RESUMEN
Prostate cancer (PCa) is the second most common cancer in men, causing more than 300,000 deaths every year worldwide. Due to their superior cell-killing ability and the relative simplicity of their preparation, immunotoxin molecules have great potential in the clinical treatment of cancer, and several such molecules have been approved for clinical application. In this study, we adopted a relatively simple strategy based on a single-domain antibody (sdAb) and an improved Pseudomonas exotoxin A (PE) toxin (PE24X7) to prepare a safer immunotoxin against prostate-specific membrane antigen (PSMA) for PCa treatment. The designed anti-PSMA immunotoxin, JVM-PE24X7, was conveniently prepared in its soluble form in an Escherichia coli (E. coli) system, avoiding the complex renaturation process needed for immunotoxin preparation by the conventional strategy. The product was very stable and showed a very strong ability to bind the PSMA receptor. Cytotoxicity assays showed that this molecule at a very low concentration could kill PSMA-positive PCa cells, with an EC50 value (concentration at which the cell viability decreased by 50%) of 15.3 pM against PSMA-positive LNCaP cells. Moreover, this molecule showed very good killing selectivity between PSMA-positive and PSMA-negative cells, with a selection ratio of more than 300-fold. Animal studies showed that this molecule at a very low dosage (5 × 0.5 mg/kg once every three days) completely inhibited the growth of PCa tumors, and the maximum tolerable dose (MTD) was more than 15 mg/kg, indicating its very potent tumor-treatment ability and a wide therapeutic window. Use of the new PE toxin, PE24X7, as the effector moiety significantly reduced off-target toxicity and improved the therapeutic window of the immunotoxin. The above results demonstrate that the designed anti-PSMA immunotoxin, JVM-PE24X7, has good application value for the treatment of PCa.
Asunto(s)
Adenocarcinoma/tratamiento farmacológico , Antígenos de Neoplasias/inmunología , Antineoplásicos Inmunológicos/uso terapéutico , Glutamato Carboxipeptidasa II/antagonistas & inhibidores , Inmunotoxinas/uso terapéutico , Terapia Molecular Dirigida , Neoplasias de la Próstata/tratamiento farmacológico , Anticuerpos de Dominio Único/uso terapéutico , Animales , Especificidad de Anticuerpos , Reacciones Antígeno-Anticuerpo , Antígenos de Superficie/inmunología , Antineoplásicos Inmunológicos/toxicidad , Línea Celular Tumoral , Ensayos de Selección de Medicamentos Antitumorales , Glutamato Carboxipeptidasa II/inmunología , Humanos , Inmunotoxinas/toxicidad , Masculino , Dosis Máxima Tolerada , Ratones , Ratones Endogámicos NOD , Ratones SCID , Modelos Moleculares , Conformación Proteica , Proteínas Recombinantes de Fusión/uso terapéutico , Proteínas Recombinantes de Fusión/toxicidad , Anticuerpos de Dominio Único/toxicidad , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Aflibercept and arsenic trioxide drugs apply a cytotoxic effect on some human cancer cell lines. However, no more study has followed the effects of both drugs, especially arsenic trioxide, on oral squamous cell carcinoma (OCC). We used three OCC lines as a model to show the effect of these drugs on the genetically complex disease and investigate its targeted therapy. In this study, three human OCC cell lines were used from different patients. We treated cell lines with both medications to detect the effect and relevant molecular basis. First, methyl thiazolyl tetrazolium (MTT) assay was performed to detect the cytotoxicity effect and cell growth. Second, flow cytometry, gene and protein expression were performed to evaluate the anti-angiogenic effect on OCC lines. Next apoptosis was analyzed by flow cytometry. Finally, clonogenesis capacity and cell migration were assessed by colony formation assay and wound healing, respectively. Aflibercept had no cytotoxic effect on the three OCC cell lines but decreased cell growth rate. Arsenic trioxide had a significant cytotoxic effect on three cell lines. Our results demonstrated that both drugs significantly decreased endoglin, VEGFA, and VEGFB expression. In addition, Migration and colony formation assays confirmed that these drugs have significant anti-proliferative and anti-migration effect on oral carcinoma cells. These results revealed that both medications might be a potential drug for the management of oral cancer patients.
Asunto(s)
Trióxido de Arsénico , Carcinoma de Células Escamosas , Neoplasias de la Boca , Receptores de Factores de Crecimiento Endotelial Vascular , Proteínas Recombinantes de Fusión , Antineoplásicos/farmacología , Antineoplásicos/toxicidad , Apoptosis/efectos de los fármacos , Trióxido de Arsénico/farmacología , Trióxido de Arsénico/toxicidad , Carcinoma de Células Escamosas/tratamiento farmacológico , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Endoglina/efectos de los fármacos , Endoglina/metabolismo , Humanos , Neoplasias de la Boca/tratamiento farmacológico , Proteínas Recombinantes de Fusión/farmacología , Proteínas Recombinantes de Fusión/toxicidad , Carcinoma de Células Escamosas de Cabeza y Cuello/tratamiento farmacológico , Factor A de Crecimiento Endotelial Vascular/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/metabolismo , Factor B de Crecimiento Endotelial Vascular/efectos de los fármacos , Factor B de Crecimiento Endotelial Vascular/metabolismoRESUMEN
In two proof-of-concept studies, we established and validated the Bacterial Growth Inhibition Screen (BGIS), which explores recombinant protein toxicity in Escherichia coli as a largely overlooked and alternative means for basic characterization of functional eukaryotic protein domains. By applying BGIS, we identified an unrecognized RNA-interacting domain in the DEK oncoprotein (this study) and successfully combined BGIS with random mutagenesis as a screening tool for loss-of-function mutants of the DNA modulating domain of DEK [1]. Collectively, our findings shed new light on the phenomenon of recombinant protein toxicity in E. coli. Given the easy and rapid implementation and wide applicability, BGIS will extend the repertoire of basic methods for the identification, analysis and unbiased manipulation of proteins.
Asunto(s)
Escherichia coli/efectos de los fármacos , Escherichia coli/crecimiento & desarrollo , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/toxicidad , Pruebas de Toxicidad/métodos , Animales , Sesgo , Biocatálisis , Proteínas Cromosómicas no Histona/química , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Proteínas Cromosómicas no Histona/toxicidad , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/toxicidad , Escherichia coli/genética , Humanos , Mutación con Pérdida de Función , Proteínas Oncogénicas/química , Proteínas Oncogénicas/genética , Proteínas Oncogénicas/metabolismo , Proteínas Oncogénicas/toxicidad , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/toxicidad , Proteínas de Unión a Poli-ADP-Ribosa/química , Proteínas de Unión a Poli-ADP-Ribosa/genética , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/toxicidad , Dominios Proteicos/genética , ARN/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/toxicidad , Receptores de la Familia Eph/química , Receptores de la Familia Eph/genética , Receptores de la Familia Eph/metabolismo , Receptores de la Familia Eph/toxicidad , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Reproducibilidad de los Resultados , Factores de Tiempo , Pruebas de Toxicidad/normasRESUMEN
Objective: The expression of therapeutic proteins in plant oil body bioreactors has attracted much attention. But its safety is not yet clear. This article determines the risk of safety after using the drug. Methods: The oil body-linked oleosin-hEGF microgel emulsion (OBEME) was prepared by mixing the xanthan gum with suitable concentrations in an appropriate proportion. Skin irritation and sensitization reaction were investigated in rats and guinea pigs using OBEME as test article.Results: The OBEME did not produce dermal erythema/eschar or oedema responses. The dermal subacute and subchronic toxicity of OBEME were evaluated in accordance with OECD guidelines. Compared with the control group, the basic physical signs, such as weight, feed, drinking, excretion, and behaviour of experimental animals, were not abnormal. In addition, no abnormality was found in haematological parameters, biochemical indexes, relative organ weight, and histopathological observation of organs, and there was no significant difference compared with normal saline treatment group. Therefore, we conclude that OBEME has no toxic effects and is safe and reliable to be used for topical application.
Asunto(s)
Portadores de Fármacos/toxicidad , Factor de Crecimiento Epidérmico/toxicidad , Proteínas de Plantas/toxicidad , Proteínas Recombinantes de Fusión/toxicidad , Piel/efectos de los fármacos , Administración Cutánea , Animales , Reactores Biológicos/efectos adversos , Carthamus tinctorius/genética , Dermatitis por Contacto/diagnóstico , Dermatitis por Contacto/etiología , Dermatitis por Contacto/patología , Portadores de Fármacos/química , Evaluación Preclínica de Medicamentos , Emulsiones , Factor de Crecimiento Epidérmico/administración & dosificación , Factor de Crecimiento Epidérmico/genética , Eritema/inducido químicamente , Eritema/diagnóstico , Cobayas , Humanos , Gotas Lipídicas/química , Masculino , Microgeles , Proteínas de Plantas/administración & dosificación , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente , Ratas , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/genética , Piel/inmunología , Piel/lesiones , Piel/patología , Pruebas de Toxicidad Aguda/métodos , Pruebas de Toxicidad Subaguda/métodos , Pruebas de Toxicidad Subcrónica/métodos , Cicatrización de Heridas/efectos de los fármacosRESUMEN
This study was conducted to establish the toxicological profile of combination treatment with therapeutic HPV DNA vaccines (GX-188E) and the long-acting form of recombinant human interleukin-7 fused with hybrid Fc (IL-7hyFc). GX-188E was administered intramuscularly by electroporation with or without IL-7hyFc intravaginally once per 2 weeks for 8 weeks (five times) in female Sprague-Dawley rats. Because up-regulation of immune responses and migration of antigen-specific T cells in cervicoviginal tissue were predicted as therapeutic effects, we distinguished adverse effects from therapeutic effects based on the severity of the systemic immune response, reversibility of lymphoid tissue changes, target tissue damage, and off-target immune responses. We observed that the number of neutrophils was increased, and the number of lymphocytes was decreased in the blood. Further, myofiber degeneration, necrosis, fibroplasia, and cell infiltration were observed at the GX-188E administration site. These changes were fully or partially recovered over a 4-week period. Analysis of lymphocytes in spleen revealed that CD4+ T cells and total T cells decreased in rats treated with GX-188E in combination with a high dose of IL-7hyFc (1.25 mg/animal). However, these changes were not considered adverse because they were transient and may have been related to electroporation-mediated DNA delivery or the local migration of lymphocytes induced by IL-7. Therefore, the potential toxicity of the combination of GX-188E and IL-7hyFc treatment was comparable to that of GX-188E treatment alone, and the no observed adverse effect level for GX-188E with IL-7hyFc was considered as 320 µg/animal for GX-188E and 1.25 mg/animal for IL-7hyFc.
Asunto(s)
Fragmentos Fc de Inmunoglobulinas/toxicidad , Interleucina-7/toxicidad , Vacunas contra Papillomavirus/toxicidad , Vacunas de ADN/toxicidad , Administración Intravaginal , Animales , Biomarcadores/sangre , Biomarcadores/orina , Relación Dosis-Respuesta a Droga , Quimioterapia Combinada , Electroporación , Femenino , Fragmentos Fc de Inmunoglobulinas/administración & dosificación , Interleucina-7/administración & dosificación , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Nivel sin Efectos Adversos Observados , Vacunas contra Papillomavirus/administración & dosificación , Ratas Sprague-Dawley , Proteínas Recombinantes de Fusión/toxicidad , Medición de Riesgo , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo , Factores de Tiempo , Vacunas de ADN/administración & dosificaciónRESUMEN
The purposes of this study was to determine the toxicological effect of repeated intravenous administration of Activin A/BMP-2 chimera (AB204) in beagle dogs for a long period of four weeks and evaluate two-week recovery. AB204 was administered at doses of 0.08, 0.16, or 0.32 mg/kg/day to three male and three female beagle dogs for 4 weeks as the experimental group. For the control group, sterile saline was administered to three male and three female beagle dogs. For the two-week recovery test, two male and two female beagle dogs were randomly selected from the control group and the 0.32 mg/kg/day administered experimental group. General symptoms, body weight, food consumption, ophthalmological examination, electrocardiogram, urinalysis, hematology and blood biochemistry, organ weights, autopsy, and histopathological examination were observed or conducted. No animals died. There was no significant difference in any parameter evaluated between the experimental group and the control group. Histopathological examination revealed compound inflammation at the administration site in both the experimental group and the control group. The inflammation disappeared during the two-week recovery. These results indicated that repetitive intravenous injection of AB204 in beagle dog for a long period of four weeks did not show any toxicity. Therefore, no observed adverse effects level (NOAEL) of AB204 was 0.32 mg/kg/day in big animal model.
Asunto(s)
Inflamación/etiología , Proteínas Recombinantes de Fusión/toxicidad , Animales , Perros , Femenino , Inflamación/patología , Inyecciones Intravenosas , Masculino , Nivel sin Efectos Adversos Observados , Proteínas Recombinantes de Fusión/administración & dosificación , Factores de TiempoRESUMEN
Age-related macular degeneration (AMD) is a leading cause of vision loss, the treatment of which may require monthly intravitreal injections. This is a burden on patients and health services, and new delivery modalities that reduce injection frequency are required. To that end, we investigated the suitability of a novel reverse thermoresponsive polymer (RTP) as an ocular drug-delivery vehicle. In this work, we detail the structure and synthesis of a novel RTP, and determine drug release curves for two drugs commonly used in the treatment of AMD, bevacizumab and aflibercept. Biocompatibility of the RTP was assessed in vitro in human and rat cell lines and in vivo following intravitreal injection in rats. Bevacizumab demonstrated a more appropriate release profile than aflibercept, with 67% released within 14 days and 78% released in total over a 183-day period. No toxic effects of RTP were seen in human or rat cells in up to 14 days of co-culture with RTP. Following intravitreal injection, intraocular pressure was unaffected by the presence of RTP and no changes in retinal function or structure were observed at 1 week or 1 month post-injection. RTP injection did not cause inflammation, gliosis or apoptosis in the retina. This work demonstrates the potential suitability of the novel RTP as a sustained-release vehicle for ocular drug delivery for anti-neovascular therapies. Optimization of polymer chemistry for optimal drug loading and release is needed.
Asunto(s)
Inhibidores de la Angiogénesis/administración & dosificación , Bevacizumab/administración & dosificación , Sistemas de Liberación de Medicamentos , Polímeros/química , Receptores de Factores de Crecimiento Endotelial Vascular/administración & dosificación , Proteínas Recombinantes de Fusión/administración & dosificación , Inhibidores de la Angiogénesis/toxicidad , Animales , Bevacizumab/toxicidad , Línea Celular , Preparaciones de Acción Retardada , Liberación de Fármacos , Humanos , Presión Intraocular , Inyecciones Intravítreas , Degeneración Macular/tratamiento farmacológico , Masculino , Ratas , Ratas Long-Evans , Proteínas Recombinantes de Fusión/toxicidad , Retina/efectos de los fármacos , Retina/metabolismo , Temperatura , Factores de TiempoRESUMEN
Although Interleukin-22 (IL-22) is produced by various leukocytes, it preferentially targets cells with epithelial origins. IL-22 exerts essential roles in modulating various tissue epithelial functions, such as innate host defense against extracellular pathogens, barrier integrity, regeneration, and wound healing. Therefore, IL-22 is thought to have therapeutic potential in treating diseases associated with infection, tissue injury or chronic tissue damage. A number of in vitro and in vivo nonclinical studies were conducted to characterize the pharmacological activity and safety parameters of UTTR1147A, an IL-22 recombinant fusion protein that links the human cytokine IL-22 with the Fc portion of a human immunoglobulin. To assess the pharmacological activity of UTTR1147A, STAT3 activation was evaluated in primary hepatocytes isolated from human, cynomolgus monkey, minipig, rat, and mouse after incubation with UTTR1147A. UTTR1147A activated STAT3 in all species evaluated, demonstrating that all were appropriate nonclinical species for toxicology studies. The nonclinical safety profile of UTTR1147A was evaluated in rats, minipigs, and cynomolgus monkeys to establish a safe clinical starting dose for humans in Phase I trials and to support clinical intravenous, subcutaneous and/or topical administration treatment regimen. Results demonstrate the cross-species translatability of the biological response in activating the IL-22 pathway as well as the translatability of findings from in vitro to in vivo systems. UTTR1147A was well tolerated in all species tested and induced the expected pharmacologic effects of epidermal hyperplasia and a transient increase in on-target acute phase proteins. These effects were all considered to be clinically predictable, manageable, monitorable, and reversible.
Asunto(s)
Hepatocitos/efectos de los fármacos , Factores Inmunológicos/farmacología , Interleucinas/toxicidad , Proteínas Recombinantes de Fusión/toxicidad , Animales , Ensayos Clínicos Fase I como Asunto , Evaluación Preclínica de Medicamentos , Femenino , Hepatocitos/metabolismo , Humanos , Interleucinas/administración & dosificación , Macaca fascicularis , Masculino , Ratones , Cultivo Primario de Células , Ratas , Proteínas Recombinantes de Fusión/administración & dosificación , Factor de Transcripción STAT3/metabolismo , Porcinos , Porcinos Enanos , Interleucina-22RESUMEN
The efficient delivery of proteins into cells is needed to fully realize the potential of protein-based therapeutics. Current protein delivery strategies generally suffer from poor endosomal escape and low tolerance for serum. Here, the genetic fusion of a supercharged polypeptide, called SCP, to a protein provides a generic method for intracellular protein delivery. It allows efficient protein endocytosis and endosomal escape and is capable of potently delivering various proteins with a range of charges, sizes, and bioactivities into the nucleus of living cells. SCP is discovered to bind directly to the nuclear import protein importin ß1 and gains access to the nucleus. Furthermore, SCP shows minimal hemolytic activity and stability in serum and lacks toxicity and immunogenicity in vivo. Effective gene editing can be achieved by SCP-mediated delivery of Cas9 protein and guide RNA. This study may provide an efficient and useful tool for the design and development of cell-nuclear-targeted drug delivery.
Asunto(s)
Péptidos de Penetración Celular/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Animales , Línea Celular Tumoral , Núcleo Celular/metabolismo , Péptidos de Penetración Celular/sangre , Péptidos de Penetración Celular/genética , Péptidos de Penetración Celular/toxicidad , Endocitosis/fisiología , Escherichia coli/genética , Femenino , Humanos , Ratones Endogámicos BALB C , Estabilidad Proteica , Proteínas Recombinantes de Fusión/sangre , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/toxicidad , beta Carioferinas/metabolismoRESUMEN
Cervical cancer, the second most common cause of cancer death in women worldwide, is significantly associated with infection of high-risk human papillomaviruses (HPVs), especially the most common genotype, HPV 16. To date, there is no established noninvasive therapy to treat cervical cancer. Methods: Here, we report a novel affitoxin that targets HPV16 E7 protein, one of the primary target proteins in molecular targeted therapy for HPV-induced cervical cancer. The affitoxin, ZHPV16E7 affitoxin384 was generated by fusing the modified Pseudomonas Exotoxin A (PE38KDEL) to the HPV16 E7-specific affibody. The expressed and purified ZHPV16E7 affitoxin384 was characterized using numerous methods. SPR assay, indirect immunofluorescence assay, and near-infrared (NIR) optical imaging were respectively performed to assess the targeting ability of ZHPV16E7 affitoxin384 to HPV16 E7 protein both in vitro and in vivo. Cell viability assays and SiHa tumor-bearing nude mice were used to evaluate the efficacy of ZHPV16 E7 affitoxin384 in vitro and in vivo, respectively. Results: Using in vitro methods the SPR assay and indirect immunofluorescence assay showed that ZHPV16E7 affitoxin384 targeted HPV16 E7 with high binding affinity and specificity. Significant reduction of cell viability in HPV16 positive cells was observed in the presence of ZHPV16 E7 affitoxin384. By NIR optical imaging, ZHPV16 E7 affitoxin384 specifically targeted HPV16 positive tumors in vivo. ZHPV16E7 affitoxin384 showed significant in vivo antitumor efficacy in two kinds of tumor-bearing nude mouse models. Conclusions: ZHPV16E7 affitoxin384 is a potent anti-cervical cancer therapeutic agent that could be effective against HPV16 positive tumors in humans.
Asunto(s)
Antineoplásicos/administración & dosificación , Inmunoterapia/métodos , Inmunotoxinas/administración & dosificación , Terapia Molecular Dirigida/métodos , Proteínas E7 de Papillomavirus/antagonistas & inhibidores , Neoplasias del Cuello Uterino/tratamiento farmacológico , Animales , Anticuerpos Antivirales/genética , Anticuerpos Antivirales/metabolismo , Antineoplásicos/farmacología , Toxinas Bacterianas/genética , Toxinas Bacterianas/toxicidad , Línea Celular Tumoral , Supervivencia Celular , Modelos Animales de Enfermedad , Femenino , Humanos , Inmunotoxinas/genética , Inmunotoxinas/farmacología , Ratones , Ratones Desnudos , Pseudomonas/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/toxicidad , Resultado del TratamientoRESUMEN
Bacillus thuringiensis Vip3A protein has been widely used for crop protection and for delay resistance to existing insecticidal Cry toxins. During current study, a fusion between vip3Aa16 and the toxic core sequence of cry1Ac was constructed in pHT Blue plasmid. Vip3Aa16-Cry1Ac protein was expressed in the supernatant of B. thuringiensis with a size of about 150â¯kDa. Bioassays tested on Ephestia kuehniella showed that the use of the chimera toxin as biopesticide improved the toxicity to reach 90%⯱â¯2 with an enhancement of 20% compared to the single Vip3Aa16 protein. The findings indicated that the fusion protein design opens new ways to enhance Vip3A toxicity against lepidopteran species and could avoiding insect tolerance of B. thuringiensis delta-endotoxins. Through computational study, we have predicted for the first time the whole 3D structure of a Vip3A toxin. We showed that Vip3Aa16 structure is composed by three domains like Cry toxins: an N-terminal domain containing hemolysin like fold as well as two others Carbohydrate Binding Module (CBM)-like domains. Molecular docking analysis of the chimera toxin and the single Vip3Aa16 protein against specific insect receptors revealed that residues of CBM like domains are clearly involved in the binding of the toxin to receptors.
Asunto(s)
Proteínas Bacterianas/genética , Endotoxinas/química , Endotoxinas/toxicidad , Proteínas Hemolisinas/genética , Simulación del Acoplamiento Molecular , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/toxicidad , Secuencia de Aminoácidos , Toxinas de Bacillus thuringiensis , Endotoxinas/genética , Conformación Proteica , Proteínas Recombinantes de Fusión/genéticaRESUMEN
TDP-43-mediated proteinopathy is a key factor in the pathology of amyotrophic lateral sclerosis (ALS). A potential underlying mechanism is dysregulation of the cytoskeleton. Here we investigate the effects of expressing TDP-43 wild-type and M337V and Q331K mutant isoforms on cytoskeletal integrity and function, using rat cortical neurons in vitro. We find that TDP-43 protein becomes mislocalised in axons over 24-72 hours in culture, with protein aggregation occurring at later timepoints (144 hours). Quantitation of cell viability showed toxicity of both wild-type and mutant constructs which increased over time, especially of the Q331K mutant isoform. Analysis of the effects of TDP-43 on axonal integrity showed that TDP-43-transfected neurons had shorter axons than control cells, and that growth cone sizes were smaller. Axonal transport dynamics were also impaired by transfection with TDP-43 constructs. Taken together these data show that TDP-43 mislocalisation into axons precedes cell death in cortical neurons, and that cytoskeletal structure and function is impaired by expression of either TDP-43 wild-type or mutant constructs in vitro. These data suggest that dysregulation of cytoskeletal and neuronal integrity is an important mechanism for TDP-43-mediated proteinopathy.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Neuronas Motoras/metabolismo , Proteinopatías TDP-43/etiología , Esclerosis Amiotrófica Lateral/etiología , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Animales , Transporte Axonal/efectos de los fármacos , Transporte Axonal/fisiología , Axones/metabolismo , Axones/patología , Muerte Celular , Células Cultivadas , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/toxicidad , Conos de Crecimiento/metabolismo , Conos de Crecimiento/patología , Humanos , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/patología , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteínas Mutantes/toxicidad , Neurotoxinas/genética , Neurotoxinas/metabolismo , Neurotoxinas/toxicidad , Agregación Patológica de Proteínas/etiología , Agregación Patológica de Proteínas/genética , Agregación Patológica de Proteínas/metabolismo , Ratas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/toxicidad , Proteinopatías TDP-43/genética , Proteinopatías TDP-43/metabolismo , TransfecciónRESUMEN
We previously demonstrated that patients with multiple sclerosis (MS) of high serum Sema4A levels are resistant to IFN-ß therapy. To further elucidate the role of serum Sema4A as a biomarker for therapeutic stratification in MS patients, it is important to clarify the efficacy of other disease-modifying drugs (DMD) in those with high serum Sema4A levels. Thus, in this study we investigated whether fingolimod has beneficial effects on MS patients with high Sema4A levels. We retrospectively analyzed annualized relapse rate (ARR) and Expanded Disability Status Scale (EDSS) change in 56 relapsing-remitting multiple sclerosis (RRMS) patients who had been treated with fingolimod, including those who switched from IFN-ß therapy. The levels of Sema4A in the sera were measured by sandwich ELISA. The implications of Sema4A on the efficacy of fingolimod were investigated by administering recombinant Sema4A-Fc and fingolimod to mice with experimental autoimmune encephalomyelitis (EAE). Retrospective analysis of MS cohort (17 high Sema4A and 39 low Sema4A) demonstrated the effectiveness of fingolimod in those with high serum Sema4A levels, showing reduction of ARR (from 1.21 to 0.12) and EDSS progression (from 0.50 to 0.04). Consistent with this observation, improvement in the disease severity of EAE mice receiving recombinant Sema4A-Fc was also observed after fingolimod treatment. These data suggest that fingolimod could serve as a candidate DMD for managing the disease activity of MS patients with high Sema4A levels.
Asunto(s)
Antirreumáticos/uso terapéutico , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Clorhidrato de Fingolimod/uso terapéutico , Esclerosis Múltiple Recurrente-Remitente/tratamiento farmacológico , Semaforinas/sangre , Adulto , Animales , Biomarcadores , Progresión de la Enfermedad , Evaluación de Medicamentos , Evaluación Preclínica de Medicamentos , Resistencia a Medicamentos , Sustitución de Medicamentos , Encefalomielitis Autoinmune Experimental/sangre , Femenino , Humanos , Fragmentos Fc de Inmunoglobulinas/genética , Inmunoglobulina G/genética , Interferón beta/uso terapéutico , Recuento de Linfocitos , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Esclerosis Múltiple Recurrente-Remitente/sangre , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/toxicidad , Estudios Retrospectivos , Semaforinas/genética , Semaforinas/toxicidad , Índice de Severidad de la Enfermedad , Organismos Libres de Patógenos Específicos , Resultado del TratamientoRESUMEN
Scorpion long-chain insect neurotoxins have important potential application value in agricultural pest control. The difficulty of obtaining natural toxins is the major obstacle preventing analyses of their insecticidal activity against more agricultural insect pests. Here we cloned the insect neurotoxin BjαIT gene into the pET32 expression vector and expressed the resulting thioredoxin (Trx)-BjαIT fusion protein in Escherichia coli. Soluble Trx-BjαIT was expressed at a high level when induced at 18 °C with 0.1 mM isopropyl ß-d-1-thiogalactopyranoside, and it was purified by Ni2+-nitriloacetic acid affinity chromatography. After cleaving the Trx tag with recombinant enterokinase, the digestion products were purified by CM Sepharose FF ion-exchange chromatography, and 1.5 mg of purified recombinant BjαIT (rBjαIT) was obtained from 100 ml of induced bacterial cells. Injecting rBjαIT induced obvious neurotoxic symptoms and led to death in locust (Locusta migratoria) larvae. Dietary toxicity was not observed in locusts. The results demonstrate that active rBjαIT could be obtained efficiently from an E. coli expression system, which is helpful for determining its insecticidal activity against agricultural insect pests.
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Larva/efectos de los fármacos , Locusta migratoria/efectos de los fármacos , Proteínas Recombinantes de Fusión/biosíntesis , Venenos de Escorpión/biosíntesis , Escorpiones/química , Animales , Cromatografía por Intercambio Iónico/métodos , Clonación Molecular , Enteropeptidasa/química , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Insecticidas/aislamiento & purificación , Insecticidas/metabolismo , Insecticidas/toxicidad , Isopropil Tiogalactósido/farmacología , Larva/fisiología , Locusta migratoria/fisiología , Plásmidos/química , Plásmidos/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/toxicidad , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Venenos de Escorpión/genética , Venenos de Escorpión/aislamiento & purificación , Venenos de Escorpión/toxicidad , Solubilidad , Tiorredoxinas/genética , Tiorredoxinas/metabolismoRESUMEN
This study was performed for the purpose to evaluate the effect of activin A/BMP-2 chimera (AB204) on cardiovascular system and toxicological effect in beagle dogs. When administered AB204 at the dose of 0.32 mg/kg via intravenous injection in beagle dogs, there were no changes in systolic, diastolic and mean blood pressure as well as in pulse rate, in addition that there were no differences in ORS complex, PR interval, R-R interval, QT interval and QTcV interval on the electrocardiography. Also, when administered AB204 at the doses of 0.25 and 0.5 mg/kg/day via repetitive intravenous injection for 2 weeks, it did not cause any significant changes in general symptoms, weight, food intake, ophthalmologic abnormality, urine, hematology, serum biochemistry, organ weight and autopsy values. Therefore, AB204 did not affect cardiovascular functions including blood pressure, pulse rate and ECG, when administered at the dose of ≤0.32 mg/kg via single intravenous injection in male beagle dogs. When it was administered at the dose of 0.5 mg/kg repetitive intravenous injection for 2 weeks, it did not show any toxicity.
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Sistema Cardiovascular/efectos de los fármacos , Proteínas Recombinantes de Fusión/toxicidad , Animales , Biomarcadores/sangre , Presión Sanguínea/efectos de los fármacos , Sistema Cardiovascular/patología , Sistema Cardiovascular/fisiopatología , Perros , Esquema de Medicación , Frecuencia Cardíaca/efectos de los fármacos , Inyecciones Intravenosas , Masculino , Proteínas Recombinantes de Fusión/administración & dosificación , Medición de Riesgo , Factores de Tiempo , Pruebas de ToxicidadRESUMEN
Scorpion α-toxins are polypeptides that inhibit voltage-gated sodium channel inactivation. They are divided into mammal, insect and α-like toxins based on their relative activity toward different phyla. Several factors are currently known to influence the selectivity, which are not just particular amino acid residues but also general physical, chemical, and topological properties of toxin structural modules. The objective of this study was to change the selectivity profile of a chosen broadly active α-like toxin, BeM9 from Mesobuthus eupeus, toward mammal-selective. Based on the available information on what determines scorpion α-toxin selectivity, we designed and produced msBeM9, a BeM9 derivative, which was verified to be exclusively active toward mammalian sodium channels and, most importantly, toward the Nav 1.2 isoform expressed in the brain.
Asunto(s)
Canal de Sodio Activado por Voltaje NAV1.2/química , Neurotoxinas/química , Oocitos/efectos de los fármacos , Proteínas Recombinantes de Fusión/química , Venenos de Escorpión/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Humanos , Insectos/efectos de los fármacos , Insectos/metabolismo , Ratones , Modelos Moleculares , Canal de Sodio Activado por Voltaje NAV1.2/metabolismo , Neurotoxinas/biosíntesis , Neurotoxinas/genética , Neurotoxinas/toxicidad , Oocitos/citología , Oocitos/metabolismo , Unión Proteica , Ingeniería de Proteínas , Dominios y Motivos de Interacción de Proteínas , Estructura Secundaria de Proteína , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/toxicidad , Venenos de Escorpión/biosíntesis , Venenos de Escorpión/genética , Venenos de Escorpión/toxicidad , Escorpiones/química , Escorpiones/patogenicidad , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Relación Estructura-Actividad , Especificidad por Sustrato , Tiorredoxinas/biosíntesis , Tiorredoxinas/química , Tiorredoxinas/genética , Xenopus laevisRESUMEN
BACKGROUND: MP4-induced experimental autoimmune encephalomyelitis (EAE) is a mouse model of multiple sclerosis (MS), which enables targeted research on B cells, currently much discussed protagonists in MS pathogenesis. Here, we used this model to study the impact of the S1P1 receptor modulator FTY720 (fingolimod) on the autoreactive B cell and antibody response both in the periphery and the central nervous system (CNS). METHODS: MP4-immunized mice were treated orally with FTY720 for 30 days at the peak of disease or 50 days after EAE onset. The subsequent disease course was monitored and the MP4-specific B cell/antibody response was measured by ELISPOT and ELISA. RNA sequencing was performed to determine any effects on B cell-relevant gene expression. S1P1 receptor expression by peripheral T and B cells, B cell subset distribution in the spleen and B cell infiltration into the CNS were studied by flow cytometry. The formation of B cell aggregates and of tertiary lymphoid organs (TLOs) was evaluated by histology and immunohistochemistry. Potential direct effects of FTY720 on B cell aggregation were studied in vitro. RESULTS: FTY720 significantly attenuated clinical EAE when treatment was initiated at the peak of EAE. While there was a significant reduction in the number of T cells in the blood after FTY720 treatment, B cells were only slightly diminished. Yet, there was evidence for the modulation of B cell receptor-mediated signaling upon FTY720 treatment. In addition, we detected a significant increase in the percentage of B220+ B cells in the spleen both in acute and chronic EAE. Whereas acute treatment completely abrogated B cell aggregate formation in the CNS, the numbers of infiltrating B cells and plasma cells were comparable between vehicle- and FTY720-treated mice. In addition, there was no effect on already developed aggregates in chronic EAE. In vitro B cell aggregation assays suggested the absence of a direct effect of FTY720 on B cell aggregation. However, FTY720 impacted the evolution of B cell aggregates into TLOs. CONCLUSIONS: The data suggest differential effects of FTY720 on the B cell compartment in MP4-induced EAE.
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Linfocitos B/efectos de los fármacos , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Encefalomielitis Autoinmune Experimental/patología , Clorhidrato de Fingolimod/uso terapéutico , Inmunosupresores/uso terapéutico , Animales , Antígenos CD19/metabolismo , Linfocitos B/metabolismo , Linfocitos T CD4-Positivos/patología , Proteínas de Unión al Calcio/metabolismo , Agregación Celular/efectos de los fármacos , Sistema Nervioso Central/patología , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/sangre , Encefalomielitis Autoinmune Experimental/inducido químicamente , Ensayo de Immunospot Ligado a Enzimas , Femenino , Citometría de Flujo , Adyuvante de Freund/toxicidad , Ganglios Linfáticos/patología , Ratones , Proteína Básica de Mielina/inmunología , Proteína Básica de Mielina/toxicidad , Proteína Proteolipídica de la Mielina/inmunología , Proteína Proteolipídica de la Mielina/toxicidad , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/toxicidad , Bazo/patología , Factores de TiempoRESUMEN
Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system (CNS) in young adults that has serious negative socioeconomic effects. In addition to symptoms caused by CNS pathology, the majority of MS patients frequently exhibit gastrointestinal dysfunction, which was previously either explained by the presence of spinal cord lesions or not directly linked to the autoimmune etiology of the disease. Here, we studied the enteric nervous system (ENS) in a B cell- and antibody-dependent mouse model of MS by immunohistochemistry and electron microscopy at different stages of the disease. ENS degeneration was evident prior to the development of CNS lesions and the onset of neurological deficits in mice. The pathology was antibody mediated and caused a significant decrease in gastrointestinal motility, which was associated with ENS gliosis and neuronal loss. We identified autoantibodies against four potential target antigens derived from enteric glia and/or neurons by immunoprecipitation and mass spectrometry. Antibodies against three of the target antigens were also present in the plasma of MS patients as confirmed by ELISA. The analysis of human colon resectates provided evidence of gliosis and ENS degeneration in MS patients compared to non-MS controls. For the first time, this study establishes a pathomechanistic link between the well-established autoimmune attack on the CNS and ENS pathology in MS, which might provide a paradigm shift in our current understanding of the immunopathogenesis of the disease with broad diagnostic and therapeutic implications.
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Autoanticuerpos/sangre , Enfermedades Gastrointestinales/etiología , Esclerosis Múltiple , Animales , Sistema Nervioso Central/metabolismo , Sistema Nervioso Central/patología , Citocinas/metabolismo , Modelos Animales de Enfermedad , Sistema Nervioso Entérico/metabolismo , Sistema Nervioso Entérico/patología , Sistema Nervioso Entérico/ultraestructura , Femenino , Adyuvante de Freund/toxicidad , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Esclerosis Múltiple/complicaciones , Esclerosis Múltiple/inmunología , Esclerosis Múltiple/patología , Músculo Liso/patología , Músculo Liso/ultraestructura , Proteína Básica de Mielina/inmunología , Proteína Básica de Mielina/metabolismo , Proteína Básica de Mielina/toxicidad , Glicoproteína Mielina-Oligodendrócito/inmunología , Glicoproteína Mielina-Oligodendrócito/toxicidad , Plexo Mientérico/patología , Plexo Mientérico/ultraestructura , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/toxicidad , Tubulina (Proteína)/metabolismoRESUMEN
Ontak® is a FDA-approved diphtheria toxin-based recombinant fusion toxin for treatment of human CD25+ cutaneous T cell lymphoma (CTCL). However, it has been discontinued clinically due to the production issue related to the bacterial expression system with difficult purification. Recently we have developed monovalent and bivalent human IL-2 fusion toxins targeting human CD25+ cells using advanced unique diphtheria toxin resistant yeast Pichia Pastoris expression system. In vitro efficacy characterization using human CD25+ HUT102/6TG cells demonstrated that both monovalent and bivalent isoforms are potent and the bivalent isoform is approximately two logs more potent than the monovalent isoform. In this study, we further assessed the in vivo efficacy of the human IL-2 fusion toxins using human CD25+ HUT102/6TG tumor-bearing NSG mouse model. The data demonstrated that both monovalent and bivalent human IL-2 fusion toxins significantly prolonged the survival of the human CD25+ tumor-bearing NSG mice in a dose-dependent manner. Then we further assessed the residual tumor cells from the HUT102/6TG tumor-bearing NSG mice using the residual tumor cell bearing NSG mouse model. The results demonstrated that the residual tumor cells were still sensitive to the continual treatment with the human IL-2 fusion toxin. This yeast-expressed human IL-2 fusion toxin will be a promising candidate to replace the clinically discontinued Ontak®.
Asunto(s)
Antineoplásicos/farmacología , Toxina Diftérica/farmacología , Inmunoconjugados/farmacología , Interleucina-2/farmacología , Linfoma Cutáneo de Células T/tratamiento farmacológico , Neoplasias Cutáneas/tratamiento farmacológico , Animales , Antineoplásicos/metabolismo , Antineoplásicos/toxicidad , Línea Celular Tumoral , Toxina Diftérica/toxicidad , Relación Dosis-Respuesta a Droga , Humanos , Inmunoconjugados/genética , Inmunoconjugados/metabolismo , Inmunoconjugados/toxicidad , Interleucina-2/biosíntesis , Interleucina-2/genética , Interleucina-2/toxicidad , Subunidad alfa del Receptor de Interleucina-2/inmunología , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Linfoma Cutáneo de Células T/inmunología , Linfoma Cutáneo de Células T/patología , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Pichia/genética , Pichia/metabolismo , Proteínas Recombinantes de Fusión/farmacología , Proteínas Recombinantes de Fusión/toxicidad , Retirada de Medicamento por Seguridad , Neoplasias Cutáneas/inmunología , Neoplasias Cutáneas/patología , Factores de Tiempo , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Haemophilus parasuis is the causative agent of Glasser's disease in pigs. Cytolethal distending toxin (CDT) is an important virulence factor of H. parasuis. It is composed of three subunits: CdtA, CdtB and CdtC and all were successfully expressed in soluble form in Escherichia coli when the signal peptides were removed. Purified CdtB had DNase activity, i.e. caused DNA double strand damage, in vitro and in vivo prior to cell arrest and apoptosis. Flow cytometry analysis showed CdtB alone could induce cell cycle arrest and apoptosis in PK-15 porcine kidney and pulmonary alveolar macrophage (PAM) cells, which could be enhanced by CdtA or/and CdtC. CDT holotoxin could lead to significant cell distension, G2 arrest and apoptotic death in PK-15 and PAM cells. The apoptosis induced by CDT holotoxin was significantly inhibited by pifithrin-α, which indicates that it is p53-dependent. The results suggest that H. parasuis CDT holotoxin is a major virulence factor.