RESUMEN
OBJECTIVES: To compare different approaches for the expression of an anti-PCSK9 biosimilar monoclonal antibody (mAb) in CHO cells using IRES-mediated tricistronic plasmid vectors combining different signal peptides, IRES elements and selection markers. RESULTS: Transient transfection indicated a similar level of secreted mAb 48 h post-transfection for all constructs. However, transfections carried out with circular plasmids showed a higher expression than with linearized plasmids. After two months under selection pressure, only part of the transfected pools recovered. The cultures co-transfected using two antibiotics as selection markers for double selection did not recover. Growth, metabolism and mAb production profiles of the only part of the transfected pools recovered resulting stable pools were compared and the stable pool transfected with circular L1-LC-IRES-H7-HC-IRES-NEO plasmid was chosen for further studies, due to higher cell growth and mAb production. Critical quality attributes of the protein A-purified mAb such as purity, homogeneity, binding affinity to PCSK9, and amino acid sequence were assessed confirming the success of the approach adopted in this study. CONCLUSIONS: The expression platform proposed showed to be efficient to produce a high-quality anti-PCSK9 mAb in stable CHO cell pools and provides benchmarks for fast production of different mAbs for characterization, formulation studies and pre-clinical investigation.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Biosimilares Farmacéuticos/farmacología , Sitios Internos de Entrada al Ribosoma/genética , Proproteína Convertasa 9/genética , Secuencia de Aminoácidos/genética , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/farmacología , Células CHO , Cricetulus/genética , Expresión Génica/efectos de los fármacos , Vectores Genéticos/genética , Vectores Genéticos/farmacología , Humanos , Sitios Internos de Entrada al Ribosoma/efectos de los fármacos , Plásmidos/genética , Plásmidos/farmacología , Proproteína Convertasa 9/inmunología , Proproteína Convertasa 9/farmacología , TransfecciónRESUMEN
Apesar de incomuns, as enfermidades oculares em roedores têm grande importância clínica, já que podem provocar dor e alterar o bem-estar desse tipo de paciente. A proptose de globo ocular pode ocorrer pela característica anatômica dos roedores, cuja esclera é fina, ou ainda devido a manejo de contenção inadequado. O objetivo deste trabalho é relatar a conduta terapêutica aplicada a um hamster- chinês (Cricetulus griseus) macho, filhote, que apresentava protrusão de globo ocular esquerdo de etiologia desconhecida. O paciente foi submetido ao procedimento cirúrgico de enucleação transconjuntival, em que os cuidados perioperatórios e os procedimentos anestésicos adequados foram essenciais, ressaltando a importância do conhecimento sobre uma espécie ainda incomum na rotina clínica.
Ophthalmic diseases in rodents are uncommon, but clinically importante. Eye diseases can cause pain and compromise the well-being of these patients. Roptosis of the eyeball may occur due to anatomical characteristics of rodents, which have a thin scler; and inadequate clinical management can also result in proptosis. We report the treatment of left eyeball proptosis of unknown etiology in a male cub Chinese hamster (Cricetulus griseus). Transconjunctival enucleation with adequate anesthetic protocol and perioperative care,resulted in a successful outcome. We emphasize the importance of understanding the peculiarities of a species still infrequently seen in clinical routine of small animal practice.
A pesar de ser poco frecuentes, las enfermedades oculares en los roedores son de gran importancia clínica, ya que pueden causar dolor y alterar el bienestar de este tipo de pacientes. La proptosis del globo ocular se puede producir por las características anatómicas de los roedores, que tienen una esclera fina, o debido un manejo inadecuado durante la contención. El objetivo de este estudio es relatar el enfoque terapéutico aplicado a un hámster chino (Cricetulus griseus) macho, jóven, que se presentó a consulta con una protrusión del globo ocular izquierdo de origen desconocido. Al paciente se le realizó una enucleación transconjuntival, previo manejo detallado esencial de los cuidados perioperatorios y la anestesia. Se debe destacar la importancia del conocimiento de esta especie poco común durante la atención clínica diaria.
Asunto(s)
Masculino , Animales , Anestésicos , Cricetulus/genética , Enucleación del Ojo/rehabilitación , Enucleación del Ojo/veterinariaRESUMEN
Apesar de incomuns, as enfermidades oculares em roedores têm grande importância clínica, já que podem provocar dor e alterar o bem-estar desse tipo de paciente. A proptose de globo ocular pode ocorrer pela característica anatômica dos roedores, cuja esclera é fina, ou ainda devido a manejo de contenção inadequado. O objetivo deste trabalho é relatar a conduta terapêutica aplicada a um hamster- chinês (Cricetulus griseus) macho, filhote, que apresentava protrusão de globo ocular esquerdo de etiologia desconhecida. O paciente foi submetido ao procedimento cirúrgico de enucleação transconjuntival, em que os cuidados perioperatórios e os procedimentos anestésicos adequados foram essenciais, ressaltando a importância do conhecimento sobre uma espécie ainda incomum na rotina clínica.(AU)
Ophthalmic diseases in rodents are uncommon, but clinically importante. Eye diseases can cause pain and compromise the well-being of these patients. Roptosis of the eyeball may occur due to anatomical characteristics of rodents, which have a thin scler; and inadequate clinical management can also result in proptosis. We report the treatment of left eyeball proptosis of unknown etiology in a male cub Chinese hamster (Cricetulus griseus). Transconjunctival enucleation with adequate anesthetic protocol and perioperative care,resulted in a successful outcome. We emphasize the importance of understanding the peculiarities of a species still infrequently seen in clinical routine of small animal practice.(AU)
A pesar de ser poco frecuentes, las enfermedades oculares en los roedores son de gran importancia clínica, ya que pueden causar dolor y alterar el bienestar de este tipo de pacientes. La proptosis del globo ocular se puede producir por las características anatómicas de los roedores, que tienen una esclera fina, o debido un manejo inadecuado durante la contención. El objetivo de este estudio es relatar el enfoque terapéutico aplicado a un hámster chino (Cricetulus griseus) macho, jóven, que se presentó a consulta con una protrusión del globo ocular izquierdo de origen desconocido. Al paciente se le realizó una enucleación transconjuntival, previo manejo detallado esencial de los cuidados perioperatorios y la anestesia. Se debe destacar la importancia del conocimiento de esta especie poco común durante la atención clínica diaria.(AU)
Asunto(s)
Animales , Masculino , Cricetulus/genética , Enucleación del Ojo/rehabilitación , Enucleación del Ojo/veterinaria , AnestésicosRESUMEN
HMGB-like proteins are architectural chromatin factors, and their function is heavily dependent on their ability to interact with DNA (especially non-canonical DNA structures). HMGB1 is involved in many DNA processes, and dysregulation of HMGB protein expression has profound effects on cellular transcription, resulting in severe developmental defects as well as cancer. During DNA replication, elements that form the origin are still not well defined in metazoans. Sites with A (adenine) or T (thymine) repeats cause intrinsic curvatures in the DNA and are described to be involved in the replication machinery by providing binding sites to replication proteins. As a result, the DNA molecule shows intrinsically bent DNA sites, caused by periodic repeats of 2 or more As/Ts (dA/dT) as well as intrinsically non-bent DNA sites (INBDs), due to a succession of curvatures that cancel each other. In the present study, we mapped 11 INBDSs present in the AMPD2 gene that are related to each replication origin (oriGNAI3, oriC, oriB, and oriA). Following characterization of INBDSs, we tested the ability of HMGB1 to bind to the bent (b1, b2, b4a, b4b, b5, b6, b7, and b8) and non-bent DNA fragments (nb7, nb11, nb1, nb2, nb4, and nb5) via electrophoretic mobility shift assays. All fragments showed efficient binding to HMGB1. However, the non-bent DNA fragments nb2, nb4, and nb5 showed slightly reduced binding efficiency.
Asunto(s)
AMP Desaminasa/genética , Replicación del ADN/genética , Proteínas de Unión al ADN/genética , Proteína HMGB1/genética , AMP Desaminasa/química , Animales , Sitios de Unión , Cromatina/química , Cromatina/genética , Cricetulus/genética , ADN/química , ADN/genética , Proteínas de Unión al ADN/química , Proteína HMGB1/química , Conformación de Ácido Nucleico , Unión Proteica , Origen de Réplica/genéticaRESUMEN
We have developed analytical methods to measure the biological functions of pVGI.1(VEGF2), a naked plasmid DNA product containing vascular endothelial growth factor 2 used in clinical trials for coronary artery diseases (CAD) and peripheral artery diseases (PAD). After being injected into muscles, vascular endothelial growth factor 2 (VEGF-2), presumably expressed in muscle tissues, binds to the endothelial cell receptors VEGFR2 or VEGFR3, triggering the downstream responses including cell proliferation and vascularization. As it is important to make sure clinical material is biological active, we developed a quantitative assay first to measure the receptor binding activity of the pVGI.1(VEGF2) gene product expressed by the transfected host cells, and then a qualitative assay to confirm the cell proliferation promoting activity of the expressed protein. In both assays the signals were plotted directly against input DNA concentrations used to transfect the host cells. We confirmed specificity for both assays. In addition, we demonstrated acceptable levels of spike recovery (86.7-116 percent), precision (largest relative standard deviation (RSD)=19.3 percent), linearity and range (60-140 percent relative potency, 15 - 35 ug/mL) for the quantitative assay. We intend to use the potency assays for routine lot release and stability studies.
Asunto(s)
Masculino , Adulto , Bovinos , Cricetinae , Plásmidos , Factor A de Crecimiento Endotelial Vascular , Cricetulus/genética , Células Endoteliales , /métodos , Proliferación Celular , Ensayo de Inmunoadsorción Enzimática/métodos , Estudios de Evaluación como AsuntoRESUMEN
INTRODUCCIÓN: Actualmente, el efecto genotóxico de los antidiabéticos orales es desconocido, motivo por el cual es necesario investigar el tema. MATERIAL Y MÉTODOS: Modelo experimental, para evaluar el efecto genotóxico de los antidiabéticos orales mediante el Test de Intercambio de Cromátides Hermanas (ICH) y el Test de Micronúcleos (MN). Se empleó como material células de la línea celular de ovario de hámster chino (CHO-Kl) Cricetulus griseus y tabletas de glibenclamida y metformina. Las células de la línea celular fueron cultivadas en Ham's F10 suplementado con 2 % estreptomicina-penicilina, 0,5 % L-glutamina y 10% suero bovino fetal (PBS) 20. Las células fueron agrupadas y tratadas con Glibenclamida 5 mg, Metformina 850 mg, Glibenclamida + Metformina 5/850 mg y el grupo control. Los resultados se valoraron en base al Test de Evaluación Genotóxica. RESULTADOS: Se observó un incremento significativo de ICH (p< 0.05) en el grupo de la terapia combinada (8.03 ± 0.17) con respecto al grupo control y a la metformina (6.91 ± 0.19; 6.94 ± 0.45) respectivamente; además se observó un incremento significativo de MN en el grupo de la terapia combinada de glibenclamida con metformina (14.44 ± 0.93), glibenclamida sola (10.18 ± 0.93) Ymetformina sola (7.10 ± 1.04) con respecto al grupo control (3.93 ± 0.46). CONCLUSIÓN: La glibenclamida más metformina produce efecto genotóxico evidenciado por medio de las pruebas ICH y el Test de MN.
INTRODUCTION: Actually, the genotoxic effect of the oral anti-diabetics is unknown, so its important to investigate about it. MATERIAL AND METHODS: We used the experimental design, in order to evaluate the existence of genotoxic effect of the oral antidiabetics by using Sister Chromatid Exchanges (SCE) and Micronucleus assays (MN) techniques. The material was chinese hamster's ovaryan cell line Cricetulus griseus and pills of glibenclamide and metformin. The chinese hamster's ovaryan cellline was grown in Ham's F10 supplemented with streptomycin penicillin 2%, L glutamina 0.5 % and bovine fetal serum 10 % (PBS) 20. Cells were grouped and treated with Glibenclamide 5 mg, Metformin 850 mg, Glibenclamide + Metformine 5/850 mg and the group control. The results were evaluated with the test of Genotoxic assessment. RESULTS: We observed a significant in crease (8,03 ± 0,17) in the treatment with the combined therapy regarding the group control and to the metformin respectively (6.91 ± 0.19; 6.94 ± 0.45), also there were a significant increase of micronucleas in the therapy combined of glibenclamide with metformin (14.44 ± 0.93), glibenclamide alone (10.18 ± 0.93) and metformin alone (7.10 ± 1.04) regarding to the group control (3.93 ± 0.46). CONCLUSION: There is genotoxic effect in the group of combinated therapy (Glibenclamide + Metformine) demonstrated using the of Sister Chromatid Exchanges (SCE) and Micronucleus assays (MN) techniques.