BackgroundThe Ala-Pro-rich O-glycoprotein known as the 45/47 kDa or APA antigen from Mycobacterium tuberculosis is an immunodominant adhesin restricted to mycobacterium genus and has been proposed as an alternative candidate to generate a new vaccine against tuberculosis or for diagnosis kits. In this work, the recombinant O-glycoprotein APA was produced by the non-pathogenic filamentous bacteria Streptomyces lividans, evaluating three different culture conditions. This strain is known for its ability to produce heterologous proteins in a shorter time compared to M. tuberculosis.ResultsThree different shake flask geometries were used to provide different shear and oxygenation conditions; and the impact of those conditions on the morphology of S. lividans and the production of rAPA was characterized and evaluated. Small unbranched free filaments and mycelial clumps were found in baffled and coiled shake flasks, but one order of magnitude larger pellets were found in conventional shake flasks. The production of rAPA is around 3 times higher in small mycelia than in larger pellets, most probably due to difficulties in mass transfer inside pellets. Moreover, there are four putative sites of O-mannosylation in native APA, one of which is located at the carboxy-terminal region. The carbohydrate composition of this site was determined for rAPA by mass spectrometry analysis, and was found to contain different glycoforms depending on culture conditions. Up to two mannoses residues were found in cultures carried out in conventional shake flasks, and up to five mannoses residues were determined in coiled and baffled shake flasks.ConclusionsThe shear and/or oxygenation parameters determine the bacterial morphology, the productivity, and the O-mannosylation of rAPA in S. lividans. As demonstrated here, culture conditions have to be carefully controlled in order to obtain recombinant O-glycosylated proteins with similar "quality" in bacteria, particularly, if the protein activity depends on the glycosylation pattern. Furthermore, it will be an interesting exercise to determine the effect of shear and oxygen in shake flasks, to obtain evidences that may be useful in scaling-up these processes to bioreactors. Another approach will be using lab-scale bioreactors under well-controlled conditions, and study the impact of those on rAPA productivity and quality.
Food-derived bioactive peptides are reported as beneficial and safe for human health. Glycomacropeptide (GMP) is a milk-protein-derived peptide that, in addition to its nutritional value, retains many biological properties and has therapeutic effects in several inflammatory disorders. GMP was shown under in vitro and in vivo conditions to exert a number of activities that regulate the physiology of important body systems, namely the gastrointestinal, endocrine, and immune systems. This review represents a comprehensive compilation summarizing the current knowledge and updated information on the major biological properties associated with GMP. GMP bioactivity is addressed with special attention on mechanisms of action, signaling pathways involved, and structural characteristics implicated. In addition, the results of various studies dealing with the effects of GMP on models of inflammatory diseases are reviewed and discussed.
Mast cells (MCs) are strategically located in tissues close to the external environment, being one of the first immune cells to interact with invading pathogens. They are long living effector cells equipped with different receptors that allow microbial recognition. Once activated, MCs release numerous biologically active mediators in the site of pathogen contact, which induce vascular endothelium modification, inflammation development and extracellular matrix remodeling. Efficient and direct antimicrobial mechanisms of MCs involve phagocytosis with oxidative and non-oxidative microbial destruction, extracellular trap formation, and the release of antimicrobial substances. MCs also contribute to host defense through the attraction and activation of phagocytic and inflammatory cells, shaping the innate and adaptive immune responses. However, as part of their response to pathogens and under an impaired, sustained, or systemic activation, MCs may contribute to tissue damage. This review will focus on the current knowledge about direct and indirect contribution of MCs to pathogen clearance. Antimicrobial mechanisms of MCs are addressed with special attention to signaling pathways involved and molecular weapons implicated. The role of MCs in a dysregulated host response that can increase morbidity and mortality is also reviewed and discussed, highlighting the complexity of MCs biology in the context of host-pathogen interactions.
Nonsteroidal anti-inflammatory drug (NSAID)-induced enteropathy is considered a serious and increasing clinical problem without available treatment. Glycomacropeptide (GMP) is a 64-amino acid peptide derived from milk κ-casein with numerous biological activities. The aim of this study was to investigate the protective effect of GMP on NSAID enteropathy in rats. Enteropathy was induced by seven days oral indomethacin administration. Rats were orally GMP treated from seven days previous and during the establishment of the enteropathy model. Changes in metabolism, hematological and biochemical blood alterations, intestinal inflammation and oxidative damage were analyzed. Integrity barrier markers, macroscopic intestinal damage and survival rate were also evaluated. GMP treatment prevented anorexia and weight loss in animals. Furthermore, prophylaxis with GMP ameliorated the decline in hemoglobin, hematocrit, albumin and total protein levels. The treatment had no therapeutic efficacy on the decrease of occludin and mucin (MUC)-2 expression in intestinal tissue. However, GMP markedly decreased neutrophil infiltration, and CXCL1, interleukin-1β and inducible nitric oxide synthase expression. Nitric oxide production and lipid hydroperoxide level in the small intestine were also diminished. These beneficial effects were mirrored by preventing ulcer development and increasing animal survival. These results suggest that GMP may protect against NSAID enteropathy through anti-inflammatory and antioxidant properties.
Glycomacropeptide (GMP) is a bioactive peptide derived from milk κ-casein with immune-modulatory and anti-inflammatory properties. Food allergy (FA) is an adverse immune reaction with a broad spectrum of manifestations. Allergen intake induces persistent intestinal inflammation and tissue damage. In this study, the anti-allergic activity of GMP was evaluated using a rat ovalbumin (OVA)-induced FA model with gastrointestinal manifestation. Rats were orally GMP treated from 3 days prior and during FA development. The severity of food anaphylaxis and diarrheal episodes, antibody production and histamine level were measured. Histopathological changes, inflammation and predominant cytokine profile at intestine were analyzed. Oral GMP intake decreased clinical signs and diarrhea severity induced by allergen, with a significant reduction in intestinal edema and expression level of IL-1β and TNF-α. Prophylaxis with GMP also diminished serum anti-OVA IgE and IgG1, and histamine levels. GMP treatment markedly decreased eosinophil infiltration, mast cell and goblet cell hyperplasia, total IgE expression in intestine, and prevented histological changes in villi, crypts and internal muscularis layer. The treatment effectively suppressed IL-5, IL-13 and GATA3 expression and skewed the intestinal cytokine profile toward type 1 and regulatory. These results suggest that GMP may protect against FA through down-regulating the type 2 inflammatory response.
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