Enhanced cell growth by nanoengineering zirconia to stimulate electrostatic fibronectin activation.

We address the enhanced bone growth on designed nanocrystalline zirconia implants as reported by in vivo experiments. In vitro experiments demonstrate that the activation of adhesive proteins on nanoengineered zirconia stimulates cell adhesion and growth as shown by confocal microscopy. Fibrillar fibronectin (FN) forms a matrix assembly on the nanostructured surface in the cell adhesion process. We discuss the importance of FN dimer activation due to its immobilization on the designed nanocrystalline ZrO2 implant fabricated by ion beam assisted deposition. The Monte-Carlo analysis indicates that FN activation on the surface can be promoted by selective electrostatic interactions between negatively charged ZrO2 surface patches and oppositely charged FN domains.

Enhanced initial protein adsorption on engineered nanostructured cubic zirconia.

Motivated by experimentally-observed biocompatibility enhancement of nanoengineered cubic zirconia (ZrO(2)) coatings to mesenchymal stromal cells, we have carried out computational analysis of the initial immobilization of one known structural fragment of the adhesive protein (fibronectin) on the corresponding surface. We constructed an atomistic model of the ZrO(2) nano-hillock of 3-fold symmetry based on Atom Force Microscopy and Transmission Electron Microscopy images. First principle quantum mechanical calculations show a substantial variation of electrostatic potential at the hillock due to the presence of surface features such as edges and vertexes. Using an implemented Monte Carlo simulated annealing method, we found the orientation of the immobilized protein on the ZrO(2) surface and the contribution of the amino acid residues from the protein sequence to the adsorption energy. Accounting for the variation of the dielectric permittivity at the protein-implant interface, we used a model distance-dependent dielectric function to describe the inter-atom electrostatic interactions in the adsorption potential. We found that the initial immobilization of the rigid protein fragment on the nanostructured pyramidal ZrO(2) surface is achieved with a magnitude of adsorption energy larger than that of the protein on the smooth (atomically flat) surface. The strong attractive electrostatic interactions are a major contributing factor in the enhanced adsorption at the nanostructured surface. In the case of adsorption on the flat, uncharged surface this factor is negligible. We show that the best electrostatic and steric fit of the protein to the inorganic surface corresponds to a minimum of the adsorption energy determined by the non-covalent interactions.

Effect of the ordered interfacial water layer in protein complex formation: A nonlocal electrostatic approach.

Using a nonlocal electrostatic approach that incorporates the short-range structure of the contacting media, we evaluated the electrostatic contribution to the energy of the complex formation of two model proteins. In this study, we have demonstrated that the existence of an ordered interfacial water layer at the protein-solvent interface reduces the charging energy of the proteins in the aqueous solvent, and consequently increases the electrostatic contribution to the protein binding (change in free energy upon the complex formation of two proteins). This is in contrast with the finding of the continuum electrostatic model, which suggests that electrostatic interactions are not strong enough to compensate for the unfavorable desolvation effects.

Serum bactericidal activity against Helicobacter pylori in patients with hypogammaglobulinaemia.

The two major primary antibody deficiency disorders are X-linked hypogammaglobulinaemia (XLA) and common variable immunodeficiency (CVID). CVID patients have an elevated risk for gastric cancer and extra-nodal marginal zone lymphoma. Both diseases are associated with Helicobacter pylori infection. We investigated whether antibody deficiency leads to defective serum bactericidal activity against H. pylori. We also investigated the correlation with immunoglobulin (Ig)M levels and observed the terminal complement complex (TCC) activity. Sera of 13 CVID patients (four H. pylori positive), one patient with hyper-IgM syndrome, one patient with Good syndrome (both H. pylori positive), five XLA patients, four H. pylori seropositive controls, four H. pylori seronegative controls and a sample of pooled human serum (PHS) were incubated in vitro with bacterial suspensions of H. pylori for 30 min. After 72 h of culture, colony-forming units were counted. TCC formation was measured by enzyme-linked immunosorbent assay. We found that normal human serum is bactericidal for H. pylori, whereas heat-inactivated serum shows hardly any killing of H. pylori. Serum (1%) of hypogammaglobulinaemia patients has a decreased bactericidal activity against H. pylori. Helicobacter pylori-positive (HP(+)) normal individuals show more than 90% killing of H. pylori, whereas CVID patients show 35% killing (P = 0.007) and XLA patients only 19% (P = 0.003). Serum (1%) of HP(+) volunteers showed significantly better killing compared with serum of H. pylori-negative (HP(-)) volunteers (P = 0.034). No correlation between (substituted) IgG levels and serum bactericidal activity was found, but a weak correlation between total serum IgM and serum bactericidal activity was found. In conclusion, serum bactericidal activity against H. pylori is decreased in patients with hypogammaglobulinaemia. Heat treatment of the serum abolished the bactericidal capacity, indicating that complement activity is essential for the bactericidal effect.

Analysis of vacA, cagA, and IS605 genotypes and those determined by PCR amplification of DNA between repetitive sequences of Helicobacter pylori strains isolated from patients with nonulcer dyspepsia or mucosa-associated lymphoid tissue lymphoma.

The vacA s and m genotypes and the presence of cagA and IS605 were determined in Helicobacter pylori strains from patients with mono- and multiple infections. Surprisingly, these genetic markers were not associated with nonulcer dyspepsia or mucosa-associated lymphoid tissue lymphoma. The presence of cagA correlated with the presence of the vacA s1 allele (P < 0.05), whereas the presence of IS605 was associated with the presence of the s2 allele (P < 0.05).

Helicobacter pylori-associated gastritis in mice is host and strain specific.

The vacA and cagA geno- and phenotypes of two mouse-adapted strains of Helicobacter pylori, SS1 and SPM326, were determined. The SS1 strain, which had the cagA+ and vacA s2-m2 genotype, induced neither vacuole formation in HeLa cells nor interleukin-8 (IL-8) production in KATO III cells. In contrast, H. pylori SPM326, with the cagA+ and vacA s1b-m1 genotype, induced vacuoles as well as IL-8 production in vitro. Furthermore, a spontaneous mutant of SPM326, which produced a vacuolating cytotoxin but was not able to induce IL-8 production (SPM326/IL-8(-)), was detected. C57Bl/6 and BALB/c mice were infected with these three strains to investigate the colonization pattern and the effect on the immune response in vivo. The SS1 strain colonized the stomachs of all mice in large numbers which remained constant over time. Colonization with the SPM326/IL-8(+) and SPM326/IL-8(-) strains was lesser, or even absent, and decreased over time. At 5 weeks postinoculation all three H. pylori strains induced a mild increase of neutrophil count in the gastric corpus of C57Bl/6 mice, which disappeared by 12 weeks. At both 5 and 12 weeks postinoculation C57Bl/6 mice colonized with SPM326/IL-8(+) showed an increased expression of major histocompatibility complex (MHC) class II antigen in the cardia which was accompanied by an increased number of T cells. C57Bl/6 mice that were infected with SS1 and SPM326/IL-8(-) did not show chronic inflammation. BALB/c mice colonized with SS1 and SPM326/IL-8(-) also showed an increase in neutrophil count at 5 weeks, which normalized again by 12 weeks postinoculation. At this time point SS1-infected mice showed inflammation in the corpus and antrum. At these sites an increased expression of MHC class II antigens and an increased number of T cells were observed. Although small lymphoid follicles were already observed 5 weeks after inoculation with SS1, their incidence as well as their number was increased at 12 weeks. These results show that inflammation induced by H. pylori depends both on the bacterial strain and the host.

The inflammatory response in CD1 mice shortly after infection with a CagA+/VacA+ Helicobacter pylori strain.

To investigate the early events of Helicobacter pylori infection in a mouse model, CD1 mice were infected with a type I (CagA+/VacA+) H. pylori strain. Up to 4 weeks after infection the majority of gastric tissue biopsies were positive in culture. Immunohistochemical analysis showed that inflammatory changes started to occur after 3 weeks. Four weeks after infection a significant increase in T cells was observed in the cardia/corpus region of the stomachs of infected mice. These T cells were CD4+ and CD8+, and they were located in an area with increased expression of MHC class II antigens. In 50% of the infected mice also an increased number of mast cells was seen. Furthermore, aggregates of B and T cells were present in the submucosa. Characterization of cytokines by immunohistochemistry showed an increase in IL-5-secreting cells in the inflamed area of the infected stomach. No difference was observed between interferon-gamma (IFN-gamma)-, IL-4- and IL-10-secreting cells in control and infected mice. These results suggest that no polarized T-helper cell response was present at this early phase of infection. Infection with H. pylori also induced a serum response and especially IgG was increased after 4 weeks of infection. However, no particular increase in IgG1, IgG2a or IgG3 isotype was observed. Part of the serum antibodies was directed against lipopolysaccharide (LPS), but no evidence for anti-Lewis antibodies or antibodies against epitopes on the gastric mucosa was found.

Local cellular immune response in the acute phase of gastritis in mice induced chemically and by Helicobacter pylori.

Gastritis was induced in mice by oral administration of acetic acid 5%, a cagA positive Helicobacter pylori strain, or both. The induction of a mild gastritis by acetic acid before inoculation with H. pylori resulted in a slight but not significantly decreased colonisation rate. To study the initial stage of inflammation, the presence of gastric lymphoid and non-lymphoid cells was studied by immunohistochemistry during the first 2 weeks after induction of gastritis. Treatment with acetic acid alone or in combination with H. pylori resulted in an increase in the number of neutrophils in the mucosa and submucosa, without evident epithelial damage. The influx of neutrophils was most prominent in the mice that received a combined treatment of acetic acid and H. pylori. Macrophages were also increased in both acetic acid and acetic acid plus H. pylori-treated groups, although a different kinetic pattern was present in these groups. In mice infected with H. pylori alone, only a slight but not significant increase in neutrophils and macrophages was observed. The early presence of lymphoid aggregates in the gastric mucosa of mice in which colonisation was shown with H. pylori was remarkable. This phenomenon was not seen in control mice, in mice that received acetic acid alone or when colonisation was not shown. These data suggest that gastritis induced by a chemical agent such as acetic acid occurs by a different mechanism than gastritis induced by H. pylori and that the continued presence of H. pylori is required for local lymphocyte activation.

Genomic DNA fingerprinting of clinical isolates of Helicobacter pylori by REP-PCR and restriction fragment end-labelling.

Genetic diversity of 32 Helicobacter pylori strains isolated from patients with gastritis, gastric or duodenal ulcer, carcinoma, or lymphoma was determined by repetitive sequence element polymerase chain reaction (REP-PCR), and by the new typing method restriction fragment end-labelling (RFEL). Furthermore, these two methods were used to investigate a possible correlation between clinical symptoms and the genetic background of Helicobacter pylori. Both REP-PCR and RFEL revealed 31 different patterns for the 32 strains tested, but the pair of isolates with identical REP-PCR patterns was not the same as the pair of isolates with identical RFEL patterns. Computer-assisted analysis of the DNA fingerprints was used to determine similarity coefficients. This analysis revealed no clustering of disease-specific strains by any of the two methods.

Neutrophil-activating protein mediates adhesion of Helicobacter pylori to sulfated carbohydrates on high-molecular-weight salivary mucin.

The in vitro binding of surface-exposed material and outer membrane proteins of Helicobacter pylori to high-molecular-weight salivary mucin was studied. We identified a 16-kDa surface protein which adhered to high-molecular-weight salivary mucin. This protein binds specifically to sulfated oligosaccharide structures such as sulfo-Lewis a, sulfogalactose and sulfo-N-acetyl-glucosamine on mucin. Sequence analysis of the protein proved that it was identical to the N-terminal amino acid sequence of neutrophil-activating protein. Moreover, this adhesin was able to bind to Lewis x blood group antigen.

Sulfated glycans on oral mucin as receptors for Helicobacter pylori.

Helicobacter pylori is able to colonize gastric epithelia, causing chronic active gastritis, gastric and duodenal ulcers and presumably gastric malignancies. Attempts to identify the natural reservoir for this microorganism other than the stomach have been unsuccessful. It is suspected that H. pylori can be transmitted orally, since the microorganism has been detected at various sites of the oral cavity. The aim of the present study was to determine whether H. pylori can bind to salivary mucins, which in vivo coat the oral epithelia, and characterize further the interaction. Binding of salivary mucins and of synthetic oligosaccharides was studied in ELISA and immunoblotting, using specific mono- and polyclonal antibodies, and synthetic neoglycoconjugates. H. pylori bound most avidly to a highly sulfated subpopulation of high molecular weight salivary mucins, secreted from the palatine salivary glands, and with less avidity to mucin species secreted by the sublingual and submandibular salivary glands, which are less sulfated. Binding was strongly enhanced upon decreasing pH from 6.0 to 5.0. Using synthetic polyacrylamide coupled oligosaccharides it was found that SO3-3-Gal and the SO3-3-Lewis(a) blood group antigen bound to H. pylori. In contrast, binding of sialylated Lewis(a) and Lewis(b) antigens was much weaker. This study indicates that sulfated oligosaccharides on salivary mucins may provide receptor structures for adhesion of H. pylori to oral surfaces.

Mechanisms of resistance to piperacillin of Enterobacter cloacae strains differ by antibiotic prescription policy.

During a study of piperacillin resistance among aerobic Gram-negative bacteria, 18 resistant strains of Enterobacter cloacae were obtained from a General Hospital in Rotterdam and 13 from a University Hospital in Amsterdam. The patterns of antibiotic susceptibilities were different: the Amsterdam strains were generally resistant to penicillins, the third generation cephalosporins and temocillin, whereas the Rotterdam strains were more often sensitive to the third generation cephalosporins and temocillin but more resistant to penicillins. Isoelectric focusing and substrate profiles showed the presence of chromosomal Class 1 beta-lactamase in ten of the Amsterdam strains: in three strains a plasmid mediated TEM-1 enzyme was detected. In contrast 15 of the 18 Rotterdam strains possessed a plasmid mediated beta-lactamase, ten of which were TEM-2. Eight of the ten strains with the TEM-2 enzyme harboured a transferable plasmid coding for resistance to piperacillin. Endonuclease analysis of plasmid DNA from these eight strains revealed an identical pattern in seven strains. Different selective pressures were operative in each hospital. In Amsterdam the general use of cefotaxim and piperacillin favoured emergence of strains with derepressed chromosomal Class 1 beta-lactamase, whereas in Rotterdam the use of cefuroxime favoured the spread of a plasmid, encoding TEM-2 beta-lactamase.

Phagocytosis and killing of suspended and adhered bacteria by peritoneal cells after dialysis.

OBJECTIVE: To determine the effect of dialysis fluid containing various glucose concentrations on the phagocytosis and killing of Staphylococcus aureus by rat peritoneal cells under conditions mimicking the in vivo situation. DESIGN: Phagocytosis and killing were evaluated by quantitation of the killing capacity of macrophages after in vivo phagocytosis of the bacteria as well as by an in vitro flow cytometric assay of the phagocytosis and killing of adhered bacteria by peritoneal cells. ANIMALS: Male Wistar rats. MAIN OUTCOME MEASURE: It was expected that the intraperitoneal administration of dialysis fluid would impair the capacity of peritoneal cells to eliminate bacteria. RESULTS: The first test revealed no effects of glucose concentration or dwell time on the killing of phagocytosed bacteria by macrophages, median percentages ranging between 29% and 64%. In the second series of experiments no effect of glucose concentration on the phagocytosis and killing of adhered bacteria was found either; however, longer dwell times significantly enhanced both the phagocytosis (at a dwell time of 1 hour, under 20%; at dwell times of 4 or 18 hours, above 20%, p < 0.02) and the killing (at a dwell time of 1 hour, under 53%; at dwell times of 4 and 18 hours, above 70%, p < 0.01). CONCLUSIONS: Glucose concentration has no effect on the phagocytosis and killing of Staphylococcus aureus, whereas the dwell time significantly enhances both of these functional capacities of peritoneal cells if the bacteria are adhered to surfaces.

Effect of glucose in dialysis fluid on antibacterial defence in the peritoneal cavity.

To study the effect of glucose concentration and dwell time of dialysis fluid on peritoneal antibacterial defence, an experimental infection with Staphylococcus aureus was induced in rats. For this purpose rats were inoculated intraperitoneally with Staphylococcus aureus at different intervals after the administration of various dialysis fluids. Twenty-four hours later the numbers of bacteria and cells in the peritoneal cavity were determined. The number of bacteria was correlated positively with the glucose concentration. Furthermore, an inverse correlation between dwell time and the number of bacteria was observed. Neither finding could be attributed to a glucose-dependent growth of the bacteria or disruption of the killing capacity of peritoneal cells in vitro. A glucose-dependent increase in the volume of the peritoneal fluid could partially explain the differences found in vivo. It is concluded that the glucose in dialysis fluid impairs antibacterial defence in the peritoneal cavity and that longer dwell times enhance this defence.

Effect of an acidic environment on the susceptibility of Helicobacter pylori to trospectomycin and other antimicrobial agents.

The susceptibility of 30 clinical isolates of Helicobacter pylori to trospectomycin, ampicillin, metronidazole, clarithromycin, azithromycin and clindamycin under varying pH conditions was evaluated. An acidic environment was shown to affect unfavourably the activity of all the antimicrobial agents tested. This pH effect was most marked for the two macrolides and for clindamycin.

Presence of Helicobacter pylori in the oral cavity, oesophagus, stomach and faeces of patients with gastritis.

The presence of Helicobacter pylori in the oral cavity (6 sites), oesophagus, stomach and bowel of 20 dyspeptic patients was investigated. Samples were cultured on three selective media and analyzed by 16S rDNA polymerase chain reaction (PCR) and southern hybridization. Helicobacter pylori DNA was detected by PCR from oral-cavity samples of three (20%) and from faeces samples of only one (7%) of the patients whose stomach biopsies were positive for Helicobacter pylori. When culture was used, the microorganism's rate of recovery from the oral cavity and faeces was 13% and 7%, respectively. One patient had a Helicobacter pylori-like organism in samples collected from the tongue and palate. Both strains were urease, catalase and oxidase positive and grew microaerophilically but were negative on PCR analysis. This demonstrates the possibility of false identification of Helicobacter pylori by use of routine enzyme reactions. Interestingly, specimens collected from the cheeks of three patients were positive for Helicobacter pylori by PCR analysis. This is the first instance of detection of this microorganism in the cheek.

Effect of interferon-gamma in dialysis fluid on peritoneal defence in rats.

BACKGROUND: A major drawback of continuous ambulatory peritoneal dialysis (CAPD) is the occurrence of peritoneal infection. This might be explained by a non-optimal phagocytic capacity of peritoneal cells which can be improved by stimulating factors. AIM: To investigate the effect of addition of interferon-gamma (IFN) to dialysis fluid with various glucose concentrations or to saline (as control) on the peritoneal defence against Staphylococcus aureus in an experimental dialysis model in rats. METHODS: Twenty-four hours after the administration of either dialysis fluid containing various glucose concentrations or saline with or without IFN, bacteria were injected intraperitoneally. At the time of the bacterial infection and 24 h later cellular and bacterial parameters were studied. RESULTS: The addition of IFN to dialysis fluid or saline resulted in a significant (P < 0.01) increase in the number of peritoneal macrophages at the time of infection; this was accompanied by a significant increase in both the number of Ia-positive peritoneal macrophages (P < 0.01) and the production of nitrite by macrophages (P < 0.05) at the time. IFN in dialysis fluid as well as in saline significantly (P < 0.01) reduced the recovery of bacteria from the peritoneal cavity 24 h after infection. Only the absence of IFN glucose increased the recovery of bacteria from the peritoneal cavity at the same time. CONCLUSION: In this experimental model the addition of IFN to dialysis fluid lowered the recovery of staphylococci from the peritoneal cavity by means of activation of an increased number of macrophages.

[Symptomatic bacteremia after periodontal treatment]. / Symptomatische bacteriëmie na parodontale behandeling.

Symptomatic bacteremia after dental treatment in an apparently healthy patient is an indication for preventive antibiotic treatment. This article describes the case of a patient with clinical symptoms of bacteremia after periodontal treatment, in whom the blood cultures remained positive after oral administration of amoxicillin (Chlamoxyl). Intramuscular administration of penicillin (Bicilline) was able to prevent the clinical symptoms as well as the bacteremia.

The role of neuraminidase in haemagglutination and adherence to colon WiDr cells by Bacteroides fragilis.

The role of neuraminidase in haemagglutination and adherence to colon WiDr cells by eight strains of Bacteroides fragilis and four strains of oral black-pigmented gram-negative anaerobes was studied. Neuraminidase treatment resulted in a very small increase of haemagglutination by some of the strains but had no effect on adherence to WiDr cells by all bacterial strains tested except one strain of Prevotella intermedia (HG 110). Inhibition of neuraminidase had no effect on haemagglutination or adherence, nor was any correlation found between haemagglutinating ability and neuraminidase activity in the B. fragilis strain. The results indicated that haemagglutination and adherence of B. fragilis to WiDr cells were not mediated by neuraminidase.