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Cellular models and cytotoxicity of pinnatoxin-G and extracts of the dinoflagellate Vulcanodinium rugosum recently isolated from the French mediterranean lagoon of Ingril
Abstracts / Toxicon 75 (2013) 205–223
dependent or Ca2+-activated K+ channels. Here, we have studied the venom of the Moroccan Buthus occitanus Paris (BoP), collected in the Rif Mountains near Chefchaouen, in order to find new peptides which could enlarge our structure-function relationship knowledge on the Kv1.3 blocker Kaliotoxin (KTX) family (alpha-Ktx3.1). Since more a decade, KTX is widely used by international investigators because it exhibits a quite sharp specificity and a high-affinity for the Kv1.3 channels, which is not only a neuronal channel but also a therapeutic target for diverse autoimmune diseases such as multiple sclerosis, type-1 diabetes, and rheumatoid arthritis. The scorpion Buthus occitanus is widely spread in North Africa and Middle East. Its classification into different sub-species is particularly elusive because of poorly distinctive characters, but previous analysis and lethality tests have shown that the venom composition greatly varied from sub-species. The BoP venom was first investigated using HPLC and MALDI-TOF-MS. The HPLC fractions were then screened by ELISA tests with antibodies raised against KTX. At least three components toxic in mice by intracerebroventricular injection were recognized by these antibodies, and were further characterized pharmacologically by competition with 125I-KTX bound to its specific binding sites on rat brain synaptosomes. A single component (4161 Da) inhibited in full the binding of 125 I-KTX with high-affinity (IC50 ¼ 0.1 nM), while the two other components were almost unable to displace the 125 I-KTX from its sites (IC50 > 100 nM). These toxins were fully sequenced using Edman's degradation. The high-affinity (BoP-KTX) ligand shares 86% sequence similarity with KTX and was classified as a-KTx3.14 toxin according to the classification previously published. The two others peptides (4093 Da and 4121 Da, respectively) differ only by Arg versus Lys. Their amino acid sequences were very similar to Martentoxin purified from the Chinese scorpion Buthus martenzi Karch (81% and 78%, respectively), which is as a BKCa and Kv1.3 blocker. Accordingly, they belong to the a-KTx16 family. http://dx.doi.org/10.1016/j.toxicon.2013.08.028
Protein chip technology for rapid and sensitive detection of botulinum neurotoxin activity G. Ferracci a, Y. Maulet b, C. Mazuet c, M.R. Popoff c, O. El Far b, M. Seagar b, C. Lévêque b a
Aix-Marseille Université, CNRS, CRN2M UMR7286, Faculté de Médecine, Bd Pierre Dramard, 13344 Marseille cedex 15, France b Aix-Marseille Université, INSERM, UNIS UMR1072, Faculté de Médecine, Bd Pierre Dramard, 13344 Marseille cedex 15, France c Institut Pasteur, CNR Bactéries anaérobies et botulisme, Unité Bactéries anaérobies et toxines, 25-28 rue du Docteur Roux, 75724 Paris cedex 15, France
Botulinum neurotoxins (BoNTs) are dangerous pathogens and powerful therapeutic agents. They have highly specific intra-cellular endoprotease activity directed towards synaptic vesicle or plasma membrane SNAREs, and induce blockade of synaptic transmission. Surveillance and diagnostics require rapid methods for BoNT detection. Our goal is to develop in vitro SPR (Surface Plasmon Resonance) methods for the detection of these toxins in order to replace the current LD50 reference method applied in animals.
215
Monoclonal antibodies recognizing specifically the BoNT/A or B cleavage products have been generated to assay the enzymatic activity of BoNTs using native or recombinant substrates. SPR methods provide rapid and label-free onestep procedures for routine assay of BoNTs near or below femtomolar concentrations. These assays are sensitive enough to detect the neurotoxin in sera from patients with botulism, but not in healthy controls or patients with other neurological diseases. Furthermore, we have developed cellbased assays that constitute the first method to date to rapidly quantify BoNT activity and could be particularly useful to detect neutralizing anti-toxin antibodies in patients treated with BoNTs and/or to screen for potential inhibitors. http://dx.doi.org/10.1016/j.toxicon.2013.08.029
Cellular models and cytotoxicity of pinnatoxin-G and extracts of the dinoflagellate Vulcanodinium rugosum recently isolated from the French mediterranean lagoon of Ingril M. Geiger a, b, G. Deslanglois c, K. Hogeveen c, V. Fessard c, E. Abadie d, T. Leprêtre a, b, F. Hervé a, V. Séchet a, R. Aráoz e, J. Molgó e, O. Grovel b, Y.F. Pouchus b, P. Hess a a
Ifremer, Laboratoire Phycotoxines, Centre Atlantique, 44311 Nantes Cedex, France b LUNAM, Université de Nantes, MMS EA2160, Faculté de Pharmacie, 44035 Nantes, France c ANSES, Unité de toxicologie des contaminants, 35302 Fougères, France d Ifremer, Laboratoire Environnement Ressource – Languedoc Roussillon, 34203 Sète, France e CNRS, Lab. Neurobiologie & Développement, UPR 3294, 91198 Gif sur Yvette, France
Pinnatoxins (PnTXs) are fast-acting neurotoxins that may accumulate in shellfish. While PnTxs are known since 1990, Vulcanodinium rugosum, the producing organism, has only been identified in 2011. PnTX-G induces strong neurological effects in mice; however, to date no human intoxication through consumption of contaminated shellfish has been confirmed. The present study aimed at clarifying the cytotoxicity of PnTX-G and V. rugosum extracts. PnTX-G was obtained from NRCC. A cytotoxicity study was carried out using different cell lines and incubation times: KB (72 h), Caco-2 (24 h) and Neuro2A (24 h). V. rugosum was grown in batch at 22 C (8 to 9 L of L1 medium, made up in Mediterranean Sea water of 40 psu). Crude extracts of V. rugosum exhibited significant cytotoxicity, with IC50s around 200 ng mL1 for KB cells and 326 ng mL-1 for Neuro2A cells. No reduction of cell viability was observed for pure PnTX-G up to 32 ng mL1 (Caco-2 cells), and up to 100 ng mL1 (KB cells). Crude extracts were purified using liquid-liquid partitioning and fractionated on open-column silica chromatography. Cell-based assays revealed strong activity of fractions containing no PnTX-G, suggesting the presence of cytotoxic compounds other than PnTX-G in V. rugosum. Trans-epithelial electrical resistance (TEER) of differentiated Caco2 cells was used as a model of the intestinal barrier. Purified PnTX-G did not affect TEER and was easily transported across the epithelial barrier within 24 h. V. rugosum crude and purified extracts strongly
216
Abstracts / Toxicon 75 (2013) 205–223
affected the cellular barrier also indicating the presence of other bioactive compounds. http://dx.doi.org/10.1016/j.toxicon.2013.08.030
Study on the usefulness and limitations of a cytotoxicity bio-assay using KB cells to detect lipophilic toxins in shellfish matrices M. Geiger a, b, S. Brochard b, V. Séchet a, O. Grovel b, Y.F. Pouchus b, P. Hess a a
Ifremer, EMP/PHYC Laboratory, Rue de l'Ile d'Yeu, 44311 Nantes, France University of Nantes, L'UNAM University, University of Nantes, MMS, BP53508, 44035 Nantes, France b
Mouse bioassays have been widely used to detect shellfish toxins. Due to major drawbacks, a general trend exists to move away from this assay. Thus, the development of alternative methods becomes necessary for the detection of emerging toxins. Previous studies demonstrated potential of cell-based assays for detecting lipophilic toxins. The present work aimed at complementing these studies through critical assessment of a cytotoxicity assay based on KB cells. Cellbased assays have no ethical concern, and present different advantages: ease of use, low requirement of laboratory facilities, low extract consumption and possibility of highthroughput. Moreover, KB cells are robust, with a low sensitivity to variations of culture conditions. Studies were carried out at different levels of matrix: pure toxins, algal and mussel extracts. KB cells were capable of discrimination between toxic and non-toxic micro-algae, with kinetic studies suggesting a minimum exposure of 48 h, even for pure toxins. Matrix effects of mussels were evaluated with crude and purified extracts of mussel hepatopancreas. Maximum concentrations of matrix not interfering with the assay were determined for different levels of purification. Even when extracts were partially purified, matrix effects observed represent a major drawback of this assay. When respecting maximum matrix concentration, the LOD for OA was around the regulatory limit while the LOD for AZA1 was 100-fold below the regulatory limit. Low sample consumption makes the use of this test attractive for the search of unknown toxins. However, matrix effects in shellfish and large difference in LODs between toxins are significant obstacles for regular use in surveillance programmes.
known to humans, and is responsible for the severe neurological illness known as tetanus characterized by generalized muscle spasms and rigid paralysis. TeNT is known to enter motor nerve terminals of the neuromuscular junction and is retrogradely transported along the motoneuron axons to the cell body. TeNT is transcytosed into adjacent inhibitory interneurons where it blocks the release of inhibitory neuro-transmitters (i.e., gammaaminobutyric acid and glycine), leading to spastic paralysis. Vaccination is the best way to prevent tetanus disease, but its protection does not last forever. Adults should get a tetanus shot, or booster, every 10 years. In the present work we have studied the efficacy of fully human recombinant monoclonal antibodies (Mabs) directed to TeNT, and obtained from B-cells of a recently immunized blood donor, using “single cell PCR” method. This approach entails the isolation of B-cells by a cell sorter coupled to a cytometer using a B-cell selective marker, anti-CD19 associated to a Bcell specific marker, C-ter fragment of TeNT-FITC-labeled. RT-PCR on single cell allowed the conversion of total RNA to cDNA, which was amplified using specific primers to select variable regions of the heavy and light chain of IgG. These variables regions were cloned into an expression vector containing the corresponding constant part of human IgG1. The expression vectors were then transiently transfected into CHO cells before purification of the expressed Mabs. Several new antitetanus Mabs were characterized in order to demonstrate their protective quality as compared to that of the therapeutic human polyclonal antibodies, Gammatetanos (LFB). Mice were injected with different doses of antibodies combined to lethal doses of TeNT. One of the tested Mabs exhibited a remarkable efficacy at nano-gram concentrations. In addition, we have tested the capacity of this human Mab to inhibit the binding and/or translocation of TeNT within motor nerve terminals. For this, we have used a fusion protein composed of the atoxic C-terminal fragment of TeNT coupled to the fluorescent probe Cy3 and isolated mouse neuromuscular preparations with or without the presence of antibodies. Confocal microscopy revealed that the fusion protein rapidly clustered inside motor nerve terminals of the neuromuscular junction under control conditions, and this internalization was markedly reduced when the novel effective human Mab was used. Comparison with Gammatetanos was also performed. Our results suggest a high therapeutic potential for this novel human monoclonal antibody.
http://dx.doi.org/10.1016/j.toxicon.2013.08.031 http://dx.doi.org/10.1016/j.toxicon.2013.08.032
In vivo and in vitro studies with a novel human monoclonal antibody against tetanus neurotoxin a
a
b
b
E. Girard , P. Villeneuve , V. Devos , A.S. Dezetter , A. Fontayne b, M.R. Popoff c, S. Jorieux b, J. Molgó a a
CNRS, Institut de Neurobiologie Alfred Fessard, laboratoire de Neurobiologie et Développement UPR3294, 91198 Gif-sur-Yvette, France b LFB Biotechnologies, Lille, France c Institut Pasteur, Département Microbiologie, Unité des Bactéries Anaérobies et Toxines, Paris, France
Tetanus neurotoxin (TeNT) synthesized by the anaerobic Clostridium tetani is one of the most deadly bacterial toxins
Development of potent small-molecule inhibitors of Shiga toxin N. Gupta a, R. Noël b, V. Pons b, A. Goudet a, J. Martinez a, D. Garcia-Castillo c, A. Michau a, L. Johannes c, D. Gillet a, J. Barbier a, J.C. Cintrat b a CEA, Institute of Biology and Technology (iBiTec-S), Service d'Ingénierie Moléculaire des Protéines (SIMOPRO), 91191 Gif-sur-Yvette, France b CEA, Institute of Biology and Technology (iBiTec-S), Service de Chimie Biorganique et Moléculaire (SCBM), Gif-sur-Yvette 91191, France c Traffic, Signaling, and Delivery Laboratory, Centre de Recherche, Institut Curie, 26 rue d'Ulm, 75248 Paris Cedex 05, France
dependent or Ca2+-activated K+ channels. Here, we have studied the venom of the Moroccan Buthus occitanus Paris (BoP), collected in the Rif Mountains near Chefchaouen, in order to find new peptides which could enlarge our structure-function relationship knowledge on the Kv1.3 blocker Kaliotoxin (KTX) family (alpha-Ktx3.1). Since more a decade, KTX is widely used by international investigators because it exhibits a quite sharp specificity and a high-affinity for the Kv1.3 channels, which is not only a neuronal channel but also a therapeutic target for diverse autoimmune diseases such as multiple sclerosis, type-1 diabetes, and rheumatoid arthritis. The scorpion Buthus occitanus is widely spread in North Africa and Middle East. Its classification into different sub-species is particularly elusive because of poorly distinctive characters, but previous analysis and lethality tests have shown that the venom composition greatly varied from sub-species. The BoP venom was first investigated using HPLC and MALDI-TOF-MS. The HPLC fractions were then screened by ELISA tests with antibodies raised against KTX. At least three components toxic in mice by intracerebroventricular injection were recognized by these antibodies, and were further characterized pharmacologically by competition with 125I-KTX bound to its specific binding sites on rat brain synaptosomes. A single component (4161 Da) inhibited in full the binding of 125 I-KTX with high-affinity (IC50 ¼ 0.1 nM), while the two other components were almost unable to displace the 125 I-KTX from its sites (IC50 > 100 nM). These toxins were fully sequenced using Edman's degradation. The high-affinity (BoP-KTX) ligand shares 86% sequence similarity with KTX and was classified as a-KTx3.14 toxin according to the classification previously published. The two others peptides (4093 Da and 4121 Da, respectively) differ only by Arg versus Lys. Their amino acid sequences were very similar to Martentoxin purified from the Chinese scorpion Buthus martenzi Karch (81% and 78%, respectively), which is as a BKCa and Kv1.3 blocker. Accordingly, they belong to the a-KTx16 family. http://dx.doi.org/10.1016/j.toxicon.2013.08.028
Protein chip technology for rapid and sensitive detection of botulinum neurotoxin activity G. Ferracci a, Y. Maulet b, C. Mazuet c, M.R. Popoff c, O. El Far b, M. Seagar b, C. Lévêque b a
Aix-Marseille Université, CNRS, CRN2M UMR7286, Faculté de Médecine, Bd Pierre Dramard, 13344 Marseille cedex 15, France b Aix-Marseille Université, INSERM, UNIS UMR1072, Faculté de Médecine, Bd Pierre Dramard, 13344 Marseille cedex 15, France c Institut Pasteur, CNR Bactéries anaérobies et botulisme, Unité Bactéries anaérobies et toxines, 25-28 rue du Docteur Roux, 75724 Paris cedex 15, France
Botulinum neurotoxins (BoNTs) are dangerous pathogens and powerful therapeutic agents. They have highly specific intra-cellular endoprotease activity directed towards synaptic vesicle or plasma membrane SNAREs, and induce blockade of synaptic transmission. Surveillance and diagnostics require rapid methods for BoNT detection. Our goal is to develop in vitro SPR (Surface Plasmon Resonance) methods for the detection of these toxins in order to replace the current LD50 reference method applied in animals.
215
Monoclonal antibodies recognizing specifically the BoNT/A or B cleavage products have been generated to assay the enzymatic activity of BoNTs using native or recombinant substrates. SPR methods provide rapid and label-free onestep procedures for routine assay of BoNTs near or below femtomolar concentrations. These assays are sensitive enough to detect the neurotoxin in sera from patients with botulism, but not in healthy controls or patients with other neurological diseases. Furthermore, we have developed cellbased assays that constitute the first method to date to rapidly quantify BoNT activity and could be particularly useful to detect neutralizing anti-toxin antibodies in patients treated with BoNTs and/or to screen for potential inhibitors. http://dx.doi.org/10.1016/j.toxicon.2013.08.029
Cellular models and cytotoxicity of pinnatoxin-G and extracts of the dinoflagellate Vulcanodinium rugosum recently isolated from the French mediterranean lagoon of Ingril M. Geiger a, b, G. Deslanglois c, K. Hogeveen c, V. Fessard c, E. Abadie d, T. Leprêtre a, b, F. Hervé a, V. Séchet a, R. Aráoz e, J. Molgó e, O. Grovel b, Y.F. Pouchus b, P. Hess a a
Ifremer, Laboratoire Phycotoxines, Centre Atlantique, 44311 Nantes Cedex, France b LUNAM, Université de Nantes, MMS EA2160, Faculté de Pharmacie, 44035 Nantes, France c ANSES, Unité de toxicologie des contaminants, 35302 Fougères, France d Ifremer, Laboratoire Environnement Ressource – Languedoc Roussillon, 34203 Sète, France e CNRS, Lab. Neurobiologie & Développement, UPR 3294, 91198 Gif sur Yvette, France
Pinnatoxins (PnTXs) are fast-acting neurotoxins that may accumulate in shellfish. While PnTxs are known since 1990, Vulcanodinium rugosum, the producing organism, has only been identified in 2011. PnTX-G induces strong neurological effects in mice; however, to date no human intoxication through consumption of contaminated shellfish has been confirmed. The present study aimed at clarifying the cytotoxicity of PnTX-G and V. rugosum extracts. PnTX-G was obtained from NRCC. A cytotoxicity study was carried out using different cell lines and incubation times: KB (72 h), Caco-2 (24 h) and Neuro2A (24 h). V. rugosum was grown in batch at 22 C (8 to 9 L of L1 medium, made up in Mediterranean Sea water of 40 psu). Crude extracts of V. rugosum exhibited significant cytotoxicity, with IC50s around 200 ng mL1 for KB cells and 326 ng mL-1 for Neuro2A cells. No reduction of cell viability was observed for pure PnTX-G up to 32 ng mL1 (Caco-2 cells), and up to 100 ng mL1 (KB cells). Crude extracts were purified using liquid-liquid partitioning and fractionated on open-column silica chromatography. Cell-based assays revealed strong activity of fractions containing no PnTX-G, suggesting the presence of cytotoxic compounds other than PnTX-G in V. rugosum. Trans-epithelial electrical resistance (TEER) of differentiated Caco2 cells was used as a model of the intestinal barrier. Purified PnTX-G did not affect TEER and was easily transported across the epithelial barrier within 24 h. V. rugosum crude and purified extracts strongly
216
Abstracts / Toxicon 75 (2013) 205–223
affected the cellular barrier also indicating the presence of other bioactive compounds. http://dx.doi.org/10.1016/j.toxicon.2013.08.030
Study on the usefulness and limitations of a cytotoxicity bio-assay using KB cells to detect lipophilic toxins in shellfish matrices M. Geiger a, b, S. Brochard b, V. Séchet a, O. Grovel b, Y.F. Pouchus b, P. Hess a a
Ifremer, EMP/PHYC Laboratory, Rue de l'Ile d'Yeu, 44311 Nantes, France University of Nantes, L'UNAM University, University of Nantes, MMS, BP53508, 44035 Nantes, France b
Mouse bioassays have been widely used to detect shellfish toxins. Due to major drawbacks, a general trend exists to move away from this assay. Thus, the development of alternative methods becomes necessary for the detection of emerging toxins. Previous studies demonstrated potential of cell-based assays for detecting lipophilic toxins. The present work aimed at complementing these studies through critical assessment of a cytotoxicity assay based on KB cells. Cellbased assays have no ethical concern, and present different advantages: ease of use, low requirement of laboratory facilities, low extract consumption and possibility of highthroughput. Moreover, KB cells are robust, with a low sensitivity to variations of culture conditions. Studies were carried out at different levels of matrix: pure toxins, algal and mussel extracts. KB cells were capable of discrimination between toxic and non-toxic micro-algae, with kinetic studies suggesting a minimum exposure of 48 h, even for pure toxins. Matrix effects of mussels were evaluated with crude and purified extracts of mussel hepatopancreas. Maximum concentrations of matrix not interfering with the assay were determined for different levels of purification. Even when extracts were partially purified, matrix effects observed represent a major drawback of this assay. When respecting maximum matrix concentration, the LOD for OA was around the regulatory limit while the LOD for AZA1 was 100-fold below the regulatory limit. Low sample consumption makes the use of this test attractive for the search of unknown toxins. However, matrix effects in shellfish and large difference in LODs between toxins are significant obstacles for regular use in surveillance programmes.
known to humans, and is responsible for the severe neurological illness known as tetanus characterized by generalized muscle spasms and rigid paralysis. TeNT is known to enter motor nerve terminals of the neuromuscular junction and is retrogradely transported along the motoneuron axons to the cell body. TeNT is transcytosed into adjacent inhibitory interneurons where it blocks the release of inhibitory neuro-transmitters (i.e., gammaaminobutyric acid and glycine), leading to spastic paralysis. Vaccination is the best way to prevent tetanus disease, but its protection does not last forever. Adults should get a tetanus shot, or booster, every 10 years. In the present work we have studied the efficacy of fully human recombinant monoclonal antibodies (Mabs) directed to TeNT, and obtained from B-cells of a recently immunized blood donor, using “single cell PCR” method. This approach entails the isolation of B-cells by a cell sorter coupled to a cytometer using a B-cell selective marker, anti-CD19 associated to a Bcell specific marker, C-ter fragment of TeNT-FITC-labeled. RT-PCR on single cell allowed the conversion of total RNA to cDNA, which was amplified using specific primers to select variable regions of the heavy and light chain of IgG. These variables regions were cloned into an expression vector containing the corresponding constant part of human IgG1. The expression vectors were then transiently transfected into CHO cells before purification of the expressed Mabs. Several new antitetanus Mabs were characterized in order to demonstrate their protective quality as compared to that of the therapeutic human polyclonal antibodies, Gammatetanos (LFB). Mice were injected with different doses of antibodies combined to lethal doses of TeNT. One of the tested Mabs exhibited a remarkable efficacy at nano-gram concentrations. In addition, we have tested the capacity of this human Mab to inhibit the binding and/or translocation of TeNT within motor nerve terminals. For this, we have used a fusion protein composed of the atoxic C-terminal fragment of TeNT coupled to the fluorescent probe Cy3 and isolated mouse neuromuscular preparations with or without the presence of antibodies. Confocal microscopy revealed that the fusion protein rapidly clustered inside motor nerve terminals of the neuromuscular junction under control conditions, and this internalization was markedly reduced when the novel effective human Mab was used. Comparison with Gammatetanos was also performed. Our results suggest a high therapeutic potential for this novel human monoclonal antibody.
http://dx.doi.org/10.1016/j.toxicon.2013.08.031 http://dx.doi.org/10.1016/j.toxicon.2013.08.032
In vivo and in vitro studies with a novel human monoclonal antibody against tetanus neurotoxin a
a
b
b
E. Girard , P. Villeneuve , V. Devos , A.S. Dezetter , A. Fontayne b, M.R. Popoff c, S. Jorieux b, J. Molgó a a
CNRS, Institut de Neurobiologie Alfred Fessard, laboratoire de Neurobiologie et Développement UPR3294, 91198 Gif-sur-Yvette, France b LFB Biotechnologies, Lille, France c Institut Pasteur, Département Microbiologie, Unité des Bactéries Anaérobies et Toxines, Paris, France
Tetanus neurotoxin (TeNT) synthesized by the anaerobic Clostridium tetani is one of the most deadly bacterial toxins
Development of potent small-molecule inhibitors of Shiga toxin N. Gupta a, R. Noël b, V. Pons b, A. Goudet a, J. Martinez a, D. Garcia-Castillo c, A. Michau a, L. Johannes c, D. Gillet a, J. Barbier a, J.C. Cintrat b a CEA, Institute of Biology and Technology (iBiTec-S), Service d'Ingénierie Moléculaire des Protéines (SIMOPRO), 91191 Gif-sur-Yvette, France b CEA, Institute of Biology and Technology (iBiTec-S), Service de Chimie Biorganique et Moléculaire (SCBM), Gif-sur-Yvette 91191, France c Traffic, Signaling, and Delivery Laboratory, Centre de Recherche, Institut Curie, 26 rue d'Ulm, 75248 Paris Cedex 05, France