BACKGROUND: Lactoferrin (LF) is an 80 kDa glycoprotein which is known for its effects against bacteria, viruses and other pathogens. It also has a high potential in nutrition therapy and welfare of people and a variety of animals, including piglets. The ability to bind lipopolysaccharide (LPS) is one of the described anti-inflammatory mechanisms of LF. Previous studies suggested that cells can be stimulated even by LPS-free LF. Therefore, the aim of our study was to bring additional information about this possibility. Porcine monocyte derived macrophages (MDMF) and human embryonic kidney (HEK) cells were stimulated with unpurified LF in complex with LPS and with purified LF without bound LPS. RESULTS: Both cell types were stimulated with unpurified as well as purified LF. On the other hand, neither HEK0 cells not expressing any TLR nor HEK4a cells transfected with TLR4 produced any pro-inflammatory cytokine transcripts after stimulation with purified LF. This suggests that purified LF without LPS stimulates cells via another receptor than TLR4. An alternative, TLR4-independent, pathway was further confirmed by analyses of the NF-kappa-B-inducing kinase (NIK) activation. Western blot analyses showed NIK which activates different NFκB subunits compared to LF-LPS signaling via TLR4. Though, this confirmed an alternative pathway which is used by the purified LF free of LPS. This stimulation of MDMF led to low, but significant amounts of pro-inflammatory cytokines, which can be considered as a positive stimulation of the immune system. CONCLUSION: Our results suggest that LF's ability is not only to bind LPS, but LF itself may be a stimulant of pro-inflammatory pathways.
- MeSH
- cytokiny genetika metabolismus MeSH
- HEK293 buňky MeSH
- intracelulární signální peptidy a proteiny metabolismus MeSH
- laktoferrin izolace a purifikace farmakologie MeSH
- lidé MeSH
- lipopolysacharidy farmakologie MeSH
- makrofágy účinky léků MeSH
- prasata MeSH
- protein-serin-threoninkinasy metabolismus MeSH
- regulace genové exprese účinky léků MeSH
- signální transdukce účinky léků MeSH
- toll-like receptor 4 genetika metabolismus MeSH
- vazba proteinů MeSH
- zánět MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Birth is an event that exposes the newborn baby to a high concentration of free radicals (reactive oxygen species (ROS)/reactive nitrogen species (RNS)), which can contribute to several diseases. The balanced equilibrium between ROS/RNS and the antioxidant system of the newborn is a key factor in preventing a plethora of diseases. The antioxidant system involves endogenous and exogenous molecules, from vitamins (A, E, C) to enzymes (glutathione peroxidase, superoxide dismutase), metals (copper, zinc, and selenium) and other molecules (coenzyme Q10, melatonin) that can act in a synergistic manner to deactivate free radicals. A competent antioxidant system of the baby is strongly dependent on the intake of free radical deactivating molecules from feeding, either maternal or formula milk, with several studies pointing that breast milk has more powerful antioxidant effects on lowering the infant's oxidative status. An improved understanding of the antioxidant molecules, their mechanism of action, and the relationships between them, are key factors to comprehend all the potential benefits of human breastfeeding in this matter. The purpose of this review is to describe different research efforts and methodologies of evaluation of TAS in human milk, and to evaluate and summarize the contribution of different antioxidant molecules.
- Klíčová slova
- TAS/TAC (total antioxidant system/total antioxidant capacity) - (celková antioxidační kapacita lidského mléka),
- MeSH
- antioxidancia farmakologie metabolismus MeSH
- ceruloplasmin imunologie izolace a purifikace metabolismus MeSH
- farmakologické účinky - molekulární mechanismy MeSH
- glutathionperoxidasa imunologie izolace a purifikace metabolismus MeSH
- kaseiny imunologie izolace a purifikace metabolismus MeSH
- katalasa imunologie izolace a purifikace metabolismus MeSH
- kojenec MeSH
- kojení * MeSH
- kovy imunologie izolace a purifikace metabolismus MeSH
- kyselina askorbová MeSH
- laktoferrin imunologie izolace a purifikace metabolismus MeSH
- lidé MeSH
- mateřské mléko * enzymologie imunologie metabolismus MeSH
- melatonin imunologie izolace a purifikace metabolismus MeSH
- mléčné bílkoviny imunologie izolace a purifikace metabolismus MeSH
- ochranné látky farmakologie metabolismus MeSH
- oxidační stres fyziologie imunologie MeSH
- proteiny imunologie izolace a purifikace metabolismus MeSH
- statistika jako téma MeSH
- stopové prvky imunologie izolace a purifikace metabolismus MeSH
- superoxiddismutasa imunologie izolace a purifikace metabolismus MeSH
- ubichinon imunologie izolace a purifikace metabolismus MeSH
- vitamin A MeSH
- vitamin E MeSH
- volné radikály antagonisté a inhibitory metabolismus škodlivé účinky MeSH
- Check Tag
- kojenec MeSH
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- přehledy MeSH
Lactoferrin (LF) is approximately 80 kDa iron-binding protein, which is important part of saliva and other body fluids. Due to its ability to bind metal ions, it has many biologically important functions. In this study, a method for the isolation of LF from a biological sample using robotically prepared antibody-modified paramagnetic particles was developed using robotic pipetting station. The method consisted of the following optimised steps. Protein G was bound on the paramagnetic particles, on which goat antibody (10 μg) was linked. LF was subsequently added to microtitration plate, which had affinity to goat antibody and the interaction lasted for 30 min. We found that the highest signals were obtained using the combination of goat antibody 1:3000, murine antibody 1:5000 and conjugate 1:1500. Horseradish peroxidase reducing 3,3,5,5-tetramethylbenzidine (TMB) was linked to the merged complex. The resulted product of this reaction was subsequently analysed spectrometrically with detection limit (3 S/N) as 5 ng/mL. In addition, we also determined TMB by stopped flow injection analysis with electrochemical detection. The limit of detection (3 S/N) was estimated as 0.1 μg/mL. To compare spectrometric and electrochemical approach for detection of TMB, calibration range of bead-LF-antibodies complex was prepared and was determined using a least-squares correlation with coefficient R² higher than 0.95, indicating a very good agreement of the results obtained.
- MeSH
- design vybavení MeSH
- dospělí MeSH
- elektrochemické techniky přístrojové vybavení MeSH
- imobilizační protilátky chemie MeSH
- laktoferrin analýza izolace a purifikace MeSH
- lidé MeSH
- limita detekce MeSH
- magnety chemie MeSH
- průtoková injekční analýza přístrojové vybavení MeSH
- sliny chemie MeSH
- zvířata MeSH
- Check Tag
- dospělí MeSH
- lidé MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
