Papers by Maurice Stassen
or visit the DOI to the publisher's website. • The final author version and the galley proof are ... more or visit the DOI to the publisher's website. • The final author version and the galley proof are versions of the publication after peer review. • The final published version features the final layout of the paper including the volume, issue and page numbers. Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal. If the publication is distributed under the terms of Article 25fa of the Dutch Copyright Act, indicated by the "Taverne" license above, please follow below link for the End User
Increased expression of rapsyn in muscles prevents acetylcholine receptor loss in experimental au... more Increased expression of rapsyn in muscles prevents acetylcholine receptor loss in experimental autoimmune myasthenia gravis
Increased expression of rapsyn in muscles prevents acetylcholine receptor loss in experimental au... more Increased expression of rapsyn in muscles prevents acetylcholine receptor loss in experimental autoimmune myasthenia gravis
Abs to U1 RNA are frequently found in patients suffering from systemic lupus erythematosus overla... more Abs to U1 RNA are frequently found in patients suffering from systemic lupus erythematosus overlap syndromes and Ab titers correlate with disease activity. We describe the isolation of the first human anti-U1 RNA autoantibodies from a combinatorial IgG library made from the bone marrow of a systemic lupus erythematosus patient. With the use of phage display technology, two anti-U1 RNA singlechain variable fragment (scFv) Abs were selected. Both high affinity anti-U1 RNA Ab fragments (K d ϳ 1 nM) recognize stem II of U1 RNA and were derived from the same heavy chain gene (VH3-11) and the same (3r) light chain gene although somatic mutations, predominantly present in the complementarity-determining regions, are different. Experiments, in which the heavy chain genes of both anti-U1 RNA scFvs were reshuffled with the original light chain repertoire of the patient resulted, after selection on stem loop II, in a large number of RNA-binding Ab fragments. All these stem loop II-specific RNA binding clones used a similar, but not identical, 3r light chain. When scFvs were selected from the reshuffled libraries by stem loop IV, representing the other autoantigenic site of U1 RNA, most selected Ab clones did react with stem loop IV, but no longer with stem loop II. The stem loop IV-reactive Ab clones contained different, not 3r-related, light chains. These results point to a major role for the light chain in determining the sequence specificity of these disease-related anti-U1 RNA Abs. The possibility that secondary light chain rearrangements are involved in this autoimmune response is discussed.
RNA, 1998
This is the first study in which the complex of a monoclonal autoantibody fragment and its target... more This is the first study in which the complex of a monoclonal autoantibody fragment and its target, stem loop II of U1 snRNA, was investigated with enzymatic and chemical probing. A phage display antibody library derived from bone marrow cells of an SLE patient was used for selection of scFvs specific for stem loop II. The scFv specificity was tested by RNA immunoprecipitation and nitrocellulose filter binding competition experiments. Immunofluorescence data and immunoprecipitation of U1 snRNPs containing U1A protein, pointed to an scFv binding site different from the U1A binding site. The scFv binding site on stem loop II was determined by footprinting experiments using RNase A, RNase V1, and hydroxyl radicals. The results show that the binding site covers three sequence elements on the RNA, one on the 59 strand of the stem and two on the 39 strand. Hypersensitivity of three loop nucleotides suggests a conformational change of the RNA upon antibody binding. A three-dimensional representation of stem loop II reveals a juxtapositioning of the three protected regions on one side of the helix, spanning approximately one helical turn. The location of the scFv binding site on stem loop II is in full agreement with the finding that both the U1A protein and the scFv are able to bind stem loop II simultaneously. As a consequence, this recombinant monoclonal anti-U1 snRNA scFv might be very useful in studies on U1 snRNPs and its involvement in cellular processes like splicing.
The Journal of Immunology
Abs to U1 RNA are frequently found in patients suffering from systemic lupus erythematosus overla... more Abs to U1 RNA are frequently found in patients suffering from systemic lupus erythematosus overlap syndromes and Ab titers correlate with disease activity. We describe the isolation of the first human anti-U1 RNA autoantibodies from a combinatorial IgG library made from the bone marrow of a systemic lupus erythematosus patient. With the use of phage display technology, two anti-U1 RNA single-chain variable fragment (scFv) Abs were selected. Both high affinity anti-U1 RNA Ab fragments (Kd ∼ 1 nM) recognize stem II of U1 RNA and were derived from the same heavy chain gene (VH3–11) and the same λ (3r) light chain gene although somatic mutations, predominantly present in the complementarity-determining regions, are different. Experiments, in which the heavy chain genes of both anti-U1 RNA scFvs were reshuffled with the original light chain repertoire of the patient resulted, after selection on stem loop II, in a large number of RNA-binding Ab fragments. All these stem loop II-specific R...
Chinese journal of microbiology and immunology, 2005
重症肌无力(myasthenia gravis,MG)是由乙酰胆碱受体(acetylcholine receptor,AchR) 抗体介导的自身免疫病.AchR抗体与神经肌肉接头处的AchR结合... more 重症肌无力(myasthenia gravis,MG)是由乙酰胆碱受体(acetylcholine receptor,AchR) 抗体介导的自身免疫病.AchR抗体与神经肌肉接头处的AchR结合,通过抗原调变、激活补体导致突触后膜裂解而使AchR数量减少,结果出现肌肉收缩无力等症状.MG的动物模型--实验性自身免疫性MG(experimental autoimmune MG,EAMG)可经主动免疫动物富含AchR的电鳐(Torpedo)电器官制备的AchR(tAchR)诱导出。
Chinese journal of immunology, 2009
Objective:To construct a monovalent IgG antibody gene for specific immunotherapy of myasthenia gr... more Objective:To construct a monovalent IgG antibody gene for specific immunotherapy of myasthenia gravis.Methods:A pathogenic anti-human acetylcholine receptor(AChR) antibody IgG637,previously developed in our laboratory,was mutated in the site of K322A to remove the complement binding activity,and 4 amino acids(PLKP) were deleted in complemetary determining region 3(CDR3) to remove the specific antigen binding activity by site-directed mutagenesis technology.The genes of mutant IgG637/K322A and IgG637/K322A/CDR3ΔPLKP were then transformed respectively into E.coli XL1-Blue for cloning,sequencing and furthermore transinfected into mammalian cell line CHO-k1 for expression.The complement C3-binding activity of expressed product IgG637/K322A was determined in ELISA,and the human AChR-binding activity of IgG637/K322A/CDR3ΔPLKP in RIA.The mutant antibody genes were further undergone mutation of knob(T366Y) and hole(Y407T) to favor the heterodimerization of H chain in making monovalent IgG antibody.Results:The mutant IgG637/K322A was not able to bind complement C3 in ELISA,and the mutant IgG637/K322A/CDR3ΔPLKP was not able to bind the specific antigen human AChR in RIA.The correct mutations of knob and hole were observed by sequencing.Conclusion:The genes of monovalent IgG anti-AChR antibody without complement-binding activity are successfully developed from a pathogenic anti-AChR antibody IgG637.
Journal of the Neurological Sciences, 2002
Myasthenia gravis is an autoimmune disease associated with antibodies directed to the postsynapti... more Myasthenia gravis is an autoimmune disease associated with antibodies directed to the postsynaptic acetylcholine receptor. These antibodies reduce the number of receptors. Autoantibodies against AChR and other muscle antigens can be used for the diagnosis of myasthenia gravis and related disorders. The origin and the role of these antibodies in the disease are discussed. Experimental autoimmune myasthenia gravis, an experimental model closely mimicking the disease, has provided answers to many questions about the role of antibodies, complement macrophages and AChR anchor proteins. Genetically modified anti-AChR antibodies may also be used in the future to treat myasthenia.
European Journal of Anaesthesiology, 2005
In myasthenic patients, the time course of action of non-depolarizing neuromuscular blocking agen... more In myasthenic patients, the time course of action of non-depolarizing neuromuscular blocking agents is prolonged and the sensitivity is increased. We used our antegrade perfused rat peroneal nerve anterior tibialis muscle model to investigate if this altered time course of effect and sensitivity can be explained by the decreased acetylcholine receptor concentration that is caused by the disease. Functional acetylcholine receptors were reduced by administration of alpha-bungarotoxin or by injecting monoclonal antibodies against rat acetylcholine receptors (experimental autoimmune myasthenia gravis). After induction of anaesthesia, the model was set up and perfusion of the tibialis anterior muscle with blood was started. After stabilization of the twitch, rocuronium or pancuronium were infused until 90% block was obtained. Twitch data and infusion data were recorded and used to calculate the time course of effect and potency. The potency of neuromuscular blocking agents was increased and the offset of the neuromuscular block was prolonged in both the alpha-bungarotoxin groups and the experimental autoimmune myasthenia gravis groups compared to controls. This study shows that the increased sensitivity to neuromuscular-blocking agents in myasthenia gravis can be accounted for by a decreased number of acetylcholine receptors. It also shows that the antegrade perfused rat peroneal nerve anterior tibialis muscle model is a suitable model to study the effects of myasthenia gravis on the time course of effect of neuromuscular blocking agents.
Brain, 2005
Myasthenia gravis is usually caused by autoantibodies to the acetylcholine receptor (AChR). The A... more Myasthenia gravis is usually caused by autoantibodies to the acetylcholine receptor (AChR). The AChR is clustered and anchored in the postsynaptic membrane of the neuromuscular junction (NMJ) by a cytoplasmic protein called rapsyn. We previously showed that resistance to experimental autoimmune myasthenia gravis (EAMG) in aged rats correlates with increased rapsyn concentration at the NMJ. It is possible, therefore, that endogenous rapsyn expression may be an important determinant of AChR loss and neuromuscular transmission failure in the human disease, and that upregulation of rapsyn expression could be used therapeutically. To examine first a potential therapeutic application of rapsyn upregulation, we induced acute EAMG in young rats by passive transfer of AChR antibody, mAb 35, and used in vivo electroporation to over-express rapsyn unilaterally in one tibialis anterior. We looked at the compound muscle action potentials (CMAPs) in the tibialis anterior, at rapsyn and AChR expression by quantitative radioimmunoassay and immunofluorescence, and at the morphology of the NMJs, comparing the electroporated and untreated muscles, as well as the control and EAMG rats. In control rats, transfected muscle fibres had extrasynaptic rapsyn aggregates, as well as slightly increased rapsyn and AChR concentrations at the NMJ. In EAMG rats, despite deposits of the membrane attack complex, the rapsyn-overexpressing muscles showed no decrement in the CMAPs, no loss of AChR, and the majority had normal postsynaptic folds, whereas endplates of untreated muscles showed typical AChR loss and morphological damage. These data suggest not only that increasing rapsyn expression could be a potential treatment for selected muscles of myasthenia gravis patients, but also lend support to the hypothesis that individual differences in innate rapsyn expression could be a factor in determining disease severity.
Autoimmunity, 2002
Pathogenic anti-acetylcholine receptor (AChR) antibodies in myasthenia gravis (MG) and the corres... more Pathogenic anti-acetylcholine receptor (AChR) antibodies in myasthenia gravis (MG) and the corresponding animal model, experimental autoimmune myasthenia gravis (EAMG), principally recognize the main immunogenic region (MIR) of the AChR. Bivalent anti-MIR antibodies binding to the f -subunits of AChR result in AChR loss by antigenic modulation and complement activation. Monovalent Fab and single-chain variable fragments (scFv) of pathogenic anti-AChR antibodies can interfere with AChR binding of the pathogenic antibodies. In the present study, scFv637 was constructed from its parental Fab637, previously isolated from a thymus-derived phage display library with specificity toward anti-MIR of human AChR (hAChR), by PCR amplification. Bacterial produced scFv637 was able to bind to hAChR in standard precipitation radioimmunoassay (RIA). ScFv637 also bound to monkey AChR in situ on monkey neuromuscular junctions as showed in immunohistochemical staining. Furthermore, scFv637 was capable of inhibiting the binding of its intact IgG637 and anti-MIR mAb35 binding to hAChR up to 32.9 and 73.0%, respectively demonstrated in a competitive ELISA, and of MG patient sera from up to 45.5% in a competitive RIA. Therefore, scFv637, easier for manipulation in improvement of affinity and stability compared with its parental Fab637, may serve as an alternative candidate for specific immunotherapy in MG.
Annals of the New York Academy of Sciences, 2003
Annals of the New York Academy of Sciences, 2003
Objective To construct human pathogenic anti-acetylcholine receptor (AchR) autoantibody to induce... more Objective To construct human pathogenic anti-acetylcholine receptor (AchR) autoantibody to induce an animal model of myasthenia gravis (MG) . Methods Antigen binding fragment (Fab637) of antibody against AchR, derived from the thymus of MG patients, was cloned into a mammalian expressed vector pIgG1, baring intact antibody IgG1 gene, to construct a recombinant vector (pIgG1-637) containing an intact human anti-AchR autoantibody (IgG1-637) gene. The pIgG1-637 was then transfected into CHO-k1 cell line for the expression of antibody IgG1-637. IgG1-637 was isolated and purified from a stablely expressed cell line screened by MSX (L-methionine sulfoxmine), and injected into rhesus monkeys to determine its pathogenicity by measuring muscle weakness and compound muscle action potential (CMAP) of the monkeys. Results The CHO-k1 cell line with highest expression of antibody IgG1-637 was selected by 20μmol/L MSX, and the yield of the antibody was 17.4μg/ml cell culture. Three monkeys, receiving injection of 1.7, 1.7 and 5.0 mg/kg IgG1-637 respectively, developed clinical symptoms of muscle weakness and decreased CMAP within 1 week after the first injection. Conclusion The constructed intact IgG1-637 from Fab637 is an anti-AchR autoantibody which can be used to induce EAMG in monkeys.
Anesthesiology, 2003
Background In myasthenic patients, the sensitivity for nondepolarizing relaxants is increased and... more Background In myasthenic patients, the sensitivity for nondepolarizing relaxants is increased and the time course of effect is prolonged due to a reduced number of functional acetylcholine receptors at the neuromuscular junction. The authors investigated both the performance of the link model proposed by Sheiner and a pharmacodynamic-pharmacokinetic model taking into account the number of unbound acetylcholine receptors in myasthenic pigs. Methods After obtaining the approval of the Animal Experiments Committee of their institution, the authors studied eight myasthenic pigs and eight control pigs. Myasthenia gravis was induced by injecting Torpedo acetylcholine receptors in weeks 1 and 4. On the day of the experiments, the pigs were anesthetized and intubated, and the appropriate muscles and nerves were prepared for the measurements. Rocuronium was administered by infusion to reach 90% twitch height block. Arterial blood was sampled during onset and offset of effect, and the plasma ...
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Papers by Maurice Stassen