RESUMO
Melanocytic neoplasms originate from melanocytes and melanoma, the malignant form, is a common canine neoplasm and the most aggressive human skin cancer. Despite many similarities between these neoplasms in both species, only a limited number of studies have approached these entities in a comparative manner. Therefore, this review compares benign and malignant melanocytic neoplasms in dogs and humans, exclusively those arising in the haired skin, with regard to their clinicopathological, immunohistochemical and molecular aspects. Shared features include spontaneous occurrence, macroscopic features and microscopic findings when comparing human skin melanoma in the advanced/invasive stage and canine cutaneous melanoma, immunohistochemical markers and several histopathological prognostic factors. Differences include the apparent absence of active mutations in the BRAF gene in canine cutaneous melanoma and less aggressive clinical behaviour in dogs than in humans. Further studies are required to elucidate the aetiology and genetic development pathways of canine cutaneous melanocytic neoplasms. Evaluation of the applicability of histopathological prognostic parameters commonly used in humans for dogs are also needed. The similarities between the species and the recent findings regarding genetic mutations in canine cutaneous melanomas suggest the potential utility of dogs as a natural model for human melanomas that are not related to ultraviolet radiation.
Assuntos
Doenças do Cão , Imuno-Histoquímica , Melanoma , Neoplasias Cutâneas , Cães , Neoplasias Cutâneas/veterinária , Neoplasias Cutâneas/patologia , Animais , Doenças do Cão/patologia , Melanoma/veterinária , Melanoma/patologia , Humanos , Biomarcadores Tumorais , Melanoma Maligno CutâneoRESUMO
BACKGROUND: Bovine herpesvirus type 5 (BoHV-5), frequently lethal in cattle, is associated with significant agricultural economic losses due to neurological disease. Cattle and rabbits are frequently used as models to study the biology and pathogenesis of BoHV-5 infection. In particular, neural invasion and proliferation are two of the factors important in BoHV-5 infection. The present study investigated the potential of bovine Wharton's jelly mesenchymal stromal cells (bWJ-MSCs) to differentiate into a neuronal phenotype and support robust BoHV-5 replication. RESULTS: Upon inducing differentiation within a defined neuronal specific medium, most bWJ-MSCs acquired the distinctive neuronal morphological features and stained positively for the neuronal/glial markers MAP2 (neuronal microtubule associated protein 2), N200 (neurofilament 200), NT3 (neutrophin 3), tau and GFAP (glial fibrillary acidic protein). Expression of nestin, N200, ß-tubulin III (TuJI) and GFAP was further demonstrated by reverse transcriptase polymerase chain reaction (RT-PCR). Following BoHV-5 inoculation, there were low rates of cell detachment, good cell viability at 96 h post-infection (p.i.), and small vesicles developed along neuronal branches. Levels of BoHV-5 antigens and DNA were associated with the peak in viral titres at 72 h p.i. BoHV-5 glycoprotein C mRNA expression was significantly correlated with production of progeny virus at 72 h p.i. (p < 0.05). CONCLUSION: The results demonstrated the ability of bWJ-MSCs to differentiate into a neuronal phenotype in vitro and support productive BoHV-5 replication. These findings constitute a remarkable contribution to the in vitro study of neurotropic viruses. This work may pave the way for bWJ-MSCs to be used as an alternative to animal models in the study of BoHV-5 biology.
Assuntos
Bovinos , Herpesvirus Bovino 5/fisiologia , Neurônios/virologia , Geleia de Wharton/citologia , Animais , Biomarcadores , Sobrevivência Celular , Citometria de Fluxo , Regulação da Expressão Gênica/fisiologia , Células-Tronco Mesenquimais/fisiologia , Células-Tronco Mesenquimais/virologia , Neurônios/citologia , RNA/genética , RNA/metabolismo , Células EstromaisRESUMO
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
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
Canine transmissible venereal tumor (CTVT) is a neoplasm transmitted by the physical transfer of viable tumor cells by direct contact with injured skin and/or mucous tissue. These cells can transpose across histocompatibility barriers into unrelated hosts. This review focuses on the biology of apoptosis and the interaction of proteins involved in this process, as well as p53, p63 and the antiapoptotic protein Bcl-2. As such, this disease offer unique opportunity to study the biology of transplantable tumours and the interaction of proteins involved in apoptosis process and the prognosis of CTVT.(AU)
Assuntos
Animais , Cães , Tumores Venéreos Veterinários/diagnóstico , Apoptose/fisiologia , Neoplasias/veterinária , Sarcoma/diagnóstico , CãesRESUMO
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
A sensitive reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the rapid visual detection of turkey coronavirus (TCoV) infection. The reaction is performed in one step in a single tube at 65 °C for 45 min, with hydroxynaphthol blue (HNB) dye added prior to amplification. The detection limit of the RT-LAMP assay was approximately 10(2) EID(50/50 µl) TCoV genome, and no cross-reaction with other avian viruses was observed. The assay was evaluated further in tissue suspensions prepared from the ileum and ileum-caecal junctions of infected turkey embryos; 100% of these samples were positive in the RT-LAMP assay. All individual feces samples collected in the field were considered positive by both conventional RT-PCR and RT-LAMP. In conclusion, RT-LAMP with HNB dye was shown to be a sensitive, simple assay for the rapid diagnosis of TCoV infection, either directly from feces or in association with virus isolation methods.
Assuntos
Coronavirus do Peru/genética , Fezes/virologia , Naftalenossulfonatos/química , RNA Viral/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Temperatura , Perus/virologia , Animais , Corantes , Especificidade de Órgãos , Sensibilidade e EspecificidadeRESUMO
An in situ polymerase chain reaction (IS-PCR) hybridisation assay was carried out on the brains of 20 cattle infected naturally with bovine herpesvirus type 5 (BoHV-5). Sections from the olfactory bulb and the frontal cortex of each sample were analysed using IS-PCR followed by hybridisation targeting the BoHV-5 US9 gene using a biotinylated primer. Each of the IS-PCR and hybridisation steps was optimised, and three different methods for detecting the virus were used. No false positive signals were observed in any negative control sample (n=20), resulting in a specificity of 100%. The results of IS-PCR hybridisation analysis of the olfactory bulb and the frontal cortex be compared directly with the results obtained using virus isolation, and the specificity and sensitivity were calculated. The most suitable method of visualisation was the peroxidase/3'-3-diaminobenzidine (DAB) detection system coupled with the use of the fluorescent dye Cy3. Using either of these methods, 80% of the positive samples (16 out of 20 samples) were identified using olfactory bulb sections. This is the first report using IS-PCR hybridisation for the direct detection of BoHV-5 DNA in clinical samples, and it provides an additional method for veterinary virology.
Assuntos
Doenças dos Bovinos/virologia , Encefalite Viral/veterinária , Infecções por Herpesviridae/veterinária , Herpesvirus Bovino 5/isolamento & purificação , Hibridização In Situ/métodos , Meningoencefalite/veterinária , Reação em Cadeia da Polimerase/métodos , Animais , Encéfalo/virologia , Bovinos , Primers do DNA/genética , Encefalite Viral/virologia , Fixadores/farmacologia , Formaldeído/farmacologia , Infecções por Herpesviridae/virologia , Herpesvirus Bovino 5/genética , Meningoencefalite/virologia , Inclusão em Parafina , Patologia Molecular/métodos , Sensibilidade e Especificidade , Fixação de TecidosRESUMO
Astroglial cells are the most abundant cells in the mammalian central nervous system, yet our knowledge about their function in bovine Herpesvirus type 5 (BoHV-5) has been limited. The aim of this study was to detect by immunohistochemistry assay the reactive astrocytes for glial fibrilary acidic protein (GFAP) and vimentin (VIM), considered intermediate filaments of the cytoskeleton, localized in olfactory bulb from natural acute cases of BoHV-5 infection. All samples were submitted to virus isolation, real-time polymerase chain reaction (RT-PCR) and in situ hybridization (ISH) technique to confirm the virus transcription and respective genome. Samples were classified into four groups according to the severity of histological lesions. Groups III and IV, which histological lesions were classified as alacia, gliosis, satellitosis, neuronophagia and neuronal necrosis, 35% (± 1.8-2.1) of the inflammatory mononuclear cells, corresponded to CD3 positive lymphocytes. In the same group, 35% (± 1.8) of astrocytes were described as reactive to GFAP and VIM proteins. An agreement of r = 1.0 (P<0.0001) was found between histological lesions, intermediate filaments expression, viral DNA and transcription and CD3 lymphocytes. However, samples with mild histological lesions, 10.8 to 14.2% of astrocytes were classified as reactive to GFAP and VIM filaments. Our findings suggest that GFAP and VIM reactive astrocytes, in primary site of virus replication, seems to play an important role in neurovirulence, in spite of many questions concerning the virus immunopathology remains unclear(AU)
Assuntos
Animais , Herpesvirus Bovino 5 , Astrócitos , Proteína Glial Fibrilar Ácida , Proteína Glial Fibrilar Ácida/farmacologia , Vimentina , Vimentina/farmacologiaRESUMO
Astroglial cells are the most abundant cells in the mammalian central nervous system, yet our knowledge about their function in bovine Herpesvirus type 5 (BoHV-5) has been limited. The aim of this study was to detect by immunohistochemistry assay the reactive astrocytes for glial fibrilary acidic protein (GFAP) and vimentin (VIM), considered intermediate filaments of the cytoskeleton, localized in olfactory bulb from natural acute cases of BoHV-5 infection. All samples were submitted to virus isolation, real-time polymerase chain reaction (RT-PCR) and in situ hybridization (ISH) technique to confirm the virus transcription and respective genome. Samples were classified into four groups according to the severity of histological lesions. Groups III and IV, which histological lesions were classified as alacia, gliosis, satellitosis, neuronophagia and neuronal necrosis, 35% (± 1.8-2.1) of the inflammatory mononuclear cells, corresponded to CD3 positive lymphocytes. In the same group, 35% (± 1.8) of astrocytes were described as reactive to GFAP and VIM proteins. An agreement of r = 1.0 (P<0.0001) was found between histological lesions, intermediate filaments expression, viral DNA and transcription and CD3 lymphocytes. However, samples with mild histological lesions, 10.8 to 14.2% of astrocytes were classified as reactive to GFAP and VIM filaments. Our findings suggest that GFAP and VIM reactive astrocytes, in primary site of virus replication, seems to play an important role in neurovirulence, in spite of many questions concerning the virus immunopathology remains unclear
Assuntos
Animais , Astrócitos , Proteína Glial Fibrilar Ácida , Proteína Glial Fibrilar Ácida/farmacologia , Vimentina , Vimentina/farmacologiaRESUMO
We exposed chicken embryos at embryonating day (ED18) to a cell-adapted very virulent strain of IBDV (ca-vvIBDV) and original vvIBDV and examined the apoptosis from infected bursa of Fabricius (BF) and thymus organs. Following ca-vvIBDV exposure, embryonic bursa showed mild cellular destruction, lower rate of apoptosis and presence of viral proteins detectable by immunohistochemistry. In contrary, original vvIBDV exposed embryos had an enhanced detectable changes in the bursa associated to an increase apoptotic events, and most of the times, total destruction of BF follicles. In thymus, viral antigen was detectable until after hatch. Positives cell signals to activated caspase-3 were intensively detect in embryos lymphoid tissues exposed to original vvIBDV observed in BF and less in thymus. No immunoreactive thymocytes were visualized in embryos exposed to ca-vvIBDV. Apoptosis changes, such as chromatin condensation, DNA fragmentation, and the appearance of apoptotic nuclear bodies, were observed in both organs. TUNEL-detected DNA was more intense in original vvIBDV infected lymphoid cells, and less apoptotic cells were detectable in attenuated strain. By sequencing analysis, e attenuation presented amino acid changes at position 222 (AââP), 256 (IâV) and 279 (DâN). One serine in the serine-rich heptapeptide (position 333) was substituted into other amino acid which is similar to the IBDV vaccine strain. Taken together our results indicate that virus attenuation interferes with caspase-3 apoptotic pathway and may play an important role in switch viral pathogenesis.
Assuntos
Animais , Galinhas/virologia , Apoptose , Vírus da Doença Infecciosa da Bursa , Caspase 3 , Tecido Linfoide/virologiaRESUMO
Reverse transcription polymerase chain reaction (RT-PCR) of turkey astrovirus (TAstV) capsid and polymerase genes was applied to the bursa of Fabricius (BF), thymus (TH), spleen (SP) and cloacal swabs (CS) of young poults with "Poult enteritis complex" (PEC). The histological lesions included atrophy, lymphoid depletion, cellular infiltration and necrosis of the BF, TH and SP, respectively. The RT-PCR reactions were positive for the polymerase gene of TAstV-2 in all 100 CSs, 7 out of 10 of BFs and 10 out of 20 THs and SPs, respectively. Five out of 10 THs and SPs samples, considered to be negative by RT-PCR, were positive when specific primers designed for the TAstV-2 capsid gene were applied. This is the first description of turkey astrovirus infection presenting PEC in Latin America.
Assuntos
Infecções por Astroviridae/veterinária , Avastrovirus/genética , Síndrome de Mortalidade do Peruzinho por Enterite/epidemiologia , Síndrome de Mortalidade do Peruzinho por Enterite/virologia , Animais , Infecções por Astroviridae/epidemiologia , Infecções por Astroviridae/patologia , Brasil/epidemiologia , Bolsa de Fabricius/virologia , Cloaca/virologia , Eletroforese em Gel de Ágar , Síndrome de Mortalidade do Peruzinho por Enterite/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Baço/virologia , Timo/virologia , PerusRESUMO
Poult enteritis complex has been incriminated as a major cause of loss among turkey poults in other countries. We have observed this in Brazil, associated with diarrhoea, loss of weight gain and, commonly, high mortality. In this study, we have used the reverse transcriptase polymerase chain reaction (RT-PCR) to detect turkey coronavirus (TCoV) in sick poults 30 to 120 days of age from a particular producer region in Brazil. The RT-PCR was applied to extracts of intestine tissue suspensions, and the respective intestinal contents, bursa of Fabrícius, faecal droppings and cloacal swabs. Primers were used to amplify the conserved 3' untranslated region of the genome, and the nucleocapsid protein gene of TCoV. Histopathological and direct immunohistochemical examinations were performed to detect TCoV antigen in infected intestine and bursa slides. All the results from stained tissues revealed lesions as described previously for TCoV infection. The direct immunohistochemical positive signal was present in all intestine slides. However, all bursa of Fabrícius tissues analysed were negative. RT-PCR findings were positive for TCoV in all faecal droppings samples, and in 27% of cloacal swabs. Finally, the best field material for TCoV diagnosis was faecal droppings and/or intestine suspensions.