamma 3 cells remained defective after stimulation with IL-15 in combination with IL-12. These data suggest that IL-15 is redundant for the development and/or survival of mature V gamma 3 cells in the fetal thymus, whereas it is essential for the localization of V gamma 3 cells in the skin. Furthermore, a possible role for IRF-1 in inducing morphological maturation of DETCs and cytolytic capacity of V gamma 3 cells is suggested.