The use of nanoparticles as a delivery system for a specific antigen could solve many limitations of mucosal vaccine applications, such as low immunogenicity, or antigen protection and stabilization. In this study, we tested the ability of nasally administered chitosan nanoparticles loaded with glycoprotein B of murine cytomegalovirus to induce an immune response in an animal model. The choice of chitosan nanoparticle type was made by in vitro evaluation of sorption efficiency and antigen release. Three types of chitosan nanoparticles were prepared: crosslinked with tripolyphosphate, coated with hyaluronic acid, and in complex with polycaprolactone. The hydrodynamic size of the nanoparticles by dynamic light scattering, zeta potential, Fourier transform infrared spectroscopy, scanning electron microscopy, stability, loading efficiency, and release kinetics with ovalbumin were evaluated. Balb/c mice were immunized intranasally using the three-dose protocol with nanoparticles, gB, and adjuvants Poly(I:C) and CpG ODN. Subsequently, the humoral and cell-mediated antigen-specific immune response was determined. On the basis of the properties of the tested nanoparticles, the cross-linked nanoparticles were considered optimal for further investigation. The results show that nanoparticles with Poly(I:C) and with gB alone raised IgG antibody levels above the negative control. In the case of mucosal IgA, only gB alone weakly induced the production of IgA antibodies compared to saline-immunized mice. The number of activated cells increased slightly in mice immunized with nanoparticles and gB compared to those immunized with gB alone or to negative control. The results demonstrated that chitosan nanoparticles could have potential in the development of mucosal vaccines.
- MeSH
- adjuvancia imunologická MeSH
- aplikace intranazální MeSH
- chitosan * chemie MeSH
- glykoproteiny MeSH
- imunizace MeSH
- imunoglobulin A MeSH
- Muromegalovirus * MeSH
- myši inbrední BALB C MeSH
- myši MeSH
- nanočástice * chemie MeSH
- slizniční imunita MeSH
- vakcíny * MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Nasal immunisation represents an innovative and perspective route of vaccine administration that provides many benefits compared to the more traditional approaches. Since most infections start on mucosal membranes, the mucosal immunisation provides a rational reason for its application. Mucosal delivery for vaccine administration (for example oral or nasal routes) could stimulate both systemic and mucosal immune responses. However, there are still some limitations that should be solved for a broader utilisation of this approach. There is still the necessity to use strongly immunogenic antigens or appropriate adjuvants for the induction of a strong immune response. The use of nanoparticles in the vaccine development could represent a promising approach for the mucosal vaccine research. Nanoparticles could thus serve as delivery vehicles providing to vaccines their unique properties, such as the antigen stabilisation and protection, serve as an adjuvant and elicit an antigen-specific immune response on the target sites.
- MeSH
- antigeny imunologie klasifikace MeSH
- aplikace slizniční MeSH
- chitosan * aplikace a dávkování chemická syntéza chemie farmakologie imunologie MeSH
- lidé MeSH
- nanočástice klasifikace terapeutické užití MeSH
- nanočásticový lékový transportní systém * terapeutické užití MeSH
- nos imunologie MeSH
- nosní sliznice imunologie MeSH
- vakcinace metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
- přehledy MeSH
This article describes the characterization and application of collagenase-based chitosan nanofiber membranes with rat burns. Electrospun chitosan nanofibers were functionalized with clostridial collagenase using carbodiimide chemistry. The immobilized collagenase was characterized by enzyme activity, kinetic constants, and dry storage stability measurements using a Pz-peptide substrate. The apparent kinetic constants KM and Vmax of immobilized collagenase showed a high affinity for the peptide substrate compared to the free enzyme. Drying of chitosan membranes with immobilized collagenase ensured 98 % stability of enzyme activity after rehydration. The effect of collagenase immobilized on chitosan nanofibers on the burn of the rat model was compared with a control treatment with chitosan nanofibers. The healing of the wound with both materials was terminated after 30 days at the same time, although the collagenase wound healed more rapidly during healing. The scar area size after the application of collagenase-containing chitosan nanofiber membranes was 31.6 % smaller than when only chitosan nanofibers were used.
- MeSH
- chitosan terapeutické užití MeSH
- Clostridium histolyticum MeSH
- enzymy imobilizované MeSH
- hojení ran * účinky léků MeSH
- krysa rodu rattus MeSH
- kůže zranění MeSH
- mikrobiální kolagenasa * metabolismus terapeutické užití MeSH
- nanovlákna terapeutické užití MeSH
- pilotní projekty MeSH
- rány a poranění farmakoterapie patologie MeSH
- stabilita enzymů MeSH
- výsledek terapie MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
The key factor in the development of antibody-based assays is to find an antibody that has an appropriate affinity, high specificity, and low cross-reactivity. However, this task is not easy to carry out since the research antibodies on the market may suffer from low specificity and reproducibility. Here, we report on a palm-sized dot blot-based device, called the affiblot, that has a specially designed lid that allows simultaneous semi-quantitative comparison of up to five antibodies from different suppliers regarding their affinity/avidity, cross-reactivity, and batch-to-batch reliability. The only required peripheral equipment is a vacuum pump, a camera, and densitometry software. The affiblot device was tested for its functionality and its measurements were compared against those obtained by standard dot blot and ELISA. The benefit over these methods, when various antibodies are evaluated, is in its simplicity. It allows easy antigen deposition, fast application and the discarding of the solutions, a compact undivided membrane, and therefore significant decrease of labor. The device was tested with specific anti-ApoE, anti-EpCAM, anti-Salmonella, anti-E. coli, and anti-Listeria antibodies from different suppliers. Their properties were compared for their ability to interact specifically with antigen and/or non-target structures and the best-suited antibody for the intended application was identified.
One of the major obstacles that limits the use of magnetic nanoparticles in biomedical applications is their potential toxicity. In the present study, we evaluated the cytotoxic effects of thiol-functionalized silica-coated iron oxide (Fe3O4@SiO2-SH) nanoparticles using human lung epithelial cells A549. We investigated the effect of Fe3O4@SiO2-SH nanoparticles on the cell viability, proliferation, cell cycle distribution, adhesion, apoptosis, and the orientation of the cytoskeletal networks, as well as on expression of proteins involved in cell death, cell survival, and cell adhesion. We demonstrated that exposure of A549 cells to Fe3O4@SiO2-SH nanoparticles resulted in severe disruption of the actin microfilaments and microtubule cytoskeleton and reduced the size of focal adhesions. Furthermore, cell adhesion was significantly affected as well as the phosphorylation of focal adhesion kinase (FAK), extracellular-signal-regulated kinase (ERK), and p38. Our findings highlight the need for in-depth cytotoxic evaluation of nanoparticles supporting their safer use, especially in biomedical applications.
- MeSH
- buněčná adheze účinky léků MeSH
- buňky A549 MeSH
- cytoskelet účinky léků MeSH
- lidé MeSH
- magnetické nanočástice oxidů železa chemie toxicita MeSH
- oxid křemičitý chemie MeSH
- proliferace buněk účinky léků MeSH
- sulfhydrylové sloučeniny chemie MeSH
- železo chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Vydání druhé opravené 117 stran : ilustrace (některé barevné) ; 30 cm
Skripta pro studenty vysokých škol. Laboratorní cvičení, která se zaměřují na imunoanalytické metody.
- MeSH
- imunoanalýza MeSH
- Konspekt
- Patologie. Klinická medicína
- Učební osnovy. Vyučovací předměty. Učebnice
- NLK Obory
- alergologie a imunologie
- NLK Publikační typ
- učebnice vysokých škol
- laboratorní cvičení
Vyd. 1. 115 s. : il. (některé barev.) ; 30 cm + CD-ROM
- MeSH
- chemické techniky analytické MeSH
- imunoanalýza klasifikace metody přístrojové vybavení MeSH
- imunochemie metody přístrojové vybavení MeSH
- klinické laboratorní techniky MeSH
- Konspekt
- Patologie. Klinická medicína
- Učební osnovy. Vyučovací předměty. Učebnice
- NLK Obory
- chemie, klinická chemie
- NLK Publikační typ
- učebnice vysokých škol
Specific allergen immunotherapy is frequently associated with adverse reactions. Several strategies are being developed to reduce the allergenicity while maintaining the therapeutic benefits. Peptide immunotherapy is one such approach. Methods for the simple and rapid identification of immunogenic epitopes of allergens (i.e. allergenic epitopes) are ongoing and could potentially lead to peptide-based vaccines. An epitope extraction technique, based on biofunctionalized magnetic microspheres self-organized under a magnetic field in a channel of a simple microfluidic device fabricated from polydimethylsiloxane, was applied in the isolation and identification of prospective allergenic epitopes. Similarly to chromatographic column separations, the easily replaceable plug of self-organized beads in the channel benefits especially from an even larger surface-to-volume ratio and an enhanced interaction of the surfaces with passing samples. Ovalbumin, the major protein of egg white and a typical representative of food allergens, was selected as the model molecule. Highly resistant ovalbumin was at first efficiently digested by a magnetic proteolytic reactor with trypsin treated with l-1-tosylamido-2-phenylethyl chloromethyl ketone and the second step, i.e. capture of allergenic epitopes from the mixture of peptides, was performed by a magnetic immunoaffinity carrier with orientedly immobilized rabbit anti-ovalbumin IgG molecules. Captured peptides were released with 0.05% trifluoroacetic acid. The elution fractions were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The peptide fragment of ovalbumin HIATNAVLFFGR (m/z: 1345.75, position: 371-382) was identified as a relevant allergenic epitope in this way. Such a microfluidic magnetic force-based epitope extraction technique applied in the epitope mapping of ovalbumin has the potential to be a significant step towards developing safe and cost-effective epitope-based vaccines.
- MeSH
- alergeny chemie imunologie MeSH
- epitopy analýza MeSH
- financování organizované MeSH
- hmotnostní spektrometrie MeSH
- imunomagnetická separace metody MeSH
- mapování epitopu metody MeSH
- mikrofluidní analytické techniky metody MeSH
- mikrosféry MeSH
- ovalbumin chemie imunologie MeSH
- potravinová alergie MeSH
- vakcíny MeSH
We report an efficient and streamlined way to improve the analysis and identification of peptides and proteins in complex mixtures of soluble proteins, cell lysates, etc. By using the shotgun proteomics methodology combined with bioaffinity purification we can remove or minimize the interference contamination of a complex tryptic digest and so avoid the time-consuming separation steps before the final MS analysis. We have proved that by means of enzymatic fragmentation (endoproteinases with Arg-C or/and Lys-C specificity) connected with the isolation of specific peptides we can obtain a simplified peptide mixture for easier identification of the entire protein. A new bioaffinity sorbent was developed for this purpose. Anhydrotrypsin (AHT), an inactive form of trypsin with an affinity for peptides with arginine (Arg) or lysine (Lys) at the C-terminus, was immobilized onto micro/nanoparticles with superparamagnetic properties (silica magnetite particles (SiMAG)-Carboxyl, Chemicell, Germany). This AHT carrier with a determined binding capacity (26.8 nmol/mg of carrier) was tested with a model peptide, human neurotensin, and the resulting MS spectra confirmed the validity of this approach.
- MeSH
- bioreaktory MeSH
- časové faktory MeSH
- chromatografie afinitní metody přístrojové vybavení MeSH
- enzymy imobilizované chemie MeSH
- financování organizované MeSH
- lidé MeSH
- ligandy MeSH
- magnetismus MeSH
- metaloendopeptidasy chemie MeSH
- nanočástice chemie MeSH
- neurotensin analýza MeSH
- oxid křemičitý MeSH
- peptidy analýza chemie MeSH
- proteomika MeSH
- reprodukovatelnost výsledků MeSH
- senzitivita a specificita MeSH
- serinové endopeptidasy chemie MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice metody MeSH
- trypsin chemie izolace a purifikace MeSH
- Check Tag
- lidé MeSH
