RESUMO
According to the United States Food and Drug Administration (FDA) agency, bacteriophage solutions targeting the serotype O157:H7 are Generally Recognized as Safe (GRAS) to control STEC during beef processing. However, outbreaks involving the "Big Six" STEC increased the industry concern about those serotypes. The objective of this study was to test the efficacy of MS bacteriophages to reduce the "Big Six" non-O157 STEC in beef. The lysing efficacy of phages isolated for each specific serotype varied from 96.2% to 99.9% in vitro. When applied to contaminated trim, reductions ranging from 0.7 to 1.3 Log of all STEC were observed in ground beef. Bacteriophages may provide an additional barrier against the "Big Six" STEC in ground beef. Results of this research provide support documentation to the FDA to extend GRAS status for bacteriophages as processing aids against all adulterant STEC.
Assuntos
Bacteriófagos , Escherichia coli O157/virologia , Produtos da Carne/microbiologia , Escherichia coli Shiga Toxigênica/virologia , Animais , Agentes de Controle Biológico , Bovinos , Microbiologia de Alimentos , Inocuidade dos Alimentos/métodos , SorogrupoRESUMO
After High Event Periods, beef subprimals are usually removed from vacuum and treated with antimicrobials. After re-packaging, subprimals are tested to verify the presence of STEC. In this study, bacteriophage and organic acids were applied on beef contaminated with STEC O157:H7 to evaluate the efficiency of industry practices. Beef samples inoculated with STEC were treated with bacteriophage, lactic acid, and peroxyacetic acid and kept under vacuum or aerobic conditions. STEC loads were evaluated 30 min and 6 h after antimicrobial applications. Under aerobic conditions for 30 min and 6 h, phage reduced STEC in beef by approximately 1.4 log whereas organic acids led to a 0.5 log reduction. Under vacuum for 30 min, bacteriophage significantly reduced STEC by 1 log. No effects were observed when samples were treated with organic acids. Under vacuum after 6 h, bacteriophage reduced STEC loads by 1.4 log, lactic acid reduced by 0.6 log, and no effects were observed when peroxyacetic acid was applied.
Assuntos
Anti-Infecciosos/farmacologia , Caudovirales/fisiologia , Escherichia coli O157/efeitos dos fármacos , Escherichia coli O157/virologia , Carne Vermelha/microbiologia , Animais , Bovinos , Manipulação de Alimentos/métodos , Microbiologia de Alimentos , Ácido Láctico/farmacologia , Ácido Peracético/farmacologia , VácuoRESUMO
The precise nature of hormones and growth factors directly responsible for cartilage maturation is still largely unclear. Since longitudinal bone growth occurs through endochondral bone formation, excess or deficiency of most hormones and growth factors strongly influences final adult height. The structure and composition of the cartilaginous extracellular matrix have a critical role in regulating the behavior of growth plate chondrocytes. Therefore, the maintenance of the three-dimensional cell-matrix interaction is necessary to study the influence of individual signaling molecules on chondrogenesis, cartilage maturation and calcification. To investigate the effects of insulin on both proliferation and induction of hypertrophy in chondrocytes in vitro we used high-density micromass cultures of chick embryonic limb mesenchymal cells. Culture medium was supplemented with 1 percent FCS + 60 ng/ml (0.01 æM) insulin and cultures were harvested at regular time points for later analysis. Proliferating cell nuclear antigen immunoreactivity was widely detected in insulin-treated cultures and persisted until day 21 and [ H]-thymidine uptake was highest on day 14. While apoptosis increased in control cultures as a function of culture time, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-labeled cells were markedly reduced in the presence of insulin. Type II collagen production, alkaline phosphatase activity and cell size were also lower in insulin-treated cultures. Our results indicate that under the influence of 60 ng/ml insulin, chick chondrocytes maintain their proliferative potential but do not become hypertrophic, suggesting that insulin can affect the regulation of chondrocyte maturation and hypertrophy, possibly through an antiapoptotic effect
Assuntos
Animais , Embrião de Galinha , Diferenciação Celular , Condrócitos , Insulina , Mesoderma , Apoptose , Técnicas de Cultura de Células , Divisão Celular , Matriz Extracelular , Extremidades , MesodermaRESUMO
The precise nature of hormones and growth factors directly responsible for cartilage maturation is still largely unclear. Since longitudinal bone growth occurs through endochondral bone formation, excess or deficiency of most hormones and growth factors strongly influences final adult height. The structure and composition of the cartilaginous extracellular matrix have a critical role in regulating the behavior of growth plate chondrocytes. Therefore, the maintenance of the three-dimensional cell-matrix interaction is necessary to study the influence of individual signaling molecules on chondrogenesis, cartilage maturation and calcification. To investigate the effects of insulin on both proliferation and induction of hypertrophy in chondrocytes in vitro we used high-density micromass cultures of chick embryonic limb mesenchymal cells. Culture medium was supplemented with 1% FCS + 60 ng/ml (0.01 microM) insulin and cultures were harvested at regular time points for later analysis. Proliferating cell nuclear antigen immunoreactivity was widely detected in insulin-treated cultures and persisted until day 21 and [ 3H]-thymidine uptake was highest on day 14. While apoptosis increased in control cultures as a function of culture time, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-labeled cells were markedly reduced in the presence of insulin. Type II collagen production, alkaline phosphatase activity and cell size were also lower in insulin-treated cultures. Our results indicate that under the influence of 60 ng/ml insulin, chick chondrocytes maintain their proliferative potential but do not become hypertrophic, suggesting that insulin can affect the regulation of chondrocyte maturation and hypertrophy, possibly through an antiapoptotic effect.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Condrócitos/efeitos dos fármacos , Insulina/farmacologia , Mesoderma/citologia , Animais , Apoptose/efeitos dos fármacos , Técnicas de Cultura de Células , Divisão Celular/efeitos dos fármacos , Embrião de Galinha , Matriz Extracelular/efeitos dos fármacos , Extremidades/embriologia , Mesoderma/efeitos dos fármacosRESUMO
Fibronectin (Fn) plays an important role in tissue remodeling during embryogenesis, wound repair, and vascular disease, and is thought to regulate cellular processes such as cell adhesion, migration, proliferation, and differentiation through specialized domains within the molecule. In addition, Fn can be alternatively spliced at three regions: extradomains EIIIA, EIIIB, and a variable segment V, potentially giving rise to functionally distinct variants of the molecule. We have previously shown a sequential expression of cellular Fn first by platelets, followed by macrophages, then mesangial cells in habu snake venom-induced proliferative glomerulonephritis (Am J Pathol 145: 585-597, 1994). These studies examined the cellular sources and glomerular localization of Fn in general but did not distinguish between the various alternatively spliced isoforms. In this study, we examine by in situ hybridization and immunohistochemistry the temporal expression and cellular sources of EIIIA, EIIIB, and V in a model of proliferation glomerulonephritis that has cell migration, proliferation, and extracellular matrix synthesis as features of tissue remodeling. Macrophages were the first cells to express Fn mRNA showing an EIIIA+, EIIIB-, and V95+ pattern beginning at 8 hours after habu snake venom injection. Migrating mesangial cells at the margins of early lesions (8 and 24 hours) did not overexpress mRNA encoding these Fn variants, but immunofluorescence microscopy revealed V95 and EIIIA protein at the margins of lesions. EIIIB was absent in lesions at this time. At 48 hours and peaking at 72 hours after habu snake venom injection, mesangial cells in central aspects of glomerular lesions expressed abundant mRNA and protein for V95 and EIIIA. EIIIB mRNA and protein was slight in the mesangium at these times. Parietal epithelial cells, particularly adjacent to glomerular lesions, also expressed abundant mRNA and protein for all three variants throughout the course of the disease, beginning at 24 hours after habu snake venom injection. Expression of mRNA and protein for all three isoforms declined by 2 weeks after habu snake venom injection. These studies show that migrating mesangial cells do not require their own synthesis of Fn and suggest that they might rely on exogenous sources of Fn, particularly V95+ and EIIIA+ forms. Commencement of enhanced expression of EIIIA and EIIIB mRNA and protein by resident glomerular cells coincided with the temporal course of cell proliferation, acquisition of alpha-smooth muscle cell actin phenotype, and matrix synthesis, suggesting that Fn isoforms have specific functions during the course of glomerular remodeling.
Assuntos
Processamento Alternativo , Fibronectinas/genética , Glomerulonefrite Membranoproliferativa/patologia , Cicatrização/fisiologia , Animais , Divisão Celular , Venenos de Crotalídeos , Matriz Extracelular/química , Matriz Extracelular/patologia , Fibronectinas/química , Glomerulonefrite Membranoproliferativa/induzido quimicamente , Glomerulonefrite Membranoproliferativa/genética , Hibridização In Situ , Masculino , RNA Mensageiro/análise , Ratos , Ratos Sprague-Dawley , Estereoisomerismo , TrimeresurusRESUMO
Pericellular proteolysis involves the plasminogen activator/plasmin system and plays an important role in cell remodeling involving cell migration and extracellular matrix turnover. Studies in this laboratory have previously characterized a model of proliferative glomerulonephritis induced by Habu snake venom (HSV) in the rat that involves cell migration, proliferation, and extracellular matrix accumulation. Because plasminogen activator-inhibitor-1 (PAI-1) has been used as a marker for cell migration as well as matrix accumulation, we were interested in examining the temporal and spatial expression and cellular sources of PAI-1 mRNA and translated protein over the course of HSV-induced proliferative glomerulonephritis. The results showed a highly localized and progressive expression of PAI-1 mRNA and translated protein by in situ hybridization and immunohistochemistry at the margins and periphery of glomerular lesions 8 and 24 hr after HSV. The expression of PAI-1 in glomerular lesions localized to the same sites as mesangial cell marker proteins, desmin and Thy-1.1, indicating that mesangial cells synthesize this important regulator proteolysis. Few cells expressed PAI-1 in the central aspects of glomerular lesions at later time intervals (48 and 72 hr) when cell proliferation and expression of extracellular matrix (fibronectin protein and mRNA) were maximal. Therefore, the expression of PAI-1 in this model was associated more with early events related to cell migration than with proliferation or extracellular matrix synthesis. These observations support the hypothesis that the plasminogen activator/plasmin system is involved in cell migration in early remodeling during glomerular disease.