Genomic mapping in outbred mice reveals overlap in genetic susceptibility for HZE ion- and γ-ray-induced tumors.

Cancer risk from galactic cosmic radiation exposure is considered a potential "showstopper" for a manned mission to Mars. Calculating the actual risks confronted by spaceflight crews is complicated by our limited understanding of the carcinogenic effects of high-charge, high-energy (HZE) ions, a radiation type for which no human exposure data exist. Using a mouse model of genetic diversity, we find that the histotype spectrum of HZE ion-induced tumors is similar to the spectra of spontaneous and γ-ray-induced tumors and that the genomic loci controlling susceptibilities overlap between groups for some tumor types. Where it occurs, this overlap indicates shared tumorigenesis mechanisms regardless of the type of radiation exposure and supports the use of human epidemiological data from γ-ray exposures to predict cancer risks from galactic cosmic rays.

The role of glycosaminoglycan binding of staphylococci in attachment to eukaryotic host cells.

Attachment of microorganisms to host cells is believed to be a critical early step in microbial pathogenesis. The aim of the study was to determine the role of the known glycosaminoglycan (GAG) binding activity of Staphylococcus aureus and coagulase-negative staphylococci (CoNS) in their attachment to six different eukaryotic cell lines. Three staphylococcal species expressing GAG binding capacity-S. aureus, S. epidermidis, and S. hemolyticus-were chosen for investigation. Six different eukaryotic cell lines, endothelial HUVEC and EA. hy 926 cells, epithelial A549 and HeLa S3 cells, fibroblasts HEL Sp 12 and macrophages J774.A1, were included. A modified ELISA with biotinylated bacteria was used for estimating the adhesion of staphylococci to each of the cell lines. Our results showed that staphylococci adhered to each of the cell lines studied, although the binding of CoNS strains to epithelial cells was lower than to the other cells. The attachment to all cell types could be partially decreased by pretreatment of the bacteria with various polysulfated agents (highest inhibition was 60%), as well as by chlorate and heparitinase treatment of the cells. These observations may suggest that at least one mode of staphylococcal attachment utilizes GAG chains present on the surface of virtually all adherent cells.

Binding of selected extracellular matrix proteins to enterococci and Streptococcus bovis of animal origin.

Thirty-three enterococcal strains and 10 Streptococcus bovis strains were investigated for their protein-binding cell surface components. Seven extracellular matrix (ECM) proteins were immobilized on Difco latex beads to detect these components on the surface of all enterococcal strains and eight non-autoaggregating S. bovis strains by a particle agglutination assay (PAA). Twenty-three selected strains were also examined in microtiter plate assays. According to the absorbance readings (A(570nm)), 11 strains were classified as nonadherent (A(570nm) < 0.1), 10 strains as weakly adherent (0.1 < A(570nm) > 0.3), and 2 strains as strongly adherent (A(570nm) > 0.3) in these assays. A direct correlation was found between the values obtained in PAA and A(570nm) readings of microtiter plate assays. Binding of (125)I-labeled bovine lactoferrin to enterococci and streptococci was in the range of 6%-30% and of (125)I-labeled human vitronectin in the range of 9%-33% to streptococci. The binding of(125)I-labeled ECM proteins to selected strains was much more effectively inhibited by sulfated carbohydrates than by non-sulfated hyaluronic acid, indicating the importance of the sulfate groups of these inhibitors. An inhibition effect of heparin on bLf binding to four selected strains was higher in comparison with fucoidan in the microtiter plates. Thirty-five out of 44 strains had agglutinated rabbit erythrocytes. However, these strains showed no ability to agglutinate bovine or sheep erythrocytes.

Binding of extracellular matrix proteins by animal strains of staphylococcal species.

All 81 strains of Staphylococcus species isolated mainly from animals express high surface hydrophobicity as a stable property upon cultivation on blood agar. Bovine lactoferrin, human vitronectin, human fibronectin, heparin, human and bovine serum albumin were immobilized on latex beads to detect protein-binding cell surface components of 67 non-autoaggregating staphylococcal strains by a particle agglutination assay. Bovine lactoferrin was bound well by 22 strains (3 or 2) while 15 strains reacted weakly (1) and 30 did not react (0) with the lactoferrin-coated latex beads. The particle agglutination assay showed similar differences among staphylococcal strains in binding other proteins with the exception of human and bovine serum albumins for which 66 of 67 strains were negative (0). The specificity of the agglutination reaction was confirmed by a particle agglutination inhibition assay by preincubating bacterial cells with the protein (lactoferrin, vitronectin, etc.) used subsequently in particle agglutination assay. Autoaggregating strains together with some non-autoaggregating strains were selected for microtitre plate assay. According to absorbance at 570 nm, 14 strains were classified as non-adherent, 16 strains as weakly adherent and 18 strains as strongly adherent to bovine lactoferrin in microtitre plate assays. A direct correlation was found between the absorbance values at 570 nm of microtitre plate binding assay and test values obtained in particle agglutination assay. Binding of bovine lactoferrin to 81 staphylococcal strains as well as of human vitronectin and human fibronectin to a selected number of these strains was studied with radiolabeled (125I-labeled) proteins. Radiolabeled bovine lactoferrin was bound common by all except four strains (7 to 39%). Staphylococcal strains isolated from diseased pigs commonly bound 125I-labeled vitronectin (21 to 42% of the vitronectin added). Binding of vitronectin and lactoferrin was efficiently inhibited by preincubating of staphylococcal cells with sulphated carbohydrate compounds as heparin, dextran sulphate and fucoidan, but not by other non-sulphated highly charged glycoconjugates such as hyaluronic acid.

In vitro anti-staphylococcal activity of heparinized biomaterials bonded with combinations of rifampicin.

Biomaterial implants in various human body tissues are highly susceptible to bacterial colonization. We report here on the coating of heparinized biomaterials with heparin binding extracellular matrix proteins giving special regard to the efficient adsorption and slow release of antibiotics. Heparin was partially degraded and the resulting fragments were covalently end-point attached to 0.5 cm long silicone biomaterial surface. Collagen type I was immobilized on the heparinized biomaterials and then cross-linked with acyl-azide or carbodiimide. Finally, the resulting biosurfaces were exposed to antibiotics, i.e. rifampicin in combination with cefuroxime, fusidic acid, ofloxacin or vancomycin, respectively. The antibiotic bonded biomaterials were evaluated for their anti-staphylococcal activity after elution in NaCl, serum or blood by measuring the zones of inhibition for S. epidermidis strain RP12. Furthermore, we examined the in-vitro colonization resistance to S. epidermidis RP12 for these combinations of rifampicin-bonded biomaterials by an ATP bioluminescence assay. The ATP measurements showed that initially adherent bacteria were eradicated from the polymer surface, for at least 24 or 48 h (fusidic acid > cefuroxime > vancomycin > ofloxacin). The anti-staphylococcal activity of rifampicin-fusidic acid bonded heparinized biomaterials seems of sufficient duration and efficacy to merit testing in an animal model.

Quantitation of bacterial adhesion to polymer surfaces by bioluminescence.

Quantitation of microbes adhering to a surface is commonly used in studies of microbial adhesion to different surfaces. We have quantified different staphylococcal strains adhering to polymer surfaces by measuring bacterial ATP (adenosine triphosphate) by bioluminescence. The method is sensitive, having a detection limit of 10(4) bacterial cells. Viable counting of bacterial cells may yield falsely low results due to the presence of "dormant" and adherent bacteria. By using bioluminescence, this can be avoided. Cells of different bacterial species and cells of strains of the same species were shown to differ significantly in their basal ATP content (8.7 x 10(-13) - 5.2 x 10(-22) MATP). The size of adherent and planktonic bacteria decreased with time (0.7 micron-->0.3 micron, 20 days). During incubation in nutrient-poor buffer ("starvation"), the ATP content of adherent bacteria decreased after 24-96 h whereas that of planktonic bacteria was stable over 20 days. The presence of human serum or plasma did not interfere significantly with the test results. Since the ATP concentration of bacterial strains of different species varies and is also influenced by the growth conditions of bacteria (solid or liquid culture medium), a species-specific standard curve has to be established for bacteria grown under the same culture conditions. We conclude that the method is a sensitive tool to quantify adherent bacteria during experiments lasting for less than 6 h and constitutes a valuable method to be used in conjunction with different microscopical techniques.