MR imaging of a subungual glomus tumor.
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Adult↗
Biomedical subjects
G Opdenakker
Publications and source records attributed to G Opdenakker.
Gelatinase B in multiple sclerosis and experimental autoimmune encephalomyelitis.
The matrix metalloproteases (MMPs) are a family of structurally related proteolytic enzymes, that are involved in various physiological and pathological processes. In the central nervous system, MMPs may contribute to proteolysis of basement membranes, extracellular matrix molecules, cytokine precursors, zymogens, cell surface molecules, and myelin components. Clipping of the latter increases the local antigenic epitope load. We explain the REGA model (Remnant Epitopes Generate Autoimmunity), which may be applied to the pathophysiology of many autoimmune diseases, including multiple sclerosis, and which consists of a tight control of the enzymatic activity of the MMPs at several levels: MMP gene transcription and MMP secretion, that are regulated by cytokines and chemokines, activation of latent zymogens by proteolysis, inhibition of enzyme activity by specific inhibitors, and glycosylation. Gelatinase B, a rather complex protease, is discussed as a prototypic MMP example. Possible applications of our understanding about the regulation of MMP activity and of the influence on disease-promotion or -limitation are reviewed.
Collagenases↗
The immunological basis of current and novel therapies of multiple sclerosis.
The etiology and the pathogenesis of multiple sclerosis are not yet known. There might be a role for genetic susceptibility, for environmental factors and for inflammatory and immunological changes. Most of the actual therapies are based on the latter two phenomena. We review here corticosteroids, interferon beta and copolymer 1 as the current drugs of choice and compare schematically the immunological basis of the mechanisms of action of these three substances with those of experimental or other treatments.
Adrenal Cortex Hormones↗
Oligosaccharides of recombinant mouse gelatinase B variants.
Gelatinase B (matrix metalloproteinase-9, MMP-9) contains three N-glycosylation sites and a Ser/Thr/Pro-rich type V collagen domain with repetitive attachment sites for O-linked sugars. Recombinant mouse gelatinase B was expressed in the yeast Pichia pastoris and the N-linked oligosaccharides of the truncated glycoprotein variants were analysed by in gel enzymatic release followed by mass spectrometry and normal phase HPLC. This technology, despite of the limiting amount of material, allowed the analysis of the formula of N- and O-linked sugars of the different glycoprotein variants. The 112/99- and 88-kDa gelatinase B forms each contained an oligomannose series (Man8GlcNAc2 to Man15GlcNAc2). Analysis of the hydrazine-released sugars showed that the O-linked oligosaccharides contained alpha1-2, alpha1-3 or alpha1-6 linked mannoses. These results were confirmed by lectin blot analysis of intact and glycosidase-treated enzyme variants.
Acetaldehyde↗
Functional comparison of two human monocyte chemotactic protein-2 isoforms, role of the amino-terminal pyroglutamic acid and processing by CD26/dipeptidyl peptidase IV.
Human Monocyte Chemotactic Protein (MCP)-2 has originally been isolated from stimulated osteosarcoma cells as a chemokine coproduced with MCP-1 and MCP-3. Here, a 5'-end extended MCP-2 cDNA was cloned from a human testis cDNA library. It encoded a 76 residue MCP-2 protein, but differed from the reported bone marrow-derived MCP-2 cDNA sequence in codon 46, which coded for a Lys instead of a Gln. This MCP-2Lys46 variant, caused by a single nucleotide polymorphism (SNP), was biologically compared with MCP-2Gln46. The coding regions were subcloned into the bacterial expression vector pHEN1, and after transformation of Escherichia coli, the two MCP-2 protein variants were recovered from the periplasm. The recombinant proteins were purified to homogeneity by heparin-Sepharose affinity chromatography and reversed-phase HPLC. Edman degradation revealed a Gln residue at the NH2 terminus instead of a pGlu. To evaluate the influence of the cyclization, this Gln was chemically converted into pGlu in both MCP-2 variants. The conversion was confirmed by electrospray mass spectrometry. rMCP-2Gln46 and rMCP-2Lys46 and the NH2-terminal cyclic counterparts were tested on monocytic cells in calcium mobilization and chemotaxis assays. No significant difference in biological activity was observed between the rMCP-2Gln46 and rMCP-2Lys46 isoforms. However, for both MCP-2 variants the NH2-terminal pyroglutamate was shown to be essential for chemotaxis, but not for calcium mobilization. NH2-terminal truncation of rMCP-2Lys46 by the serine protease CD26/dipeptidyl peptidase IV (CD26/DPP IV) resulted in the cleavage of the NH2-terminal Gln-Pro dipeptide, whereas synthetic MCP-2 with an amino-terminal pGlu remained unaffected. CD26/DPP IV-clipped rMCP-2Lys46(3-76) was almost completely inactive in both chemotaxis and signaling assays. These observations indicate that the NH2-terminal pGlu in MCP-2 is necessary for chemotactic activity but also that it protects the protein against degradation by CD26/DPP IV.
Alleles↗
Matrix metalloproteinase expression in an experimentally-induced DTH model of multiple sclerosis in the rat CNS.
In an experimentally-induced DTH model of MS, we examined mRNA and protein expression of a range of MMPs and of TNFalpha to establish the contribution that individual MMPs might make to the pathogenesis. In control rat brain, mRNA for all of the MMPs examined was detectable. However, by immunohistochemistry, only MMP-2 could be detected. In the DTH lesions, significant increases in the level of mRNA expression were observed for MMP-7, MMP-8, MMP-12, and TNFalpha. Where expression of MMP mRNA was increased, there was a corresponding increase in protein expression detected by immunohistochemistry. To determine whether the upregulated MMPs could invoke destructive events in the CNS, highly purified activated MMP-7, MMP-8, and MMP-9 were stereotaxically injected into the brain parenchyma. All provoked recruitment of leukocytes and BBB breakdown. In addition, MMPs 7 and 9 induced loss of myelin staining. In conclusion, specific MMPs are upregulated in DTH lesions; for the most part, measurement of mRNA was a predictor of increased protein expression. From our injections of MMPs, it is clear that the upregulated MMPs in the DTH lesions could participate in the disruption of the BBB, leukocyte recruitment, and tissue damage.
Animals↗
Regulation of gelatinase B (MMP-9) in leukocytes by plant lectins.
The stimulatory or inhibitory effects of plant lectins on the production of gelatinase A (MMP-2) and gelatinase B (MMP-9) by mononuclear white blood cells was investigated by substrate zymography. Leukocyte cultures from 24-h old buffy coats were spontaneously activated and produced high levels of gelatinase B. Using such cultures the suppressing activity of the Datura stramonium, Viscum album, Bauhinia purpurea, Triticum aestivum and Maackia amurensis lectins on gelatinase B induction were demonstrated. When fresh leukocyte preparations from single blood donors were used, low levels of gelatinase B were produced. The induction of gelatinase B was confirmed for concanavalin A and phytohaemagglutinin (PHA-L4). In addition, the Urtica dioica, Calystegia sepium, Convolvulus arvensis and Colchicum autumnale lectins were documented as novel and potent inducers of gelatinase B. Since high circulating gelatinase B levels are associated with specific pathologies, including shock syndromes, the acute toxicity of many lectins might be partially mediated or influenced by gelatinase induction.
Cells, Cultured↗
Amino-terminal truncation of chemokines by CD26/dipeptidyl-peptidase IV. Conversion of RANTES into a potent inhibitor of monocyte chemotaxis and HIV-1-infection.
Chemokines are key players in inflammation and infection. Natural forms of the C-X-C chemokine granulocyte chemotactic protein-2 (GCP-2) and the C-C chemokine regulated on activation normal T cell expressed and secreted (RANTES), which miss two NH2-terminal residues, including a Pro in the penultimate position, have been isolated from leukocytes or tumor cells. In chemotaxis and intracellular calcium mobilization assays, the truncation caused a reduction in the specific activity of RANTES but not of GCP-2. The serine protease CD26/dipeptidyl-peptidase IV (CD26/DPP IV) could induce this observed NH2-terminal truncation of GCP-2 and RANTES but not that of the monocyte chemotactic proteins MCP-1, MCP-2 and MCP-3. No significant difference in neutrophil activation was detected between intact and CD26/DPP IV-truncated GCP-2. In contrast to intact natural RANTES(1-68), which still chemoattracts monocytes at 10 ng/ml, CD26/DPP IV-truncated RANTES(3-68) was inactive at 300 ng/ml and behaved as a natural chemotaxis inhibitor. Compared with intact RANTES, only a 10-fold higher concentration of RANTES(3-68) induced a significant Ca2+ response. Furthermore, RANTES(3-68) inhibited infection of mononuclear cells by an M-tropic HIV-1 strain 5-fold more efficiently than intact RANTES. Thus, proteolytic processing of RANTES by CD26/DPP IV may constitute an important regulatory mechanism during anti-inflammatory and antiviral responses.
Binding, Competitive↗
Interferon-gamma gene polymorphism-associated risk for multiple sclerosis in Sardinia.
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Alleles↗
Synergistic induction of MCP-1 and -2 by IL-1beta and interferons in fibroblasts and epithelial cells.
Monocyte chemotactic protein (MCP)-1 and MCP-2, two closely related CC chemokines, are important mediators of monocyte and lymphocyte migration. These chemokines are secreted by various normal cell types, including fibroblasts, epithelial cells, and leukocytes, as well as by tumor cells. After stimulation with different cytokines and cytokine inducers the MCP-2 production levels are always lower than those of MCP-1. In human diploid fibroblasts cytokines differentially regulate chemokine induction, interleukin (IL)-1beta and interferon (IFN)-gamma being potent stimuli of MCP-1 and MCP-2, respectively. Co-stimulation of fibroblasts by 10 U/mL IL-1beta and 20 ng/mL IFN-gamma resulted in a synergistic induction of MCP-2, whereas the combined effect on MCP-1 and IL-6 production was rather additive. These findings were confirmed at the mRNA level by Northern blot analysis. In contrast, in human MG-63 fibroblastoid cells and HEp-2 epithelial cells, selected for their poor responsiveness to IL-1beta and IFN-gamma, MCP-2 as well as MCP-1 and IL-6 were synergistically induced, yielding protein levels that were increased 3- to 30-fold above the additive levels. When IFN-beta was used as a co-stimulant of IL-1beta, a similar synergistic induction of MCP-1 and MCP-2 was measured both at the protein and the mRNA level. It can be concluded that, when synergy occurred, the MCP-1 and MCP-2 expression levels reached a comparable maximum, indicative for an equal contribution of these chemokines in normal and pathological conditions.
Cell Division↗
Cytokine and protease glycosylation as a regulatory mechanism in inflammation and autoimmunity.
Cytokines are locally produced hormones that alert the innate and specific immune systems. Many cytokines induce, enhance and govern the traffic of leukocytes. An important mechanism in cell trafficking and migration through endothelial basement membranes and connective tissues is the cytokine-regulated production of matrix degrading proteases. The latter include the serine proteinases of plasminogen activation and metalloproteinases such as collagenases, stromelysins and gelatinases. Many cytokines and all known matrix proteinases are glycoproteins and thus occur as sets of glycoforms. The relation between structures and functions of these glycoproteins has already been probed extensively at the protein level but not yet at the carbohydrate level. Attached oligosaccharides target the cytokines and proteinases to specific cellular receptors and matrix binding sites. In addition, a number of cytokines possess lectin-like functions and may thus interact with carbohydrates of the host or parasites. These intermolecular interactions influence for instance the compartmentalisation, the cell- and tissue-specific distribution and the pharmacokinetics of cytokines and proteinases. Attempts were done to deduce structure-function rules for the intramolecular effects of carbohydrates on cytokines and matrix proteinases. The relatively voluminous N-linked sugars downmodulate the specific activities of enzymes and cytokines. Because in host stress reactions (infection, inflammation, trauma) N-linked glycosylation is less efficient, glycosylation may constitute an important regulatory mechanism in the cytokine network and in multi-enzyme cascades.
Animals↗
Gelatinase B in proliferative vitreoretinal disorders.
PURPOSE: To investigate whether gelatinases A and B are involved in the pathogenesis of proliferative vitreoretinal disorders. METHODS: In a prospective study of 101 consecutive patients, vitreous and paired serum samples were obtained from 38 patients with rhegmatogenous retinal detachment complicated by proliferative vitreoretinopathy, 25 patients with rhegmatogenous retinal detachment with no proliferative vitreoretinopathy, and 38 patients with proliferative diabetic retinopathy. Gelatinase activities were determined by quantitative zymography. RESULTS: All vitreous samples contained comparable levels of the constitutive gelatinase A. Inducible gelatinase B was detected in eight (32%) of 25 vitreous samples from patients with rhegmatogenous retinal detachment with no proliferative vitreoretinopathy (mean +/- SD, 319.5 +/- 521.0 scanning units), in 17 (44.7%) of 38 vitreous samples from patients with proliferative vitreoretinopathy (560.6 +/- 718.9 scanning units), and in 34 (89.5%) of 38 vitreous samples from patients with proliferative diabetic retinopathy (1,707.2 +/- 1,220.3 scanning units). The incidence of detection of gelatinase B in proliferative diabetic retinopathy cases was significantly higher than it was in rhegmatogenous retinal detachment with no proliferative vitreoretinopathy and proliferative vitreoretinopathy cases (P < .001). Gelatinase B levels in the vitreous samples of patients with proliferative diabetic retinopathy were higher than the levels found in patients with rhegmatogenous retinal detachment with no proliferative vitreoretinopathy and in patients with proliferative vitreoretinopathy (P = .0152). Gelatinase A was detected in all the tested sera, whereas none of the tested paired serum samples contained detectable gelatinase B activity. CONCLUSIONS: Gelatinase B may play an important role in extracellular matrix degradation associated with neovascularization in proliferative diabetic retinopathy.
Collagenases↗
The molecular basis of leukocytosis.
The function of any known gene is often found by DNA or protein homology scanning. Conversely, it is equally rewarding to search for the genetic basis behind a known function. Here, Ghislain Opdenakker and colleagues examine the known and possible novel genes and molecular events underlying the phenomenon of leukocytosis, one of the most common clinical manifestations of inflammatory problems.
Humans↗
Concepts and principles of O-linked glycosylation.
The biosynthesis, structures, and functions of O-glycosylation, as a complex posttranslational event, is reviewed and compared for the various types of O-glycans. Mucin-type O-glycosylation is initiated by tissue-specific addition of a GalNAc-residue to a serine or a threonine of the fully folded protein. This event is dependent on the primary, secondary, and tertiary structure of the glycoprotein. Further elongation and termination by specific transferases is highly regulated. We also describe some of the physical and biological properties that O-glycosylation confers on the protein to which the sugars are attached. These include providing the basis for rigid conformations and for protein stability. Clustering of O-glycans in Ser/Thr(/Pro)-rich domains allows glycan determinants such as sialyl Lewis X to be presented as multivalent ligands, essential for functional recognition. An additional level of regulation, imposed by exon shuffling and alternative splicing of mRNA, results in the expression of proteins that differ only by the presence or absence of Ser/Thr(/Pro)-rich domains. These domains may serve as protease-resistant spacers in cell surface glycoproteins. Further biological roles for O-glycosylation discussed include the role of isolated mucin-type O-glycans in recognition events (e.g., during fertilization and in the immune response) and in the modulation of the activity of enzymes and signaling molecules. In some cases, the O-linked oligosaccharides are necessary for glycoprotein expression and processing. In contrast to the more common mucin-type O-glycosylation, some specific types of O-glycosylation, such as the O-linked attachment of fucose and glucose, are sequon dependent. The reversible attachment of O-linked GlcNAc to cytoplasmic and nuclear proteins is thought to play a regulatory role in protein function. The recent development of novel technologies for glycan analysis promises to yield new insights in the factors that determine site occupancy, structure-function relationship, and the contribution of O-linked sugars to physiological and pathological processes. These include diseases where one or more of the O-glycan processing enzymes are aberrantly regulated or deficient, such as HEMPAS and cancer.
Animals↗
Toxicity in a double-blind, placebo-controlled pilot trial with D-penicillamine and metacycline in secondary progressive multiple sclerosis.
The serine proteinase tissue-type plasminogen activator (t-PA) and the metalloproteinase gelatinase B (MMP-9) have recently been demonstrated in MS lesions. Both enzymes are interconnected in an enzyme cascade which contributes to destruction of the blood brain barrier and demyelination and both enzymes are inhibited by D-penicillamine. Metacycline was shown in in vitro experiments to inhibit gelatinase B. The combination of peroral D-penicillamine plus metacycline was evaluated in a double-blind placebo-controlled way in two groups of 10 patients suffering from secondary progressive multiple sclerosis. The major objectives of this pilot trial were to examine the safety of this combination and the possibility of blinding, while the effect on disease progression was considered as a secondary endpoint. Over a follow-up period of 1 year and in this selected patient group, there was no significant improvement in the Expanded Disability Status Scale score (EDSS) as compared with that of the placebo-control group. Toxicity was too high to consider additional trials with this combination of metalloproteinase inhibitors. Although peroral treatment is by most MS patients acknowledged as a major improvement in treatment compliance, one has to await the development of more selective and efficacious protease inhibitors than those used in the combination therapy described here.
Adult↗
Caroli's syndrome.
We report the case of a two-year-old girl presenting with recurrent episodes of fever and hepatomegaly. High resolution ultrasound (US) and computed tomography (CT) of the liver show dilated intrahepatic bile ducts with bridge formation and intraluminal protrusions. The extrahepatic bile duct is normal. The diagnosis of irregular dilated bile ducts with signs of cholangitis and cholangiolitis is proved by liver biopsy. The histologically associated congenital liver fibrosis in our patient enter into the Caroli's syndrome. We illustrate the high resolution US and the CT findings in this case.
Bile Ducts, Intrahepatic↗
Cloning and expression in Escherichia coli of a human gelatinase B-inhibitory single-chain immunoglobulin variable fragment (scFv).
The murine monoclonal antibody REGA-3G12 selectively and specifically inhibits the activity of human gelatinase B. The cDNA fragments which encode the variable regions of the light and heavy chains were isolated by PCR-mediated cloning and sequenced. Single-chain Fv expression constructs for Escherichia coli were generated in which c-myc tag sequences were encoded. Inducible expression of the scFv and secretion to the periplasm were obtained with higher yields when the c-myc tag sequence was positioned at the amino-terminal side. The inhibitory activity of purified scFv on neutrophil gelatinase B was tested in a gelatin degradation assay and it was found to possess a similar specific activity as that of the intact monoclonal antibody and of the pepsin-clipped F(ab')2 derivative. This shows for the first time that inhibition of soluble enzymes with scFv is possible and opens new perspectives for the treatment of diseases with excessive and detrimental enzyme production in the host.
Amino Acid Sequence↗
A novel human CC chemokine PARC that is most homologous to macrophage-inflammatory protein-1 alpha/LD78 alpha and chemotactic for T lymphocytes, but not for monocytes.
By searching the expressed sequence tag (EST) database, we identified partial cDNA sequences encoding a polypeptide with significant sequence identity to the human CC chemokine macrophage-inflammatory protein-1 alpha (MIP-1 alpha)/LD78 alpha. We determined the complete cDNA sequence that contained a reading frame of 89 amino acids with 61% identity to human MIP-1 alpha/LD78 alpha. The mRNA was expressed constitutively at high levels in human lung and at low levels in some lymphoid tissues. Furthermore, the mRNA was strongly induced in several human cell lines, including monocytic U937 cells, by PMA. From these results, we designated this novel CC chemokine as PARC from pulmonary and activation-regulated chemokine. In situ hybridization analyses showed that alveolar macrophages, follicular dendritic cells in the germinal centers of regional lymph nodes, and peripheral blood monocytes stimulated with LPS express PARC mRNA. Using the human CC chemokine yeast artificial chromosome contig that we constructed recently, we mapped the PARC gene (SCYA18) within one of the two subregions of the CC chemokine gene cluster at chromosome 17q11.2. To investigate its biologic activity, the PARC protein was expressed in insect cells. PARC was chemotactic for both activated (CD3+) T cells and nonactivated (CD14-) lymphocytes, but not for monocytes or granulocytes. Binding analysis using PARC fused with alkaline phosphatase-(His)6 showed the presence of a single class of receptors for PARC on lymphocytes with a Kd of 1.9 nM and 590 sites/cell. Thus, PARC is a novel CC chemokine with a close phylogenic relationship with MIP-1 alpha/LD78 alpha, but with a highly selective activity on lymphocytes.
Amino Acid Sequence↗