RESUMO
The Editor-in-Chief has retracted this article [1]. Figures 1A, 1D and 2B (bottom right) are identical with Figures 1A, 1H and 1B respectively in another article [2] which reports a study in a different species. In addition, Table 1 contains data presented in a third article [3], which also reports a study in a different species. The Editor-in-Chief therefore no longer has confidence in the validity of the data and the conclusions drawn. Tereza C. Cardoso disagrees with this retraction. Helena L. Ferreira agrees with this retraction. Sergio E. L. da Silva, Andrea F. Garcia, Felipe E. S. Silva, Roberto Gameiro, Carolina U. F. Fabri and Dielson S. Vieira have not responded to any correspondence about this retraction.
RESUMO
To establish an association between mitochondrial dysfunction and apoptosis following infectious bronchitis virus (IBV) infection, HD11 avian macrophage cells were infected with the Massachusetts 41 (M41) strain. Our results show that the M41 strain of IBV induced cytopathic effects followed by the release of new viral particles. Elevated numbers of apoptotic cells were observed at 24, 48 and 72 h post-infection (p.i.). Viral infection was associated with mitochondrial membrane depolarization and reactive oxygen species (ROS) production at all of the examined timepoints p.i. In summary, IBV M41 replication in infected HD11 macrophages seems to induce mitochondrial bioenergy failure, acting as a respiratory chain uncoupler, without compromising viral replication.
Assuntos
Interações Hospedeiro-Patógeno , Vírus da Bronquite Infecciosa/patogenicidade , Macrófagos/virologia , Mitocôndrias/virologia , Vírion/patogenicidade , Animais , Apoptose , Linhagem Celular , Proliferação de Células , Galinhas , Vírus da Bronquite Infecciosa/crescimento & desenvolvimento , Macrófagos/metabolismo , Potencial da Membrana Mitocondrial , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Vírion/crescimento & desenvolvimento , Replicação ViralRESUMO
BACKGROUND: Bovine Herpesvirus type-5 (BoHV-5) is a neurovirulent α-Herpesvirus which is potentially pathogenic for cows and suspected to be associated with reproductive disorders. Interestingly, natural transmission of BoHV-5 by contaminated semen was recently described in Australia. Additionally, BoHV-5 was also isolated from the semen of a healthy bull in the same country and incriminated in a natural outbreak of reproductive disease after artificial insemination. In contrast with BoHV-1, experimental exposure of in vitro produced bovine embryos to BoHV-5 does not affect embryo viability and seems to inhibit some pathways of apoptosis. However, the mechanisms responsible for these phenomena are poorly understood. In this study, we examined mitochondrial activity, antioxidant protection, stress response and developmental rates of in vitro produced bovine embryos that were exposed and unexposed to BoHV-5. METHODS: For this purpose, bovine embryos produced in vitro were assayed for cell markers after experimental infection of oocytes (n = 30; five repetitions), in vitro fertilization and development. The indirect immunofluorescence was employed to measure the expression of superoxide dismutase 1 (SOD1), anti-oxidant like protein 1 (AOP-1), heat shock protein 70.1 (Hsp 70.1) and also viral antigens in embryos derived from BoHV-5 exposed and unexposed oocytes. The determination of gene transcripts of mitochondrial activity (SOD1), antioxidant protection (AOP-1) and stress response (Hsp70.1) were evaluated using the reverse transcriptase polymerase chain reaction (RT-PCR). MitoTracker Green FM, JC-1 and Hoechst 33342-staining were used to evaluate mitochondrial distribution, segregation patterns and embryos morphology. The intensity of labeling was graded semi-quantitatively and embryos considered intensively marked were used for statistical analysis. RESULTS: The quality of the produced embryos was not affected by exposure to BoHV-5. Of the 357 collected oocytes, 313 (+/- 6.5; 87.7%) were cleaved and 195 (+/- 3.2; 54.6%) blastocysts were produced without virus exposure. After exposure, 388 oocytes were cleaved into 328 (+/- 8.9, 84.5%), and these embryos produced 193 (+/- 3.2, 49.7%) blastocysts. Viral DNA corresponding to the US9 gene was only detected in embryos at day 7 after in vitro culture, and confirmed by indirect immunofluorescence assay (IFA). These results revealed significant differences (p < 0.05) between exposed and unexposed oocytes fertilized, as MitoTracker Green FM staining Fluorescence intensity of Jc-1 staining was significantly higher (p < 0.005) among exposed embryos (143 +/- 8.2). There was no significant difference between the ratios of Hoechst 33342-stained nuclei and total cells in good-quality blastocysts (in both the exposed and unexposed groups). Using IFA and reverse transcriptase polymerase chain reaction (RT-PCR) for the set of target transcripts (SOD1, AOP-1 and Hsp 70.1), there were differences in the mRNA and respective proteins between the control and exposed embryos. Only the exposed embryos produced anti-oxidant protein-like 1 (AOP-1). However, neither the control nor the exposed embryos produced the heat shock protein Hsp 70.1. Interestingly, both the control and the exposed embryos produced superoxide dismutase (SOD1), revealing intense mitochondrial activity. CONCLUSION: This is the first demonstration of SOD1 and AOP-1 production in bovine embryos exposed to BoHV-5. Intense mitochondrial activity was also observed during infection, and this occurred without interfering with the quality or number of produced embryos. These findings further our understanding on the ability of α-Herpesviruses to prevent apoptosis by modulating mitochondrial pathways.
Assuntos
Apoptose , Blastocisto/virologia , Ectogênese , Herpesvirus Bovino 5/metabolismo , Mitocôndrias/metabolismo , Peroxirredoxina III/metabolismo , Superóxido Dismutase/metabolismo , Animais , Blastocisto/metabolismo , Blastocisto/patologia , Bovinos , Doenças dos Bovinos/embriologia , Doenças dos Bovinos/metabolismo , Doenças dos Bovinos/patologia , Doenças dos Bovinos/virologia , Fase de Clivagem do Zigoto/metabolismo , Fase de Clivagem do Zigoto/patologia , Fase de Clivagem do Zigoto/virologia , Feminino , Fertilização in vitro/efeitos adversos , Fertilização in vitro/veterinária , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Infecções por Herpesviridae/embriologia , Infecções por Herpesviridae/patologia , Infecções por Herpesviridae/veterinária , Infecções por Herpesviridae/virologia , Herpesvirus Bovino 5/isolamento & purificação , Técnicas de Maturação in Vitro de Oócitos , Masculino , Mitocôndrias/enzimologia , Mitocôndrias/virologia , Oócitos/fisiologia , Oócitos/virologia , Peroxirredoxina III/genética , RNA Mensageiro/metabolismo , Superóxido Dismutase/genética , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismoRESUMO
BACKGROUND: The possibility for isolating bovine mesenchymal multipotent cells (MSCs) from fetal adnexa is an interesting prospect because of the potential for these cells to be used for biotechnological applications. Bone marrow and adipose tissue are the most common sources of MSCs derived from adult animals. However, little knowledge exists about the characteristics of these progenitors cells in the bovine species. Traditionally most cell cultures are developed in two dimensional (2D) environments. In mammalian tissue, cells connect not only to each other, but also support structures called the extracellular matrix (ECM). The three-dimensional (3D) cultures may play a potential role in cell biotechnology, especially in tissue therapy. In this study, bovine-derived umbilical cord Wharton's jelly (UC-WJ) cells were isolated, characterized and maintained under 3D-free serum condition as an alternative of stem cell source for future cell banking. RESULTS: Bovine-derived UC-WJ cells, collected individually from 5 different umbilical cords sources, were successfully cultured under serum-free conditions and were capable to support 60 consecutive passages using commercial Stemline(®) mesenchymal stem cells expansion medium. Moreover, the UC-WJ cells were differentiated into osteocytes, chondrocytes, adipocytes and neural-like cells and cultured separately. Additionally, the genes that are considered important embryonic, POU5F1 and ITSN1, and mesenchymal cell markers, CD105(+), CD29(+), CD73(+) and CD90(+) in MSCs were also expressed in five bovine-derived UC-WJ cultures. Morphology of proliferating cells typically appeared fibroblast-like spindle shape presenting the same viability and number. These characteristics were not affected during passages. There were 60 chromosomes at the metaphase, with acrocentric morphology and intense telomerase activity. Moreover, the proliferative capacity of T cells in response to a mitogen stimulus was suppressed when bovine-derived UC-WJ cells was included in the culture which demonstrated the immunossupression profile typically observed among isolated mesenchymal cells from other species. After classified the UC-WJ cells as mesenchymal stromal phenotype the in vitro 3D cultures was performed using the AlgiMatrix(®) protocol. Based on the size of spheroids (283,07 µm ± 43,10 µm) we found that three weeks of culture was the best period to growth the UC-WJ cells on 3D dimension. The initial cell density was measured and the best value was 1.5 × 10(6) cells/well. CONCLUSIONS: We described for the first time the isolation and characterization of UC-WJ cells in a serum-free condition and maintenance of primitive mesenchymal phenotype. The culture was stable under 60 consecutive passages with no genetic abnormalities and proliferating ratios. Taken together all results, it was possible to demonstrate an easy way to isolate and culture of bovine-derived UC-WJ cells under 2D and 3D serum-free condition, from fetal adnexa with a great potential in cell therapy and biotechnology.
Assuntos
Técnicas de Cultura de Células/métodos , Separação Celular/métodos , Células-Tronco Mesenquimais/citologia , Cordão Umbilical/citologia , Geleia de Wharton/citologia , Animais , Bovinos , Diferenciação Celular , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Meios de Cultura Livres de Soro/metabolismo , Feminino , Masculino , Células-Tronco Mesenquimais/metabolismo , Telomerase/metabolismo , Cordão Umbilical/embriologia , Cordão Umbilical/metabolismo , Geleia de Wharton/embriologia , Geleia de Wharton/metabolismoRESUMO
Monocrotaline (MCT) is a pyrrolizidine alkaloid present in plants of the Crotalaria species that causes cytotoxicity and genotoxicity, including hepatotoxicity in animals and humans. It is metabolized by cytochrome P-450 in the liver to the alkylating agent dehydromonocrotaline (DHM). In previous studies using isolated rat liver mitochondria, we observed that DHM, but not MCT, inhibited the activity of respiratory chain complex I and stimulated the mitochondrial permeability transition with the consequent release of cytochrome c. In this study, we evaluated the effects of MCT and DHM on isolated rat hepatocytes. DHM, but not MCT, caused inhibition of the NADH-linked mitochondrial respiration. When hepatocytes of rats pre-treated with dexamethasone were incubated with MCT (5 mM), they showed ALT leakage, impaired ATP production and decreased levels of intracellular reduced glutathione and protein thiols. In addition, MCT caused cellular death by apoptosis. The addition of fructose or dithiotreitol to the isolated rat hepatocyte suspension containing MCT prevented the ATP depletion and/or glutathione or thiol oxidation and decreased the ALT leakage and apoptosis. These results suggest that the toxic effect of MCT on hepatocytes may be caused by metabolite-induced mitochondrial energetic impairment, together with a decrease of cellular glutathione and protein thiols.
Assuntos
Apoptose/efeitos dos fármacos , Ditiotreitol/farmacologia , Frutose/farmacologia , Hepatócitos/efeitos dos fármacos , Mitocôndrias Hepáticas/efeitos dos fármacos , Monocrotalina/toxicidade , Trifosfato de Adenosina/metabolismo , Animais , Crotalaria/metabolismo , Glutationa/metabolismo , Hepatócitos/metabolismo , Masculino , Mitocôndrias Hepáticas/metabolismo , Monocrotalina/análogos & derivados , Proteínas/metabolismo , Ratos , Ratos Wistar , Compostos de Sulfidrila/metabolismoRESUMO
Infection of young poults with turkey coronavirus (TCoV) produces a syndrome characterized by acute enteritis, diarrhea, anorexia, ruffled feathers, decreased body weight gain and uneven flock growth. The objective of this study was to standardize an intestinal organ culture (IOC) in order to assess host-virus interaction related to apoptosis. For this purpose the Brazilian strain (TCoV/Brazil/2006 with GenBank accession number FJ188401), was used for infection. Infected IOC cells had mitochondrial dysfunction and initial nuclear activation with MTT value of 90.7 (± 2.4) and apoptotic factor 2.21 (± 2.1), considered statistically different from uninfected IOC cells (p > 0.05). The kinetics of TCoV antigens and viral RNA was directly correlated to annexin-V, caspases- 2 and -3, p53, BCl-2 antigens at 24, 72 and 96 h post-infection (p.i.). Morphological and biochemical features of apoptosis, such as in situ nuclear fragmentation (TUNEL and annexin-V) and DNA ladder formation were also detected in infected cells at all assayed p.i. intervals. Moreover, different from other coronaviruses, the expression of both effective caspase-2 and -3 and p53 antigens were considered lower. However, at all p.i., the BCl-2 antigens were expressed quantitatively and qualitatively as viral antigen measured by immunofluorescence microscopy analysis. Because the diagnosis of TCoV infection is only performed by infecting embryonated poult eggs, the pathological characteristics related to host-virus interaction remain unclear. This is the first report on apoptosis of TCoV infected IOC, and reveals that it may be useful immunological method to assess virus pathogenesis.(AU)
Assuntos
Animais , Perus/virologia , Cultura de Vírus/métodos , Infecções por Coronavirus/diagnóstico , Interações entre Hospedeiro e Microrganismos/fisiologia , Intestinos/virologia , CoronavirusRESUMO
The degree of genetic and pathologic variation exhibited by a turkey Coronavirus (TCoV) strain was investigated after nine serial passages in 25-day-old turkey embryos obtained from wild broad-breasted bronze breeders. In spite of spleen, liver, kidneys, cloacal bursa and thymus have been collected and analysed, the main histopathological changes were only documented in the intestine sections. Microscopic lesions were characterized as mild enteritis, low degree of enterocyte vacuolization and detachment of the intestinal villous after five consecutive passages and were considered absent in the last passages. Genealogic analysis based on S1 and S2 DNA sequences suggested that Brazilian isolate might be considered as originated from TCoV strains circulating in the United States, as 100% identity with TCoV-Gl strain. Although S1 S2 sequences from each passage revealed no significant point mutations, and no correlation could be speculate between S2 nucleotide changes and pathologic features in infected embryos. This is the first demonstration of wild turkey embryos as a model for TCoV isolation and propagation.(AU)
Assuntos
Animais , Coronavirus do Peru/patogenicidade , Enterite/diagnóstico , Enterite Transmissível dos Perus/fisiopatologia , Perus , Variação Genética , Animais SelvagensRESUMO
Meningoencephalitis by Herpesvirus type 5 (BoHV-5) in cattle has some features that are similar to those of herpetic encephalitis in humans and other animal species. Human Herpesvirus 3 (commonly known as Varicella-zoster virus 1), herpes simplex viruses (HSV), and equid Herpesvirus 1 (EHV-1) induce an intense inflammatory, vascular and cellular response. In spite of the many reports describing the histological lesions associated with natural and experimental infections, the immunopathological mechanisms for the development of neurological disorder have not been established. A total of twenty calf brains were selected from the Veterinary School, University of São Paulo State, Araçatuba, Brazil, after confirmation of BoHV-5 infection by virus isolation as well as by a molecular approach. The first part of the study characterized the microscopic lesions associated with the brain areas in the central nervous system (CNS) that tested positive in a viral US9 gene hybridization assay. The frontal cortex (Fc), parietal cortex (Pc), thalamus (T) and mesencephalon (M) were studied. Secondly, distinct pathogenesis mechanisms that take place in acute cases were investigated by an immunohistochemistry assay. This study found the frontal cortex to be the main region where intense oxidative stress phenomena (AOP-1) and synaptic protein expression (SNAP-25) were closely related to inflammatory cuffs, satellitosis and gliosis, which represent the most frequently observed neurological lesions. Moreover, MMP-9 expression was shown to be localized in the leptomeninges, in the parenchyma and around mononuclear infiltrates (p < 0.0001). These data open a new perspective in understanding the role of the AOP-1, MMP-9 and SNAP-25 proteins in mediating BoHV-5 pathogenesis and the strategies of host-virus interaction in order to invade the CNS.
Assuntos
Encéfalo/metabolismo , Doenças dos Bovinos/metabolismo , Encefalite Viral/veterinária , Infecções por Herpesviridae/veterinária , Herpesvirus Bovino 5/patogenicidade , Imuno-Histoquímica , Meningoencefalite/veterinária , Animais , Biomarcadores/análise , Encéfalo/patologia , Encéfalo/virologia , Bovinos , Doenças dos Bovinos/patologia , Doenças dos Bovinos/virologia , Encefalite Viral/metabolismo , Encefalite Viral/patologia , Encefalite Viral/virologia , Infecções por Herpesviridae/metabolismo , Infecções por Herpesviridae/patologia , Infecções por Herpesviridae/virologia , Herpesvirus Bovino 5/genética , Hibridização In Situ , Metaloproteinase 9 da Matriz/análise , Meningoencefalite/metabolismo , Meningoencefalite/patologia , Meningoencefalite/virologia , Peroxirredoxinas/análise , Proteína 25 Associada a Sinaptossoma/análise , Proteínas do Envelope Viral/genéticaRESUMO
Lantana (Lantana camara Linn.) is a noxious weed to which certain medicinal properties have been attributed, but its ingestion has been reported to be highly toxic to animals and humans, especially in the liver. The main hepatotoxin in lantana leaves is believed to be the pentacyclic triterpenoid lantadene A (LA), but the precise mechanism by which it induces hepatotoxicity has not yet been established. This work addressed the action of LA and its reduced derivative (RLA) on mitochondrial bioenergetics. At the concentration range tested (5-25 microM), RLA stimulated state-4 respiration, inhibited state-3 respiration, circumvented oligomycin-inhibited state-3 respiration, dissipated membrane potential and depleted ATP in a concentration-dependent manner. However, LA did not stimulate state-4 respiration, nor did it affect the other mitochondrial parameters to the extent of its reduced derivative. The lantadenes didn't inhibit the CCCP-uncoupled respiration but increased the ATPase activity of intact coupled mitochondria. The ATPase activity of intact uncoupled or disrupted mitochondria was not affected by the compounds. We propose, therefore, that RLA acts as a mitochondrial uncoupler of oxidative phosphorylation, a property that arises from the biotransformation (reduction) of LA, and LA acts in other mitochondrial membrane components rather than the ATP synthase affecting the mitochondrial bioenergetics. Such effects may account for the well-documented hepatoxicity of lantana.
Assuntos
Metabolismo Energético/efeitos dos fármacos , Lantana/toxicidade , Mitocôndrias Hepáticas/metabolismo , Ácido Oleanólico/análogos & derivados , Toxinas Biológicas/toxicidade , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Doença Hepática Induzida por Substâncias e Drogas/patologia , Técnicas In Vitro , Lantana/química , Masculino , Potenciais da Membrana/efeitos dos fármacos , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/enzimologia , Ácido Oleanólico/química , Ácido Oleanólico/toxicidade , Fragilidade Osmótica/efeitos dos fármacos , Fosforilação Oxidativa/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , Ratos , Ratos Wistar , Toxinas Biológicas/química , Desacopladores/química , Desacopladores/toxicidadeRESUMO
Canine distemper virus (CDV) may induce multifocal demyelination in the central nervous system of infected dogs. The present work investigated apoptosis in white and gray matter (granular layer) in the cerebellum of naturally infected dogs by the analysis of the expression of the pro-apoptotic antigens caspase 2 and 3 , b(terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL-staining) positivity, annexin-V immunodetection, and the presence of the anti-apoptotic antigens, BCl-2 and p53. Cerebellum specimens were obtained from the Laboratory of Animal Pathology, from 1995 to 2009, and the 5-µm thick fragments were stained both with hematoxylin-eosin and Shorr. All samples were diagnosed as positive for CDV genome by reverse transcriptase polymerase chain reaction targeting the nucleocapsid gene. The anti-apoptotic pathways evidenced in this study were BCl-2 and p53 proteins that were intensively detected in cerebellum of CDV positive slides (40-80% of labeled cells/mm2). In addition, the apoptosis markers annexin-V and TUNEL are directly correlated among the same samples (80 and 40% of labeled cells, respectively). This is the first description of p53 and annexin-V expression, characterized as anti-apoptotic and apoptotic proteins, involvement in canine natural cases of CDV infections. (AU)
Assuntos
Animais , Vírus da Cinomose Canina/isolamento & purificação , Caspase 2/análise , Caspase 2 , Caspase 3/análise , Caspase 3 , Proteína Supressora de Tumor p53/análise , Proteína Supressora de Tumor p53 , Anexina A5/biossíntese , Hematoxilina , Amarelo de Eosina-(YS) , Coloração e Rotulagem/métodos , Coloração e Rotulagem/veterináriaRESUMO
Canine distemper virus (CDV) may induce multifocal demyelination in the central nervous system of infected dogs. The present work investigated apoptosis in white and gray matter (granular layer) in the cerebellum of naturally infected dogs by the analysis of the expression of the pro-apoptotic antigens caspase 2 and 3 , b(terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL-staining) positivity, annexin-V immunodetection, and the presence of the anti-apoptotic antigens, BCl-2 and p53. Cerebellum specimens were obtained from the Laboratory of Animal Pathology, from 1995 to 2009, and the 5-µm thick fragments were stained both with hematoxylin-eosin and Shorr. All samples were diagnosed as positive for CDV genome by reverse transcriptase polymerase chain reaction targeting the nucleocapsid gene. The anti-apoptotic pathways evidenced in this study were BCl-2 and p53 proteins that were intensively detected in cerebellum of CDV positive slides (40-80% of labeled cells/mm2). In addition, the apoptosis markers annexin-V and TUNEL are directly correlated among the same samples (80 and 40% of labeled cells, respectively). This is the first description of p53 and annexin-V expression, characterized as anti-apoptotic and apoptotic proteins, involvement in canine natural cases of CDV infections.