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ABSTRACT: Genetically modified Salmonella typhimurium VNP20009 (VNP) is a useful vehicle for cancer therapy and vaccine development but exhibits limited tumor targeting in vivo. We engineered a novel VNP derivative that expressed carcinoembryonic antigen (CEA)-specific single chain antibody fragments (scFv) on the cell surface to increase tumor-specific targeting. There was significant scFv cell surface display visualized by flow cytometry and confocal microscopy when cells were probed with fluorescently labeled CEA. Atomic force microscopy (AFM) measurements on whole bacteria confirmed binding of unlabeled CEA to the displayed scFv. The modified VNP strain exhibited increased localization in the upper gastrointestinal tract of CEA transgenic mice and accumulated in CEA-expressing tumors. Furthermore, treatment with a single dose of the VNP derivative inhibited growth of MC38CEA tumors and was associated with local accumulation of CD3(+) T cells and CD11b(+) macrophages. The display of antibody fragments on the surface of VNP represents a novel strategy for both targeting CEA-expressing tumors and increasing the immunogenicity of Salmonella-based vaccines for cancer.
Vaccine 05/2007; 25(21):4183-92. · 3.77 Impact Factor
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ABSTRACT: In this work we studied interactions between bacterial antigens and receptors on the surface of macrophages using atomic force microscopy (AFM). We used two bacterial cell wall components: lipopolysaccharide (LPS) derived from gram-negative Escherichia coli and exopolysaccharide (EPS) derived from gram-positive Lactobacillus rhamnosus. Interactions between these bacterial antigens and immune cell receptors were studied in peritoneal macrophages derived from two strains of mice, CBA and C3H/J, in which the Toll-like receptor 4 (TLR4) is genetically disabled. We collected 500 force-distance curves for LPS-activated cells using an EPS-covered AFM tip, and for EPS-activated cells using an LPS-covered AFM tip. Nonactivated cells were tested as reference cells. The results show that LPS-primed macrophages decrease their ability to bind EPS. Surprisingly, EPS-activated macrophages maintain or even increase their ability to bind LPS. This may suggest that in vivo commensal enteric bacteria, such as lactobacilli, will enhance the defense potential of local macrophages against pathogens expressing LPS.
Nanomedicine: nanotechnology, biology, and medicine 07/2006; 2(2):82-8. · 5.44 Impact Factor
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ABSTRACT: During recent years, atomic force microscopy has become a powerful technique for studying the mechanical properties (such as stiffness, viscoelasticity, hardness and adhesion) of various biological materials. The unique combination of high-resolution imaging and operation in physiological environment made it useful in investigations of cell properties. In this work, the microscope was applied to measure the stiffness of human red blood cells (erythrocytes). Erythrocytes were attached to the poly-L-lysine-coated glass surface by fixation using 0.5% glutaraldehyde for 1 min. Different erythrocyte samples were studied: erythrocytes from patients with hemolytic anemias such as hereditary spherocytosis and glucose-6-phosphate-dehydrogenase deficiency patients with thalassemia, and patients with anisocytosis of various causes. The determined Young's modulus was compared with that obtained from measurements of erythrocytes from healthy subjects. The results showed that the Young's modulus of pathological erythrocytes was higher than in normal cells. Observed differences indicate possible changes in the organization of cell cytoskeleton associated with various diseases.
Journal of Biochemical and Biophysical Methods 04/2006; 66(1-3):1-11. · 2.33 Impact Factor
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ABSTRACT: Using atomic force microscopy (AFM) we performed dynamic force measurements of the adhesive forces in two model systems: avidin-biotin and streptavidin-biotin. In our experiments we used glutaraldehyde for immobilization of (strept)avidin on the tip and biotin on the sample surface. Such interface layers are more rigid than those usually reported in the literature for AFM studies, when (strept)avidin is coupled with biotinylated bovine albumin and biotin with agarose polymers. We determined the dependence of the rupture forces of avidin-biotin and streptavidin-biotin bonds in the range 300-9600 pN/s. The slope of a semilogarithmic plot of this relation changes at about 1700 pN/s. The existence of two different regimes indicates the presence of two activation barriers of these complexes during the dissociation process. The dissociation rates and activation energy barriers, calculated from the Bell model, for the avidin-biotin and streptavidin-biotin interactions are similar to each other for loading rates > 1700 pN/s but they are different from each other for loading rates < 1700 pN/s. In the latter case, the dissociation rates show a higher stability of the avidin-biotin complex than the streptavidin-biotin complex due to a larger outer activation barrier of 0.8 k(B)T. The bond-rupture force is about 20 pN higher for the avidin-biotin pair than for the streptavidin-biotin pair for loading rates < 1700 pN/s. These two experimental observations are in agreement with the known structural differences between the biotin binding pocket of avidin and of streptavidin.
Acta biochimica Polonica 01/2006; 53(1):93-100. · 1.49 Impact Factor
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ABSTRACT: Biological samples can be investigated with atomic force microscopy (AFM) in either of two imaging modes: contact mode or tapping mode. Applications of fluid tapping mode AFM in biology and medical sciences are constantly growing and the data obtained with this technique are improving, especially in terms of resolution. Even dynamic processes can be observed almost as they would occur in vivo. AFM is a proven imaging technique that has recently gained attention in biomaterials due to its ability to analyze surface properties, non-destructively at nanometer-level resolution in ambient air or in fluid environments.
Przegla̧d lekarski 02/2004; 61(3):192-8.
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ABSTRACT: At present, there are a lot of biological and medical research fields, where atomic force microscopy (AFM) is being successfully used. AFM has contributed to research in the investigation of DNA, RNA structure, nucleic acid--protein complexes, chromosomes, ligand-receptor binding, cell membranes, proteins, lipids, carbohydrates, living cells (yeasts, bacteria, neurons, erythrocytes, endothelial cells); it enables to monitor drug interactions with cells and tissues, to visualize changes in protein molecular structure and crystal growth. Unlike electron microscopes, samples do not need to be stained, coated or frozen. Recent work indicates that, in the near future, AFM may be employed in ultra-sensitive immunoassay detection without any kind of labeling for both qualitative and quantitative work. AFM is more than a surface--imaging tool in that force measurements can be used to probe the physical properties of the specimen, such as molecular interactions, surface hydrophobicity, surface charges, and mechanical properties.
Przegla̧d lekarski 02/2004; 61(2):126-33.
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ABSTRACT: Scanning probe microscopy (SPM) has recently become the most dynamically developing technique employed in research investigations. Atomic force microscopy (AFM) that uses a digitally-controlled scanner and a cantilever system with a measurement tip shaped as a cone with the apex radius of 2-50 nm and the application force of 10(-10) N, as well as a system of recording detectors, has allowed for identification of numerous biological structures in a nanometer scale. The paper presents a review of measurement methods that employ the atomic force microscopy and are being used in medicine and biology, along with major achievements accomplished owing to this technique by various investigators, including the authors themselves.
Przegla̧d lekarski 02/2003; 60(12):828-31.
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ABSTRACT: Taurine chloramine (TauCl) and taurine bromamine (TauBr) are the main haloamines produced by activated neutrophils. TauCl exerts both anti-inflammatory and microbicidal activities. Clinical studies showed that TauCl may be useful as an antimicrobial agent in the local treatment of infections. Much less is known about TauBr. Circumstantial evidence suggests that Propionibacterium acnes (PA) has a role in the inflammation of acne. Available topical therapies include antimicrobial agents which reduce total PA numbers and anti-inflammatory agents which suppress activity of the cells present in acne inflammatory lesions. In this study the bactericidal activities of TauBr and TauCl against PA and Staphylococcus epidermidis (SE), as a control strain, were investigated. Moreover, the influence of these haloamines on the generation of reactive oxygen species (ROS) by activated neutrophils was also tested.
TauBr and TauCl were prepared by reaction of taurine with HOBr and HOCl, respectively. The reaction was monitored by UV absorption spectra. The bactericidal activities of TauBr and TauCl were determined by the pourplate method. The generation of ROS by neutrophils was determined by luminol chemiluminescence assay.
In our experimental set-up, TauBr showed stronger antibacterial activity than TauCl. Interestingly, PA was significantly more susceptible to TauBr than SE was. Moreover, TauBr at non-cytotoxic concentrations significantly reduced ROS generation by neutrophils.
Since PA is considered to be an etiological agent in acne and ROS are closely correlated with the pathogenesis of inflammatory skin diseases, the reported data suggest that TauBr may be a good candidate for the topical therapy for acne vulgaris.
Archivum Immunologiae et Therapiae Experimentalis 54(1):61-8. · 2.54 Impact Factor
