1,721,163 research outputs found
Detection of Bovine Herpesvirus 4 (BoHV-4) DNA in the cell fraction of milk of dairy cattle with history of BoHV-4 infection
No full textEfficient expression of the bovine beta-lactoglobulin gene in transgenic mice: role of regions downstream of the promoter
No full textHeterologous Matrix Metalloproteinase Gene Promoter Activity Allows In Vivo Real-time Imaging of Bleomycin-Induced Lung Fibrosis in Transiently Transgenized Mice
Full text linkIdiopathic pulmonary fibrosis is a very common interstitial lung disease derived from chronic inflammatory insults, characterized by massive scar tissue deposition that causes the progressive loss of lung function and subsequent death for respiratory failure.Bleomycin is used as the standard agent to induce experimental pulmonary fibrosis in animal models for the study of its pathogenesis. However, to visualize the establishment of lung fibrosis after treatment, the animal sacrifice is necessary. Thus, the aim of this study was to avoid this limitation by using an innovative approach based on a double bleomycin treatment protocol, along with the in vivo images analysis of bleomycintreated mice. A reporter gene construct, containing the luciferase open reading frame under the matrix metalloproteinase-1 promoter control region, was tested on doublebleomycin-treated mice to investigate, in real time, the correlation between bleomycin treatment, inflammation, tissue remodeling and fibrosis. Bioluminescence emitted by the lungs of bleomycin-treated mice, corroborated by fluorescent molecular tomography, successfully allowed real time monitoring of fibrosis establishment. The reporter gene technology experienced in this work could represent an advanced functional approach for real time non-invasive assessment of disease evolution during therapy, in a reliable and translational living animal model.Fil: Stellari, Fabio Franco. Chiese Farmaceutici; ItaliaFil: Ruscitti, Francesca. Chiese Farmaceutici; ItaliaFil: Pompilio, Daniela. Chiese Farmaceutici; ItaliaFil: Ravanetti, Francesca. Università di Parma. Dipartimento di Scienze Medico Veterinarie; ItaliaFil: Tebaldi, Giulia. Università di Parma. Dipartimento di Scienze Medico Veterinarie; ItaliaFil: Macchi, Francesca. Università di Parma. Dipartimento di Scienze Medico Veterinarie; ItaliaFil: Verna, Andrea Elizabeth. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Chiese Farmaceutici; ItaliaFil: Villetti, Gino. Chiese Farmaceutici; ItaliaFil: Donofrio, Gaetano. Università di Parma. Dipartimento di Scienze Medico Veterinarie
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Potential of bovine herpesvirus 4 as a gene delivery vector
No full textA cloning system was developed for construction of BHV-4 recombinants and recombinant virus BHV-4EGFPΔTK containing an enhanced green fluorescent protein (EGFP) gene was constructed. The host range of BHV-4EGFPΔTK was characterized in vitro. When cell lines from various species and tissues were infected, most of the non-bovine cell lines exhibited neither cytopathic effect (CPE) nor supported viral replication, but EGFP expression was clearly observed. Next, embryonic stem cells were infected and induced to either non-specific or neural differentiation to determine whether they could survive and differentiate after BHV-4EGFPΔTK infection. Embryonic stem cells were infected successfully, as indicated by EGFP expression prior to differentiation, and EGFP expression could be detected in many differentiated cells. No CPE was noted. Therefore, BHV-4EGFPΔTK infection caused neither cell death nor interfered with non-specific or neural differentiation of embryonic stem cells. Finally, to assess the capability of BHV-4EGFPΔTK to infect post-mitotic neurons, cultures from brains of 2-weeks old mice were infected. No death of neuronal cells due to infection was observed and EGFP expression persisted for at least 15 days. Several biological characteristics of BHV-4 demonstrated previously make it a good candidate for a gene delivery vector. These include: little or no pathogenicity, unlikely oncogenicity, ability to establish persistent infection, and capability of herpesviruses to accommodate large amounts of foreign genetic material. These findings add the ability to infect several cell types coming from different animal species, usually without CPE, lack of interference with differentiation, and ability to maintain transgene expression in both undifferentiated and differentiated cell
Activation of bovine herpesvirus 4 lytic replication in a non-permissive cell line by overexpression of BoHV-4 immediate early (IE) 2 gene
No full textBovine herpesvirus 4 (BoHV-4) is a gammaherpesvirus with no clear disease association, it establishes persistent infections in its natural host, the bovine, and in an experimental host, the rabbit. BoHV-4 immediate early 2 (IE2) RNA is the less abundant, spliced, 1.8 kb RNA. The predicted amino acid sequence, of the IE2 protein, reveals that it could encode a 61 kDa protein with amino acid sequence homology to the Epstein-Barr virus (EBV) transactivator R protein and its homologues including, herpesvirus saimiri (HVS), equine herpesvirus 2 (EHV-2), murine herpesvirus 68 and Kaposi's sarcoma-associated herpesvirus (KSHV). We examined recently the interaction of BoHV-4 with a human rhabdomyosarcoma cell line, RD-4, and found that although some infectious viruses can be produced, no cytopathic effect (CPE) was observed [J. Gen. Virol. 81 (2000) 1807]. Because IE2 could play a critical role in BoHV-4 productive infection and its overexpression in RD-4 cells could switch the non-permissive RD-4 status to a permissive one. RD-4 cells expressing stably BoHV-4 IE2 gene were generated. BoHV-4 IE2 induced an increased production of infectious viral particles sufficient to obtain an apparent cytopathic effect. It is concluded that BoHV-4 IE2 is a key factor in determining the outcome of BoHV-4 infectio
Bovine Endometrial Stromal Cells Support Tumor Necrosis Factor Alpha-Induced Bovine Herpesvirus Type 4 Enhanced Replication
No full textBovine uterine infections are the most important cause of economic losses in the cattle industry. Although the etiology of uterine diseases is mainly ascribed to bacterial infection, they can also be associated with viral infection, such as bovine herpesvirus 4 (BoHV-4), which is often a secondary agent following bacteria. Besides microbial infection, many inflammatory molecules belonging to the innate immune response orchestrate the outcome of the infection. In the present study, the interaction between BoHV-4-infected bovine endometrial stromal cells and tumor necrosis factor alpha (TNF-alpha) was investigated. Bovine herpesvirus 4 possesses a special tropism toward endometrial stromal cells. For this reason, a simian virus 40 (SV40) immortalized endometrial stromal cell line (SV40BESC) was established; it was proven that it was stable, it expressed toll-like receptors (TLRs; from 1 to 10) and TNFalpha receptors I and II, and it was responsive to exogenous TNF-alpha. Further, an increase of BoHV-4 replication and cytopathic effect was observed in BoHV-4-infected and TNFalpha- treated SV40BESCs. This increase of viral replication was associated with BoHV-4 immediate early 2 (IE2) gene promoter trans-activation through the interaction of the nuclear factor KB (NFKB) with the putative NFKB-responsive elements found within BoHV-4 IE2 gene promoter, and this interaction was abolished when NFKB-responsive elements were deleted. These data shed light on two important and rather controversial issues: the role of TNF-alpha receptor, which is weakly expressed in the stromal layer of the bovine uterus, as well as the possible interactions between proinflammatory molecules, viral replication, and chronic uterine disease
A chimeric protein comprising the immunogenic domains of Mannheimia haemolytica leukotoxin and outer membrane protein PlpE induces antibodies against leukotoxin and PlpE
No full textMannheimia haemolytica is a very important pathogen of pneumonia in ruminants. Bighorn sheep (BHS, Ovis canadensis) are highly susceptible to M. haemolytica-caused pneumonia which has significantly contributed to the drastic decline of bighorn sheep population in North America. Pneumonia outbreaks in wild BHS can cause mortality as high as 90%. Leukotoxin is the critical virulence factor of M. haemolytica. In a 'proof of concept' study, an experimental vaccine containing leukotoxin and surface antigens of M. haemolytica developed by us induced 100% protection of BHS, but required multiple booster injections. Vaccination of wild BHS is difficult. But they can be vaccinated at the time of transplantation into a new habitat. Administration of booster doses, however, is impossible. Therefore, a vaccine that does not require booster doses is necessary to immunize BHS against M. haemolytica pneumonia. Herpesviruses are ideal vectors for development of such a vaccine because of their ability to undergo latency with subsequent reactivation. As the first step towards developing a herpesvirus-vectored vaccine, we constructed a chimeric protein comprising the leukotoxin-neutralizing epitopes and the immuno-dominant epitopes of the outer membrane protein PlpE. The chimeric protein was efficiently expressed in primary BHS lung cells. The immunogenicity of the chimeric protein was evaluated in mice before inoculating BHS. Mice immunized with the chimeric protein developed antibodies against M. haemolytica leukotoxin and PlpE. More importantly, the anti-leukotoxin antibodies effectively neutralized leukotoxin-induced cytotoxicity. Taken together, these results represent the successful completion of the first step towards developing a herpesvirus-vectored vaccine for controlling M. haemolytica pneumonia in BHS, and possibly other ruminants
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