Proceedings of the National Academy of Sciences, 2016
Genetic polymorphisms in coding genes play an important role when using mouse inbred strains as r... more Genetic polymorphisms in coding genes play an important role when using mouse inbred strains as research models. They have been shown to influence research results, explain phenotypical differences between inbred strains, and increase the amount of interesting gene variants present in the many available inbred lines. SPRET/Ei is an inbred strain derived from Mus spretus that has ∼1% sequence difference with the C57BL/6J reference genome. We obtained a listing of all SNPs and insertions/deletions (indels) present in SPRET/Ei from the Mouse Genomes Project (Wellcome Trust Sanger Institute) and processed these data to obtain an overview of all transcripts having nonsynonymous coding sequence variants. We identified 8,883 unique variants affecting 10,096 different transcripts from 6,328 protein-coding genes, which is about 28% of all coding genes. Because only a subset of these variants results in drastic changes in proteins, we focused on variations that are nonsense mutations that ult...
TNF is a central actor during inflammation and a well-recognized drug target for inflammatory dis... more TNF is a central actor during inflammation and a well-recognized drug target for inflammatory diseases. We found that the mouse strain SPRET/Ei, known for extreme and dominant resistance against TNF-induced shock, displays weak expression of TNF receptor 1 protein (TNFR1) but normal mRNA expression, a trait genetically linked to the major TNFR1 coding gene Tnfrsf1a and to a locus harbouring the predicted TNFR1-regulating miR-511. This miRNA is a genuine TNFR1 regulator in cells. In mice, overexpression of miR-511 down-regulates TNFR1 and protects against TNF, while anti-miR-511 up-regulates TNFR1 and sensitizes for TNF, breaking the resistance of SPRET/Ei. We found that miR-511 inhibits endotoxemia and experimental hepatitis and that this miR is strongly induced by glucocorticoids and is a true TNFR1 modulator and thus an anti-inflammatory miR. Since minimal reductions of TNFR1 have considerable effects on TNF sensitivity, we believe that at least part of the anti-inflammatory effec...
The first FDA-approved drugs were small, chemically-manufactured and highly active 13 molecules w... more The first FDA-approved drugs were small, chemically-manufactured and highly active 13 molecules with possible off-target effects. After this first successful wave of small drugs, biotechnology 14 allowed the development of protein-based medicines such as antibodies. Conventional antibodies bind 15 a specific protein and are becoming increasingly important in the therapeutic landscape. A very 16 prominent class of biologicals are the anti-TNF drugs that are applied in several inflammatory diseases 17 that are characterized by dysregulated TNF levels. Marketing of TNF inhibitors revolutionized the 18 treatment of diseases such as Crohn’s disease. However, these inhibitors also have undesired effects, 19 some of them directly associated with the inherent nature of this drug class such as immunogenicity, 20 whereas others are linked with their mechanism of action. Recently, researchers tried to design 21 innovative drugs with reduced side effects aiming to make them more effective and s...
The first FDA-approved drugs were small, chemically-manufactured and highly active molecules with... more The first FDA-approved drugs were small, chemically-manufactured and highly active molecules with possible off-target effects. After this first successful wave of small drugs, biotechnology allowed the development of protein-based medicines such as antibodies. Conventional antibodies bind a specific protein and are becoming increasingly important in the therapeutic landscape. A very prominent class of biologicals are the anti-TNF drugs that are applied in several inflammatory diseases that are characterized by dysregulated TNF levels. Marketing of TNF inhibitors revolutionized the treatment of diseases such as Crohn’s disease. However, these inhibitors also have undesired effects, some of them directly associated with the inherent nature of this drug class such as immunogenicity, whereas others are linked with their mechanism of action. Recently, researchers tried to design innovative drugs with reduced side effects aiming to make them more effective and safer. Molecules with ...
Sepsis causes very high mortality and morbidity rates and remains one of the biggest medical chal... more Sepsis causes very high mortality and morbidity rates and remains one of the biggest medical challenges. This study investigates whether plasma levels of both matrix metalloproteinase 8 and tumor necrosis factor receptor 1 are associated with sepsis severity and also investigates the therapeutic applicability of simultaneous inhibition of the two molecules in sepsis. Observational human pilot study-prospective controlled animal study. University hospital and research laboratory. Sepsis patients and C57BL/6 mice deficient for matrix metalloproteinase 8 and/or tumor necrosis factor receptor 1. Plasma and whole blood RNA were collected from 13 sepsis patients for 7 consecutive days and within 24 hours of admission to ICU. Matrix metalloproteinase 8 and tumor necrosis factor receptor 1 plasma and expression levels were determined in these patients. Mice deficient for both matrix metalloproteinase 8 and tumor necrosis factor receptor 1 were generated and subjected to endotoxemia and ceca...
Members of the Camelidae (including camels and llamas) produce, in addition to conventional antib... more Members of the Camelidae (including camels and llamas) produce, in addition to conventional antibodies (Ab), a unique type of Ab that lacks light chains. The variable antigen-binding domains derived from these Ab are named 'nanobodies' (Nbs). Nbs exert high specificity and affinity and, when properly selected, are more stable than conventional Ab. Furthermore, their toxicity and immunogenicity are both low. They are easy to produce and their modularity makes them amenable for the generation of multivalent complexes. In this review, we discuss how Nbs are being explored as therapeutics in many fields of medicine, including oncology, inflammatory, infectious and neurological diseases, and imaging. In addition, we highlight their potential for use in the diagnosis and monitoring of diseases. Finally, we provide an extended overview of Nbs that are, or have been, involved in clinical trials.
Proceedings of the National Academy of Sciences, 2016
Genetic polymorphisms in coding genes play an important role when using mouse inbred strains as r... more Genetic polymorphisms in coding genes play an important role when using mouse inbred strains as research models. They have been shown to influence research results, explain phenotypical differences between inbred strains, and increase the amount of interesting gene variants present in the many available inbred lines. SPRET/Ei is an inbred strain derived from Mus spretus that has ∼1% sequence difference with the C57BL/6J reference genome. We obtained a listing of all SNPs and insertions/deletions (indels) present in SPRET/Ei from the Mouse Genomes Project (Wellcome Trust Sanger Institute) and processed these data to obtain an overview of all transcripts having nonsynonymous coding sequence variants. We identified 8,883 unique variants affecting 10,096 different transcripts from 6,328 protein-coding genes, which is about 28% of all coding genes. Because only a subset of these variants results in drastic changes in proteins, we focused on variations that are nonsense mutations that ultimately resulted in a gain of a stop codon. These genes were identified by in silico changing the C57BL/6J coding sequences to the SPRET/Ei sequences, converting them to amino acid (AA) sequences, and comparing the AA sequences. All variants and transcripts affected were also stored in a database, which can be browsed using a SPRET/Ei M. spretus variants web tool (www.spretus.org), including a manual. We validated the tool by demonstrating the loss of function of three proteins predicted to be severely truncated, namely Fas, IRAK2, and IFNγR1.
Repetitive application of topical imiquimod is used as an experimental model for the induction of... more Repetitive application of topical imiquimod is used as an experimental model for the induction of psoriasiform skin lesions in mice. The model is characterized by several inflammatory processes, including cytokine production both locally and systemically, cellular infiltration, and splenomegaly. To investigate the production of type I interferons in response to imiquimod-containing Aldara cream, IFNβ-luciferase reporter mice were imaged in vivo and ex vivo. Type I interferons were found to be produced in the skin, but also in the intestinal system caused by unintended ingestion of imiquimod by the mice. Through the use of Elizabethan collars to prevent ingestion, these effects, including psoriasiform lesions were nearly completely prevented. Our findings reveal that topical treatment with Aldara induces a psoriasiform skin inflammation, but that its mode of action depends on ingestion of the chemical, which leads to systemic responses and affects local inflammation. Therefore, potential ingestion of topical treatments during experimental procedures should be taken into account during assessment of cutaneous inflammatory parameters in skin disease models.
A detrimental role for matrix metalloproteinase 8 (MMP8) has been identified in several pathologi... more A detrimental role for matrix metalloproteinase 8 (MMP8) has been identified in several pathological conditions, e.g. lethal hepatitis and the systemic inflammatory response syndrome. Since matrix MMP8-deficient mice are protected in the above-mentioned diseases, specific MMP8 inhibitors could be of clinical value However, targeting a specific matrix metalloproteinase remains challenging due to the strong structural homology of matrix metalloproteinases, which form a family of 25 members in mammals.. Single domain antibodies, called Nanobodies, offer a range of possibilities towards therapy since they are easy to generate, express, produce and modify, e.g. by linkage to Nanobodies directed against other target molecules. Hence, we generated small MMP8-binding Nanobodies, and established a proof-of-principle for developing Nanobodies that inhibit matrix metalloproteinase activity. Also, we demonstrated for the first time the possibility of expressing Nanobodies systemically by in vivo electroporation of the muscle and its relevance as a potential therapy in inflammatory diseases.Molecular Therapy (2016); doi:10.1038/mt.2016.2.
TNF is a central actor during inflammation and a well-recognized drug target for inflammatory dis... more TNF is a central actor during inflammation and a well-recognized drug target for inflammatory diseases. We found that the mouse strain SPRET/Ei, known for extreme and dominant resistance against TNF-induced shock, displays weak expression of TNF receptor 1 protein (TNFR1) but normal mRNA expression, a trait genetically linked to the major TNFR1 coding gene Tnfrsf1a and to a locus harbouring the predicted TNFR1-regulating miR-511. This miRNA is a genuine TNFR1 regulator in cells. In mice, overexpression of miR-511 down-regulates TNFR1 and protects against TNF, while anti-miR-511 up-regulates TNFR1 and sensitizes for TNF, breaking the resistance of SPRET/Ei. We found that miR-511 inhibits endotoxemia and experimental hepatitis and that this miR is strongly induced by glucocorticoids and is a true TNFR1 modulator and thus an anti-inflammatory miR. Since minimal reductions of TNFR1 have considerable effects on TNF sensitivity, we believe that at least part of the anti-inflammatory effec...
Proceedings of the National Academy of Sciences, 2016
Genetic polymorphisms in coding genes play an important role when using mouse inbred strains as r... more Genetic polymorphisms in coding genes play an important role when using mouse inbred strains as research models. They have been shown to influence research results, explain phenotypical differences between inbred strains, and increase the amount of interesting gene variants present in the many available inbred lines. SPRET/Ei is an inbred strain derived from Mus spretus that has ∼1% sequence difference with the C57BL/6J reference genome. We obtained a listing of all SNPs and insertions/deletions (indels) present in SPRET/Ei from the Mouse Genomes Project (Wellcome Trust Sanger Institute) and processed these data to obtain an overview of all transcripts having nonsynonymous coding sequence variants. We identified 8,883 unique variants affecting 10,096 different transcripts from 6,328 protein-coding genes, which is about 28% of all coding genes. Because only a subset of these variants results in drastic changes in proteins, we focused on variations that are nonsense mutations that ult...
TNF is a central actor during inflammation and a well-recognized drug target for inflammatory dis... more TNF is a central actor during inflammation and a well-recognized drug target for inflammatory diseases. We found that the mouse strain SPRET/Ei, known for extreme and dominant resistance against TNF-induced shock, displays weak expression of TNF receptor 1 protein (TNFR1) but normal mRNA expression, a trait genetically linked to the major TNFR1 coding gene Tnfrsf1a and to a locus harbouring the predicted TNFR1-regulating miR-511. This miRNA is a genuine TNFR1 regulator in cells. In mice, overexpression of miR-511 down-regulates TNFR1 and protects against TNF, while anti-miR-511 up-regulates TNFR1 and sensitizes for TNF, breaking the resistance of SPRET/Ei. We found that miR-511 inhibits endotoxemia and experimental hepatitis and that this miR is strongly induced by glucocorticoids and is a true TNFR1 modulator and thus an anti-inflammatory miR. Since minimal reductions of TNFR1 have considerable effects on TNF sensitivity, we believe that at least part of the anti-inflammatory effec...
The first FDA-approved drugs were small, chemically-manufactured and highly active 13 molecules w... more The first FDA-approved drugs were small, chemically-manufactured and highly active 13 molecules with possible off-target effects. After this first successful wave of small drugs, biotechnology 14 allowed the development of protein-based medicines such as antibodies. Conventional antibodies bind 15 a specific protein and are becoming increasingly important in the therapeutic landscape. A very 16 prominent class of biologicals are the anti-TNF drugs that are applied in several inflammatory diseases 17 that are characterized by dysregulated TNF levels. Marketing of TNF inhibitors revolutionized the 18 treatment of diseases such as Crohn’s disease. However, these inhibitors also have undesired effects, 19 some of them directly associated with the inherent nature of this drug class such as immunogenicity, 20 whereas others are linked with their mechanism of action. Recently, researchers tried to design 21 innovative drugs with reduced side effects aiming to make them more effective and s...
The first FDA-approved drugs were small, chemically-manufactured and highly active molecules with... more The first FDA-approved drugs were small, chemically-manufactured and highly active molecules with possible off-target effects. After this first successful wave of small drugs, biotechnology allowed the development of protein-based medicines such as antibodies. Conventional antibodies bind a specific protein and are becoming increasingly important in the therapeutic landscape. A very prominent class of biologicals are the anti-TNF drugs that are applied in several inflammatory diseases that are characterized by dysregulated TNF levels. Marketing of TNF inhibitors revolutionized the treatment of diseases such as Crohn’s disease. However, these inhibitors also have undesired effects, some of them directly associated with the inherent nature of this drug class such as immunogenicity, whereas others are linked with their mechanism of action. Recently, researchers tried to design innovative drugs with reduced side effects aiming to make them more effective and safer. Molecules with ...
Sepsis causes very high mortality and morbidity rates and remains one of the biggest medical chal... more Sepsis causes very high mortality and morbidity rates and remains one of the biggest medical challenges. This study investigates whether plasma levels of both matrix metalloproteinase 8 and tumor necrosis factor receptor 1 are associated with sepsis severity and also investigates the therapeutic applicability of simultaneous inhibition of the two molecules in sepsis. Observational human pilot study-prospective controlled animal study. University hospital and research laboratory. Sepsis patients and C57BL/6 mice deficient for matrix metalloproteinase 8 and/or tumor necrosis factor receptor 1. Plasma and whole blood RNA were collected from 13 sepsis patients for 7 consecutive days and within 24 hours of admission to ICU. Matrix metalloproteinase 8 and tumor necrosis factor receptor 1 plasma and expression levels were determined in these patients. Mice deficient for both matrix metalloproteinase 8 and tumor necrosis factor receptor 1 were generated and subjected to endotoxemia and ceca...
Members of the Camelidae (including camels and llamas) produce, in addition to conventional antib... more Members of the Camelidae (including camels and llamas) produce, in addition to conventional antibodies (Ab), a unique type of Ab that lacks light chains. The variable antigen-binding domains derived from these Ab are named 'nanobodies' (Nbs). Nbs exert high specificity and affinity and, when properly selected, are more stable than conventional Ab. Furthermore, their toxicity and immunogenicity are both low. They are easy to produce and their modularity makes them amenable for the generation of multivalent complexes. In this review, we discuss how Nbs are being explored as therapeutics in many fields of medicine, including oncology, inflammatory, infectious and neurological diseases, and imaging. In addition, we highlight their potential for use in the diagnosis and monitoring of diseases. Finally, we provide an extended overview of Nbs that are, or have been, involved in clinical trials.
Proceedings of the National Academy of Sciences, 2016
Genetic polymorphisms in coding genes play an important role when using mouse inbred strains as r... more Genetic polymorphisms in coding genes play an important role when using mouse inbred strains as research models. They have been shown to influence research results, explain phenotypical differences between inbred strains, and increase the amount of interesting gene variants present in the many available inbred lines. SPRET/Ei is an inbred strain derived from Mus spretus that has ∼1% sequence difference with the C57BL/6J reference genome. We obtained a listing of all SNPs and insertions/deletions (indels) present in SPRET/Ei from the Mouse Genomes Project (Wellcome Trust Sanger Institute) and processed these data to obtain an overview of all transcripts having nonsynonymous coding sequence variants. We identified 8,883 unique variants affecting 10,096 different transcripts from 6,328 protein-coding genes, which is about 28% of all coding genes. Because only a subset of these variants results in drastic changes in proteins, we focused on variations that are nonsense mutations that ultimately resulted in a gain of a stop codon. These genes were identified by in silico changing the C57BL/6J coding sequences to the SPRET/Ei sequences, converting them to amino acid (AA) sequences, and comparing the AA sequences. All variants and transcripts affected were also stored in a database, which can be browsed using a SPRET/Ei M. spretus variants web tool (www.spretus.org), including a manual. We validated the tool by demonstrating the loss of function of three proteins predicted to be severely truncated, namely Fas, IRAK2, and IFNγR1.
Repetitive application of topical imiquimod is used as an experimental model for the induction of... more Repetitive application of topical imiquimod is used as an experimental model for the induction of psoriasiform skin lesions in mice. The model is characterized by several inflammatory processes, including cytokine production both locally and systemically, cellular infiltration, and splenomegaly. To investigate the production of type I interferons in response to imiquimod-containing Aldara cream, IFNβ-luciferase reporter mice were imaged in vivo and ex vivo. Type I interferons were found to be produced in the skin, but also in the intestinal system caused by unintended ingestion of imiquimod by the mice. Through the use of Elizabethan collars to prevent ingestion, these effects, including psoriasiform lesions were nearly completely prevented. Our findings reveal that topical treatment with Aldara induces a psoriasiform skin inflammation, but that its mode of action depends on ingestion of the chemical, which leads to systemic responses and affects local inflammation. Therefore, potential ingestion of topical treatments during experimental procedures should be taken into account during assessment of cutaneous inflammatory parameters in skin disease models.
A detrimental role for matrix metalloproteinase 8 (MMP8) has been identified in several pathologi... more A detrimental role for matrix metalloproteinase 8 (MMP8) has been identified in several pathological conditions, e.g. lethal hepatitis and the systemic inflammatory response syndrome. Since matrix MMP8-deficient mice are protected in the above-mentioned diseases, specific MMP8 inhibitors could be of clinical value However, targeting a specific matrix metalloproteinase remains challenging due to the strong structural homology of matrix metalloproteinases, which form a family of 25 members in mammals.. Single domain antibodies, called Nanobodies, offer a range of possibilities towards therapy since they are easy to generate, express, produce and modify, e.g. by linkage to Nanobodies directed against other target molecules. Hence, we generated small MMP8-binding Nanobodies, and established a proof-of-principle for developing Nanobodies that inhibit matrix metalloproteinase activity. Also, we demonstrated for the first time the possibility of expressing Nanobodies systemically by in vivo electroporation of the muscle and its relevance as a potential therapy in inflammatory diseases.Molecular Therapy (2016); doi:10.1038/mt.2016.2.
TNF is a central actor during inflammation and a well-recognized drug target for inflammatory dis... more TNF is a central actor during inflammation and a well-recognized drug target for inflammatory diseases. We found that the mouse strain SPRET/Ei, known for extreme and dominant resistance against TNF-induced shock, displays weak expression of TNF receptor 1 protein (TNFR1) but normal mRNA expression, a trait genetically linked to the major TNFR1 coding gene Tnfrsf1a and to a locus harbouring the predicted TNFR1-regulating miR-511. This miRNA is a genuine TNFR1 regulator in cells. In mice, overexpression of miR-511 down-regulates TNFR1 and protects against TNF, while anti-miR-511 up-regulates TNFR1 and sensitizes for TNF, breaking the resistance of SPRET/Ei. We found that miR-511 inhibits endotoxemia and experimental hepatitis and that this miR is strongly induced by glucocorticoids and is a true TNFR1 modulator and thus an anti-inflammatory miR. Since minimal reductions of TNFR1 have considerable effects on TNF sensitivity, we believe that at least part of the anti-inflammatory effec...
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Papers by Sophie Steeland