RESUMO
Abstract Routine microbiological monitoring of rodent colonies in animal facilities is essential to evaluate the health status of the animals used in research studies. In the present study, animals were examined for the presence of selected microbial infections. In order to determine the contamination rates of mice and rats in Argentina, animals from 102 conventional facilities were monitored from 2012 to 2016. The most frequent bacteria isolated were Pseudomonas aeruginosa and Proteus spp. The common parasites identified were Syphacia spp. and Tritrichomonas spp. Serological assays demonstrated the highest prevalence for Mouse hepatitis virus in mice and Sialodacryoadenitis virus in rats. The results indicate that there is a high incidence of infections, so it is suggested that an efficient management system and effective sanitary barriers should be implemented in conventional facilities in Argentina in order to improve sanitary standards.
Resumen Los controles microbiológicos de rutina en colonias de roedores en bioterios son esenciales para evaluar el estado de salud de los animales que se utilizan en las investigaciones. En el presente estudio se examinaron animales de bioterios de Argentina con el objeto de detectar la presencia de infecciones microbianas seleccionadas. Con el fin de determinar los porcentajes de contaminaciones en estos individuos, se controlaron animales de 102 bioterios convencionales entre 2012 y 2016. Las bacterias más frecuentes aisladas fueron Pseudomonas aeruginosa y Proteus spp. Los parásitos comunes identificados fueron Syphacia spp. y Tritrichomonas spp. Los ensayos serológicos demostraron la mayor prevalencia del virus de hepatitis del ratón en ratones y del virus de la Syalodacryoadenitis en ratas. Los resultados indican que hay una alta incidencia de infecciones, por lo que se sugiere que se debe implementar un sistema de gestión eficiente y barreras sanitarias eficaces en instalaciones convencionales en Argentina con el objeto de mejorar los estándares sanitarios.
RESUMO
Routine microbiological monitoring of rodent colonies in animal facilities is essential to evaluate the health status of the animals used in research studies. In the present study, animals were examined for the presence of selected microbial infections. In order to determine the contamination rates of mice and rats in Argentina, animals from 102 conventional facilities were monitored from 2012 to 2016. The most frequent bacteria isolated were Pseudomonas aeruginosa and Proteus spp. The common parasites identified were Syphacia spp. and Tritrichomonas spp. Serological assays demonstrated the highest prevalence for Mouse hepatitis virus in mice and Sialodacryoadenitis virus in rats. The results indicate that there is a high incidence of infections, so it is suggested that an efficient management system and effective sanitary barriers should be implemented in conventional facilities in Argentina in order to improve sanitary standards.
Assuntos
Doenças dos Animais/microbiologia , Doenças dos Animais/parasitologia , Animais de Laboratório/microbiologia , Animais de Laboratório/parasitologia , Doenças dos Animais/epidemiologia , Animais , Argentina , Infecções Bacterianas/epidemiologia , Infecções Bacterianas/microbiologia , Infecções Bacterianas/veterinária , Feminino , Incidência , Masculino , Camundongos , Doenças Parasitárias/epidemiologia , Doenças Parasitárias/parasitologia , Ratos , Viroses/epidemiologia , Viroses/veterinária , Viroses/virologiaRESUMO
We have developed a new method for the measurement of subcutaneous tumour volume which consists in taking photographs of mice in their home cages, to refine the standard method of measurement with calipers. We consider this new method to be non-aversive, as it may be more compatible with mice behavioural preferences and, therefore, improve their welfare. Photographs are captured when mice voluntarily go into an acrylic tube containing graph paper that is later used as a scale. Tumour volumes measured with the caliper and the non-aversive photographic method were compared to those obtained by water displacement volume and weight. Behavioural and physiological changes were evaluated to assess animal welfare. Significant differences were found between measurements obtained with the caliper and the non-aversive photographic method, v. the reference volume acquired by water displacement (P < 0.001). Nevertheless, there was good consistency for these measurements when tumours were measured repeatedly, with all Intra-Class Correlation Coefficients above 0.95. Mice on which the non-aversive photographic method was employed were significantly less reluctant to establish contact with the experimenter (P < 0.001) and behaved less anxiously in a modified-Novelty Suppressed Feeding test. Particularly, statistically significant differences were found in connection with the latency to eat an almond piece (P < 0.05), the frequency of grooming (P < 0.001) and the frequency of defecation (P < 0.001). Corticosterone concentration in faeces and blood glucose were determined and no significant changes were found. Therefore, we propose the non-aversive photographic method to measure subcutaneous tumours as a way to refine methodologies in the field of experimental oncology.
Assuntos
Camundongos Nus , Fotografação/métodos , Doenças dos Roedores/diagnóstico por imagem , Neoplasias de Tecidos Moles/diagnóstico por imagem , Carga Tumoral , Animais , Feminino , Masculino , Camundongos , Organismos Livres de Patógenos EspecíficosRESUMO
In this study we developed an indirect ELISA to detect antibodies against Minute Virus of Mice (MVM) using an antigen produced from BHK-21 cells infected with a prototype strain of the virus. The optimal antigen concentration and serum dilutions were established. In order to analyze variability in the laboratory, reproducibility and repeatability within and between plates were determined. Then, a panel of 460 sera from conventional facilities and previously classified as positive or negative by the indirect fluorescent antibody assay was analyzed. The cutoff value was determined by a receiver operating characteristic (ROC) curve. The results of the indirect ELISA were compared with those of the indirect fluorescent antibody assay. The ELISA assay showed 100% sensitivity and 99% specificity. ELISA is a useful tool to be developed in standard virology laboratories and can be used for screening animals faster than the traditional indirect fluorescent antibody assay.
Assuntos
Anticorpos Antivirais , Ensaio de Imunoadsorção Enzimática , Vírus Miúdo do Camundongo , Animais , Anticorpos Antivirais/análise , Técnica Indireta de Fluorescência para Anticorpo , Camundongos , Vírus Miúdo do Camundongo/imunologia , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Osteosarcoma (OS) is the most common primary tumor of bone, occurring predominantly in the second decade of life. High-dose cytotoxic chemotherapy and surgical resection have improved prognosis, with long-term survival for patients with localized disease. Vanadium is an ultra-trace element that after being absorbed accumulates in bone. Besides, vanadium compounds have been studied during recent years to be considered as representative of a new class of non-platinum antitumor agents. Moreover, flavonoids are a wide family of polyphenolic compounds that display many interesting biological effects. Since coordination of ligands to metals can improve the pharmacological properties, we report herein, for the first time, the in vitro and in vivo effects of an oxidovanadium(IV) complex with the flavonoid chrysin on the new 3D human osteosarcoma and xenograft osteosarcoma mice models. The pharmacological results show that VOchrys inhibited the cell viability affecting the shape and volume of the spheroids and VOchrys suppressed MG-63 tumor growth in the nude mice without inducing toxicity and side effects. As a whole, the results presented herein demonstrate that the antitumor action of the complex was very promissory on human osteosarcoma models, whereby suggesting that VOchrys is a potentially good candidate for future use in alternative antitumor treatments.
Assuntos
Neoplasias Ósseas/tratamento farmacológico , Complexos de Coordenação/farmacologia , Flavonoides/farmacologia , Osteossarcoma/tratamento farmacológico , Esferoides Celulares/efeitos dos fármacos , Vanádio/farmacologia , Animais , Neoplasias Ósseas/patologia , Técnicas de Cultura de Células/métodos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Complexos de Coordenação/química , Feminino , Flavonoides/química , Humanos , Masculino , Camundongos Nus , Microscopia de Contraste de Fase , Estrutura Molecular , Osteossarcoma/patologia , Esferoides Celulares/patologia , Fatores de Tempo , Resultado do Tratamento , Vanádio/química , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Antigenic proteins whose expression is induced under iron starvation, an environmental condition that bacterial pathogens have to face during colonization, might be potential candidates for improved vaccine. By mean of immune proteomics we identified novel antigens of Bordetella pertussis maximally expressed under iron limitation. Among them, Bp1152 (named as IRP1-3) showed a particularly strong reaction with human IgG purified from pooled sera of pertussis-infected individuals. Computer analysis showed IRP1-3 as a dimeric membrane protein potentially involved in iron uptake. Experimental data revealed the surface-exposure of this protein and showed its increase under iron starvation to be independent of bacterial virulence phase. Immunization of mice with the recombinant IRP1-3 resulted in a strong antibody response. These antibodies not only recognized the native protein on bacterial surface but also promote effective bacterial phagocytosis by human PMN, a key protecting activity against this pathogen. Accordingly, IRP1-3 proved protective against B. pertussis infection in mouse model. Expression of IRP1-3 was found conserved among clinical isolates of B. pertussis and positively regulated by iron starvation in these strains. Taken together these results suggest that this protein might be an interesting novel vaccine candidate.
Assuntos
Antígenos de Bactérias/imunologia , Bordetella pertussis/imunologia , Proteínas de Membrana/imunologia , Vacina contra Coqueluche/imunologia , Animais , Anticorpos Antibacterianos/sangue , Antígenos de Bactérias/administração & dosagem , Feminino , Proteínas de Membrana/administração & dosagem , Camundongos , Camundongos Endogâmicos BALB C , Neutrófilos/imunologia , Vacina contra Coqueluche/administração & dosagem , Fagocitose , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/imunologiaRESUMO
Los biomateriales para regeneración ósea son cada vez más utilizados. Los de origen sintético son a menudo mejor aceptados por los pacientes. Entre ellos, el sulfato de calcio ha sido ampliamente usado. La continua aparición de nuevos productos indica que el material ideal aun esta por descubrirse. Desarrollar y evaluar un biomaterial a base de sulfato de calcio sólo y enriquecido con minerales. Se utilizó sulfato de calcio de grado médico y minerales óseos los cuales fueron injertados en forma de partículas en defectos óseos del fémur de 20 ratas Wistar. Al Grupo 1 se le injertó Sulfato de Calcio, al Grupo 2 Sulfato de Calcio enriquecido con minerales, y el Grupo 3 (control).Se tomaron muestras a los 30 días las cuales fueron preparadas para microscopía. Las mismas fueron digitalizadas y se evaluó la superficie de hueso regenerado, dentro del defecto. Resultados: La superficie regenerada, en mm2 para el grupo 1 fue de 9,878 (0,833), para el grupo 2 de 11,317 (0,937) y para el grupo 3 de 4,370 (0,549). Se observaron diferencias estadísticamente significativas entre los grupos (P = <0,001) -ANOVA (análisis de varianza). En las comparaciones apareadas se observaron diferencias estadísticamente significativas entre todos los grupos (P = <0,005) Holm-Sidak. El material desarrollado es bien tolerado y posibilita la regeneración completa de un defecto crítico cuando fue tratado con Sulfato de Calcio enriquecido con minerales. Pruebas clínicas son necesarias para una mejor comprensión de su posible utilización como sustituto óseo
Biomaterials for bone regeneration are increasingly used. Those of synthetic origin are often better accepted by patients. Among them, calcium sulfate has been widely used. The continuing emergence of new products indicates that the ideal material has yet to be discovered. To develop and evaluate a biomaterial based on calcium sulfate alone and enriched with minerals. We used calcium sulfate and medical grade mineral bone which were grafted in the form of particles in the femur bone defects of 20 Wistar rats. Group 1 was grafted calcium sulfate, the Group 2 enriched with calcium sulfate minerals, and Group 3 (control). Samples were taken for 30 days which were prepared for microscopy. These were digitized and evaluated the surface of regenerated bone within the defect. Results: The reclaimed area in mm2 in group 1 was 9.878 (0.833), for group 2 11.317 (0.937) and group 3, 4.370 (0.549). Statistically significant differences were observed between groups (P = <0.001)-ANOVA (analysis of variance). In paired comparisons were statistically significant differences between all groups (P = <0.005) Holm-Sidak. The developed material is well tolerated and allows the complete regeneration of a critical defect when he was treated with calcium sulfate enriched with minerals. Clinical trials are needed to better understand their potential use as bone substitute
Assuntos
Animais , Masculino , Ratos , Regeneração Óssea , Teste de Materiais/veterinária , Materiais Biocompatíveis/análise , Materiais Biocompatíveis/uso terapêutico , Transplante Ósseo/métodos , Transplante Ósseo/veterináriaRESUMO
The clinical efficacy of conditionally replicative oncolytic adenoviruses (CRAd) is still limited by the inefficient infection of the tumor mass. Since tumor growth is essentially the result of a continuous cross-talk between malignant and tumor-associated stromal cells, targeting both cell compartments may profoundly influence viral efficacy. Therefore, we developed SPARC promoter-based CRAds since the SPARC gene is expressed both in malignant cells and in tumor-associated stromal cells. These CRAds, expressing or not the Herpes Simplex thymidine kinase gene (Ad-F512 and Ad(I)-F512-TK, respectively) exerted a lytic effect on a panel of human melanoma cells expressing SPARC; but they were completely attenuated in normal cells of different origins, including fresh melanocytes, regardless of whether cells expressed or not SPARC. Interestingly, both CRAds displayed cytotoxic activity on SPARC positive-transformed human microendothelial HMEC-1 cells and WI-38 fetal fibroblasts. Both CRAds were therapeutically effective on SPARC positive-human melanoma tumors growing in nude mice but exhibited restricted efficacy in the presence of co-administered HMEC-1 or WI-38 cells. Conversely, co-administration of HMEC-1 cells enhanced the oncolytic efficacy of Ad(I)-F512-TK on SPARC-negative MIA PaCa-2 pancreatic cancer cells in vivo. Moreover, conditioned media produced by stromal cells pre-infected with the CRAds enhanced the in vitro viral oncolytic activity on pancreatic cancer cells, but not on melanoma cells. The whole data indicate that stromal cells might play an important role on the outcome of the oncolytic efficacy of conditionally replicative adenoviruses.
Assuntos
Adenoviridae/fisiologia , Neoplasias/patologia , Terapia Viral Oncolítica , Células Estromais/patologia , Animais , Linhagem Celular , Linhagem Celular Tumoral , Efeito Citopatogênico Viral , Humanos , Camundongos , Transplante de Neoplasias , Neoplasias/terapia , Osteonectina/genética , Regiões Promotoras Genéticas , Replicação ViralRESUMO
PURPOSE: A33 antigen is a membrane-bound protein expressed in intestinal epithelium that is overexpressed in 95% of primary and metastatic colorectal carcinomas but is absent in most epithelial tissues and tumor types. We hypothesized that A33 promoter might be useful in the design of a conditionally replicative adenovirus for the treatment of colorectal cancer (CRC). EXPERIMENTAL DESIGN: We cloned an A33 promoter fragment (A33Pr) that extends from -105 to +307 bp. Using luciferase activity as a reporter gene, we showed that A33Pr was active in CRC cell lines. We next constructed a conditionally replicative adenovirus named AV22EL where E1A was placed under the control of A33Pr. The tumor-specific oncolytic effect of AV22EL was investigated both in vitro and in vivo. RESULTS: AV22EL induced specific in vitro lysis of human CRC cell lines that expressed A33 and have negligible lytic capacity on cells that lacked or had minimal A33 expression, including normal human colonic cells. In vivo, a marked reduction of tumor growth and increased long-term survival rates were observed in nude mice xenografted with s.c. CRC tumors. Combination with 5-fluorouracil induced an additive effect in vitro with no toxic effects in vivo. Remarkably, AV22EL completely eliminated established hepatic metastases in >90% of mice and restored hepatic function according to biochemical parameters. Its systemic administration induced E1A expression only in the hepatic metastasis but not in normal organs. CONCLUSIONS: These data show that AV22EL is a stringently regulated and potent oncolytic agent for the treatment of CRC.
Assuntos
Adenoviridae/genética , Neoplasias do Colo/terapia , Neoplasias Hepáticas/terapia , Glicoproteínas de Membrana/genética , Terapia Viral Oncolítica , Regiões Promotoras Genéticas/genética , Adenoviridae/metabolismo , Proteínas E1A de Adenovirus/metabolismo , Animais , Antimetabólitos Antineoplásicos/farmacologia , Western Blotting , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Neoplasias da Mama/terapia , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/secundário , Carcinoma Hepatocelular/terapia , Neoplasias do Colo/patologia , Terapia Combinada , Feminino , Feto/efeitos dos fármacos , Feto/virologia , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/virologia , Fluoruracila/farmacologia , Vetores Genéticos , Humanos , Neoplasias Hepáticas/secundário , Luciferases/metabolismo , Pulmão/citologia , Pulmão/efeitos dos fármacos , Pulmão/virologia , Masculino , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Nus , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Esferoides Celulares , Replicação Viral , Ensaios Antitumorais Modelo de Xenoenxerto , beta-GalactosidaseRESUMO
The current study demonstrates the ability of an indirect enzyme-linked immunosorbent assay (iELISA) to detect antibodies against Theiler's murine encephalomyelitis virus in mice colonies. The antigen was produced from infected baby hamster kidney (BHK)-21 cells and treated with 1% Nonidet P40 in saline buffer. Control antigen was prepared following the same procedure using uninfected BHK-21 cells. The optimal antigen and serum dilutions were established. The reaction was revealed using an anti-mouse-horseradish peroxidase conjugate and 2,2'-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid). Optimized iELISA was validated by detection of antibodies in known positive and negative serum samples before testing the samples of unknown status. Performance of the iELISA was compared with the indirect fluorescent antibody test, and the cutoff value was determined by receiver operating curve. Indirect ELISA showed 100% sensitivity, 99.38% specificity, and 97.78% predictive positive value. The antigen used is easy to produce, and no special equipment is required. The iELISA developed is simple and provides a rapid and less costly tool for diagnosis and research.