RESUMO
Lysozyme is an important non-specific immune protein in human milk, modulating the immune response against bacterial infections. The aim of this study was to characterize the milk of a transgenic goat expressing a recombinant human lysozyme (rhLZ) in the milk, also testing the in vitro antibacterial activity of the rhLZ milk against pathogens of the gastrointestinal tract. Milk samples collected from Tg and non-transgenic goats (nTg) from the 3rd to the 11th week of lactation were submitted to physicochemical analyses, rhLZ semi-quantification, and to rhLZ antimicrobial activity against Micrococcus luteus, Shiguella sonnei and Enterococcus faecalis. Viability and cell migration were studied in ileum epithelial cells (IEC-18) in absence or presence of E. faecalis, Staphylococcus aureus, Escherichia coli (EPEC) and S. sonnei. The expression of ZO-1 and IL-6 genes was evaluated in IEC-18 to evaluate the effect of rhLZ milk on intestinal barrier function and intestinal inflammation. Physicochemical parameters between goat Tg and nTg milk were similar and within normal values for human consumption, with hLZ concentrations being similar between Tg (224µg/mL) and human (226µg/mL) milk. The Tg milk had bactericidal activity against M. luteus, no bactericidal effect on S. sonnei, and relative to discrete sensitivity against E. feacalis than controls. Better migrating parameters were observed in cells in culture with nTg and Tg than controls. In the presence of pathogens, the Tg milk promoted improved migrating parameters than controls, except for S. sonnei, with lower cell numbers in the presence of nTg samples and E. faecalis and S. sonnei. No differences in ZO-1 relative expression patterns were observed in cultured cells, with increased expression in IL-6 in cells exposed to nTg milk than controls, with the Tg group being similar to all groups. In conclusion, goat milk containing rhLZ demonstrated valid evidence for its potential use as a nutraceutical for improvement of health and nutrition quality in humans.
Assuntos
Antibacterianos , Fenômenos Fisiológicos Bacterianos , Trato Gastrointestinal/microbiologia , Cabras/genética , Leite , Muramidase/genética , Animais , Animais Geneticamente Modificados , Linhagem Celular , Suplementos Nutricionais , Trato Gastrointestinal/metabolismo , Humanos , Interleucina-6/genética , Muramidase/metabolismo , Ratos , Proteína da Zônula de Oclusão-1/genéticaRESUMO
BACKGROUND: Enteroaggregative Escherichia coli (EAEC) causes diarrhea, malnutrition and poor growth in children. Human breast milk decreases disease-causing bacteria by supplying nutrients and antimicrobial factors such as lysozyme. Goat milk with and without human lysozyme (HLZ) may improve the repair of intestinal barrier function damage induced by EAEC. This work investigates the effect of the milks on intestinal barrier function repair, bacterial adherence in Caco-2 and HEp-2 cells, intestinal cell proliferation, migration, viability and apoptosis in IEC-6 cells in the absence or presence of EAEC. METHODS: Rat intestinal epithelial cells (IEC-6, ATCC, Rockville, MD) were used for proliferation, migration and viability assays and human colon adenocarcinoma (Caco-2, ATCC, Rockville, MD) and human larynx carcinoma (HEp-2, ATCC, Rockville, MD) cells were used for bacterial adhesion assays. Goats expressing HLZ in their milk were generated and express HLZ in milk at concentration of 270 µg/ml. Cells were incubated with pasteurized milk from either transgenic goats expressing HLZ or non-transgenic control goats in the presence and absence of EAEC strain 042 (O44:H18). RESULTS: Cellular proliferation was significantly greater in the presence of both HLZ transgenic and control goat milk compared to cells with no milk. Cellular migration was significantly decreased in the presence of EAEC alone but was restored in the presence of milk. Milk from HLZ transgenic goats had significantly more migration compared to control milk. Both milks significantly reduced EAEC adhesion to Caco-2 cells and transgenic milk resulted in less colonization than control milk using a HEp-2 assay. Both milks had significantly increased cellular viability as well as less apoptosis in both the absence and presence of EAEC. CONCLUSIONS: These data demonstrated that goat milk is able to repair intestinal barrier function damage induced by EAEC and that goat milk with a higher concentration of lysozyme offers additional protection.
Assuntos
Escherichia coli/fisiologia , Intestinos/efeitos dos fármacos , Intestinos/patologia , Leite/enzimologia , Muramidase/farmacologia , Animais , Animais Geneticamente Modificados , Apoptose/efeitos dos fármacos , Aderência Bacteriana/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Epitélio/efeitos dos fármacos , Epitélio/microbiologia , Epitélio/patologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/isolamento & purificação , Cabras , Humanos , Técnicas In Vitro , Intestinos/microbiologia , Muramidase/genética , RatosRESUMO
Background: Transgenic animals have been generated for a variety of purposes including research tools, medical models, bioreactors (dairy animals producing human pharmaceuticals in their milk or in the egg whites of chickens) and for production agriculture (animals with increased growth, decreased environmental pollution, disease resistance), which includes the generation of animals designed to benefit human health. For example, transgenic goats expressing human lysozyme in their milk are being used as a model method to supply milk with antibacterial properties to help fight diarrheal illnesses in children. Lysozyme is a naturally occurring antimicrobial found in human milk at much higher levels than in the milk of dairy goats and cows. Lysozyme serves as part of the natural defense system against infection and also helps establish a healthy gut microbiota in the infant. We hypothesized that the presence of increased levels of lysozyme in the milk of dairy goats could offer several benefits that affect human health, including the promotion of a healthy gut microbiota and associated benefits such as improved growth and resistance to intestinal infections. Review: Research with this line of transgenic goats over the last 12 years has demonstrated that the presence and expression of the human lysozyme transgene is not detrimental to the animals themselves and that the milk can indeed act in an antimicrobial fashion when consumed by pigs, a model animal for human health, and impact the state of the intestine in a positive manner. Pigs consuming milk from human lysozyme transgenic goats had significantly lower levels of coliforms and E. coli in their intestine than did pigs consuming milk from non-transgenic control animals. In addition to bacterial changes, intestinal tissue of pigs consuming milk from lysozyme transgenic goats had a significantly larger surface area with significantly fewer intraepithelial lymphocytes and an elevated level of expression of the anti-inflammatory cytokine TGF-â1 compared to control-fed animals, all indicators of a healthier intestinal tract. Metabolite profile analysis demonstrated significant differences in the levels of 18 metabolites in the serum of pigs fed lysozyme milk with the direction of changes beneficial to the health of the animal. Finally, pigs consuming milk from lysozyme transgenic goats were more resistant to infection when challenged with an enteropathogenic E. coli, indicating a protective effect of lysozyme milk. In the semi-arid northeast of Brazil, 89 of 1000 children die before they reach the age of 2 years and 17% of these deaths can be attributed to diarrhea. The use of genetically engineered animals containing increased levels of lysozyme in their milk is a novel and simple approach to fight this problem. Work will be presented outlining the characterization of these animals and the impact of consuming the milk with the goal of one day having the milk available as a preventative or treatment agent for diarrheal illnesses. Conclusions: Genetic engineering is a viable approach to produce animal food products that can be used to improve human health. Brazil's acceptance of this technology has positioned itself at the forefront to demonstrate to the world a new tool to help fight common diarrhea and its impact on the growth and development of children.
Assuntos
Animais , Cabras/genética , Animais Geneticamente Modificados , Muramidase/análise , Leite/química , Diarreia/prevenção & controle , Engenharia Genética/veterináriaRESUMO
Background: The current status of genetic engineering animals for biomedical and human health applications, including improving the supply of animal-based food products, was reviewed. Although transgenic animals have been available for almost 30 years only one product, a drug derived from transgenic goats' milk, has been approved for use anywhere in the world. While a number of technical issues limited efficiency initially, products coming to market were hindered by the lack of a regulatory framework and compounded by opposition from anti-biotechnology groups. Review: As presented in the review, the production of genetically engineered livestock has progressed from the initial technology of pronuclear microinjection through to the wide-spread use today of somatic cell nuclear transfer-based cloning technology following the transfection of cells in culture with methods such as lipofection or electroporation. There also was significant progress in the development of systems based upon lentivirus or adeno-associated virus-based vectors. More recently, advances occurred based on transposon-mediated transgenesis. In conjunction with advances in methods to genetically engineer livestock, there are also advances, such as the use of zinc-finger nucleases and transient depletion of endogenous non-homologous recombination systems, to increase the efficiency of homologous recombination-based gene targeting. Applications range from the production of pharmaceutical proteins, silk for use in sutures and scaffolds for cellular regeneration, and xenotransplantation through to the development of transgenic animals that improve animal production and the nutritional value of animal-based food products. Both of these agricultural applications are important for maintaining the global production of sufficient meat-based products in an economically and environmentally sustainable manner in the face of a dramatically increasing human population. Conclusion: Advances in the production of genetically engineered animals and identification of useful applications have significantly improved and have set the stage for the adoption of transgenic animals. Brazil, because of recent technological developments and the adoption of regulatory guidelines is now in a strong position to benefit from genetically engineered animals and to contribute to the leadership in the adoption of transgenic animals for applications to improve human and animal health and well-being.