Biomedical subjects

S A Zaat

Publications and source records attributed to S A Zaat.

At least 19 recordsLinked to original sources

PubMed2001

Platelet microbicidal activity is an important defense factor against viridans streptococcal endocarditis.

To study the role of platelet microbicidal activity in host defense against infective endocarditis (IE) due to viridans streptococci (VS), the susceptibility to platelet releasate of blood and oral VS isolates from patients with and without IE was compared. The influence of neutralization of platelet microbicidal activity was studied in 2 experimental IE models. Resistance to platelet releasate was more prevalent among VS from blood of patients with IE than from blood of bacteremic patients without IE and among oral VS isolates. Serum from rabbits vaccinated with human platelet sonicate supernatants neutralized human and rabbit platelet-released microbicidal activity and had antibodies recognizing microbicidal proteins thrombocidin-1 and -2 and other human platelet proteins. In the 2 rabbit IE models, vaccination increased the susceptibility to experimental IE due to platelet releasate-susceptible VS. Thus, platelet-released microbicidal activity is an important host defense factor against IE due to VS.

Animals↗

PubMed2001

Mycoplasma pneumoniae P1 type 1- and type 2-specific sequences within the P1 cytadhesin gene of individual strains.

Mycoplasma pneumoniae strains traditionally are divided into two types, based on sequence variation in the P1 gene. Recently, however, we have identified 8 P1 subtypes by restriction fragment length polymorphism analysis. In the present study the P1 gene sequences of three P1 type 1 and two P1 type 2 M. pneumoniae strains were analyzed. A new P1 gene sequence in a type 1 strain with partial similarity to a recently reported variable region in the P1 gene of an M. pneumoniae type 2 strain (T. Kenri, R. Taniguchi, Y. Sasaki, N. Okazaki, M. Narita, K. Izumikawa, M. Umetsu, and T.Sasaki, Infect. Immun. 67:4557-4562, 1999) was identified. In addition, the P1 gene of the type 1 strain contained another region with nucleotide polymorphisms identical to a stretch in the P1 gene of one of our type 2 strains. These findings indicate that recombination between sequences specific for P1 type 1 and type 2 had occurred and that P1 type 1 and type 2 hybrid sequences can be present within the P1 gene of an individual strain. Identical or nearly identical variable P1 gene sequences were present in several repetitive regions outside the P1 gene locus in the genome of M. pneumoniae strain M129, implying recombination as a mechanism for generation of the P1 gene variation. Additionally, in the P1 gene sequences of four of the five strains studied, single-nucleotide polymorphisms different from the previously reported P1 type 1 and 2 characteristic sequences were identified. The polymorphic sites are candidate targets for genotyping of M. pneumoniae by direct sequencing of amplicons from clinical specimens.

Adhesins, Bacterial↗

PubMed2000

Thrombocidins, microbicidal proteins from human blood platelets, are C-terminal deletion products of CXC chemokines.

Antibacterial proteins are components of the innate immune system found in many organisms and produced by a variety of cell types. Human blood platelets contain a number of antibacterial proteins in their alpha-granules that are released upon thrombin activation. The present study was designed to purify these proteins obtained from human platelets and to characterize them chemically and biologically. Two antibacterial proteins were purified from platelet granules in a two-step protocol using cation exchange chromatography and continuous acid urea polyacrylamide gel electrophoresis and were designated thrombocidin (TC)-1 and TC-2. Characterization of these proteins using mass spectrometry and N-terminal sequencing revealed that TC-1 and TC-2 are variants of the CXC chemokines neutrophil-activating peptide-2 and connective tissue-activating peptide-III, respectively. TC-1 and TC-2 differ from these chemokines by a C-terminal truncation of 2 amino acids. Both TCs, but not neutrophil-activating peptide-2 and connective tissue-activating peptide-III, were bactericidal for Bacillus subtilis, Escherichia coli, Staphylococcus aureus, and Lactococcus lactis and fungicidal for Cryptococcus neoformans. Killing of B. subtilis by either TC appeared to be very rapid. Because TCs were unable to dissipate the membrane potential of L. lactis, the mechanism of TC-mediated killing most probably does not involve pore formation.

Amino Acid Sequence↗

PubMed2000

In vivo and in vitro release of lysozyme from cross-linked gelatin hydrogels: a model system for the delivery of antibacterial proteins from prosthetic heart valves.

Prosthetic valve endocarditis may be reduced by the local delivery of antibacterial proteins from the Dacron sewing ring of a prosthetic heart valve. Dacron discs were treated with a carbon dioxide gas plasma to improve the hydrophilicity and thereby enabling homogeneous impregnation with gelatin type B. The gelatin samples were cross-linked to different degrees using various amounts of water-soluble carbodiimide (EDC) and N-hydroxysuccinimide (NHS). Lysozyme, a model protein for antibacterial proteins, was loaded into (non)-cross-linked gelatin gels incorporated in Dacron, or adsorbed onto non-treated and gas plasma-treated Dacron. The in vivo lysozyme release was measured after subcutaneous implantation of lysozyme-loaded samples in rats. The lysozyme content of the samples, and the lysozyme level of the surrounding tissue were determined at different explantation times (ranging from 6 h up to 1 week). For cross-linked gelatin gels, the lysozyme tissue level was elevated up to 2 days after implantation. In vitro release was measured using agarose medium or phosphate buffer. Lysozyme release in buffer solution under sink conditions was in good agreement with the in vivo lysozyme release profiles, and therefore considered a good model to describe in vivo release characteristics. The release was modelled with a solution of Fick's second law of diffusion using the appropriate boundary conditions. In this way the lysozyme concentration in the gel and the surrounding tissue as a function of time and distance was obtained. The presence of cross-linked gelatin in Dacron did lead to an increased uptake of lysozyme and a delayed release during 30 h after implantation, whereas a burst release took place from Dacron, gas plasma-treated Dacron, or Dacron containing non-cross-linked gelatin.

Algorithms↗

PubMed2000

Subcutaneous abscess formation around catheters induced by viable and nonviable Staphylococcus epidermidis as well as by small amounts of bacterial cell wall components.

The use of catheters is often complicated by infection, mainly due to Staphylococcus epidermidis. Recently, a novel poly(vinylpyrrolidone)-grafted silicone elastomer catheter (SEpvp) was introduced. Less bacteria adhered to SEpvp than to conventional SE catheters in vitro. The frequency of S. epidermidis infection associated with SEpvp and SE was assessed in a rabbit model. Unexpectedly, abscesses were induced by the injection of low numbers of S. epidermidis along subcutaneously inserted SEpvp. No abscesses were seen around SE, even when very high numbers of S. epidermidis were injected. This bioincompatibility reaction observed around the SEpvp was independent of the host, bacterial strain, and method of inoculation. Abscesses were also induced by nonviable S. epidermidis and by bacterial cell wall components. Because these incompatibility reactions were not observed in the absence of bacteria, biocompatibility testing should include experiments in which the inflammatory effects of the combination of catheter and (non)viable bacteria are tested.

Abscess↗

PubMed2000

Biomaterial-associated persistence of Staphylococcus epidermidis in pericatheter macrophages.

Biomaterial surfaces may be modified to reduce bacterial adhesion. The susceptibility in mice to Staphylococcus epidermidis infection in tissue surrounding the commonly used catheter materials-silicon elastomer (SE), polyamide (PA), and their surface-modified polyvinylpyrrolidone (PVP)-grafted derivatives, SE-PVP and PA-PVP, respectively-was assessed. Abscesses developed around SE-PVP. Around SE, PA, and PA-PVP catheters, no signs of infection were observed, although mice carrying PA-PVP developed septicemia after 14-21 days. S. epidermidis was cultured from the tissue surrounding PA-PVP segments. Cells around PA-PVP segments containing large numbers of bacteria were identified as macrophages by use of immunohistochemistry and electron microscopy. This persistence of intracellular bacteria was also observed around SE-PVP, SE, and PA catheters, although to a lesser extent. The cytokine profiles around the 4 materials were different. Implanted biomaterial induces an inflammatory response favorable to the persistence of S. epidermidis. Intracellular persistence of bacteria inside macrophages may be a pivotal process in the pathogenesis of biomaterial-associated infection.

Animals↗

PubMed2000

In vivo compatibility and degradation of crosslinked gelatin gels incorporated in knitted Dacron.

Gelatin gels were applied to porous Dacron meshes with the aim of using these gels for local drug delivery. In this article, the biocompatibility and degradation of gelatin gels with different crosslink densities applied in Dacron were studied in vivo by subcutaneous implantation in rats. Dacron discs were treated with carbon dioxide gas plasma to improve hydrophilicity, and subsequently impregnated with gelatin type B. The gelatin samples were crosslinked to different extents using various amounts of water-soluble carbodiimide (EDC) and N-hydroxysuccinimide (NHS). After 6 h, 2, 5, and 10 days, and 3, 6, and 10 weeks of postimplantation, the tissue reactions and biodegradation were studied by light microscopy. The early reaction of macrophages and polymorphonuclear cells to crosslinked gelatin was similar to or milder than Dacron. Giant cell formation was predominantly aimed at Dacron fibers and was markedly reduced in the presence of a crosslinked gelatin coating. At week 10 of implantation, the crosslinked gelatin gels were still present in the Dacron matrix. The gelatin degradation was less for samples with the highest crosslink density. The gelatin gel with the lowest crosslink density showed clear cellular ingrowth, starting after 6 weeks of implantation. The intermediate and high crosslinked gelatin gels showed little or no ingrowth. In these gels, giant cells were involved in the phagocytosis of gelatin parts at week 10. Application of carbodiimide crosslinked gelatin gels in Dacron is suitable for medical applications because of the good biocompatibility of the gels and the possibility of adapting the degradation rate of gelatin to a specific application.

Amines↗

PubMed2000

In vitro and in vivo evaluation of gelatin-chondroitin sulphate hydrogels for controlled release of antibacterial proteins.

Chemically cross-linked gelatin-chondroitin sulphate (ChS) hydrogels, impregnated in Dacron, were evaluated as drug delivery systems for antibacterial proteins. The gelatin-chondroitin sulphate gels, plain or impregnated in Dacron, were cross-linked with a water-soluble carbodiimide (EDC) and N-hydroxysuccinimide (NHS). The release of lysozyme and recombinant thrombocidin (rTC-1), an antibacterial protein derived from human blood platelets, from the gelatin-ChS gels in Dacron in phosphate-buffered saline at 37 degrees C was determined, and compared to the release from gelatin gels in Dacron and plain gelatin-ChS gels. The incorporation of chondroitin sulphate into gelatin gels, caused a marked increase in lysozyme loading capacity, and a slower release rate. The relative release profiles for rTC-1 and lysozyme were equal for cross-linked gelatin as well as for cross-linked gelatin-ChS gels. Furthermore, rTC-1 showed no loss of antibacterial activity after 1 week of release. The lysozyme concentration profiles in the samples and in the surrounding medium as a function of time were calculated using mathematical solutions for Ficks second law of diffusion for a semi-infinite composite medium, which is a schematic representation of a slab in a surrounding medium. The biocompatibility and degradation of the Dacron matrices impregnated with gelatin-ChS gels was studied after implantation in subcutaneous pockets in rats. Chemically cross-linked gelatin-Ch5 gels showed a mild tissue reaction, and almost complete degradation within 18 weeks of implantation.

Animals↗

PubMed2000

Interferon-gamma protects against biomaterial-associated Staphylococcus epidermidis infection in mice.

Survival of Staphylococcus epidermidis inside macrophages has been recognized as a pivotal process in the pathogenesis of biomaterial-associated infection (BAI). Interferon (IFN)-gamma is a potent activator of macrophages. This study examined whether subcutaneous injections of IFN-gamma can reverse macrophage deactivation induced by implanted biomaterials. Mice received subcutaneous implants combined with an injection of 106 S. epidermidis to induce an experimental BAI. Subsequently, 3 groups of mice received subcutaneous injections of 25,000 IU IFN-gamma 3 times weekly, 10,000 IU IFN-gamma 3 times in 2 weeks, or saline 3 times weekly (saline control), respectively. A fourth group received no injections (control). Segments and tissues of the IFN-gamma-treated mice were significantly less (P<.05) culture positive than those of the control groups. Histologically, the high numbers of intracellularly persisting gram-positive cocci observed in the control mice were absent in the IFN-gamma-treated mice. These data indicate that IFN-gamma protects against experimental BAI.

Animals↗

PubMed2000

Antibacterial activity of antibiotic-soaked polyvinylpyrrolidone-grafted silicon elastomer hydrocephalus shunts.

If shunts, inserted for the relief of hydrocephalus, are pretreated with antimicrobials, the incidence of shunt-associated infections (SAI) may be reduced. The duration of the antibacterial activity of shunts, made from conventional silicon elastomer (SE) or from SE grafted with the hydrogel polyvinylpyrrolidone (SEpvp), which had been soaked in various antibiotics, was assessed in vitro. For any antibiotic or combination, using an arbitrary breakpoint (aBP), SEpvp remained antibacterially active for longer periods than SE. Bacterial adherence to either shunt was prevented during the period of antibacterial activity. Thus, the aBP is a good indicator of the capacity of antimicrobial-treated shunts to prevent bacterial colonization in vitro. Hydrogel-grafting of shunts may be useful in preventing SAI.

Anti-Bacterial Agents↗

PubMed2000

The effect of pseudomonas exotoxin A on cytokine production in whole blood exposed to Pseudomonas aeruginosa.

To determine the effect of Pseudomonas aeruginosa exotoxin A (P-ExA) on cytokine production, we studied cytokine release induced by heat-killed P. aeruginosa (HKPA) in human whole blood in the presence or absence of P-ExA. P-ExA (0.01-1 microgram ml(-1)) caused a dose-dependent decrease in HKPA-induced production of tumor necrosis factor alpha (TNF), interleukin (IL-) 10, IL-6 and IL-8 (all P<0.05). P-ExA-induced inhibition of IL-10, IL-6 and IL-8 release was not dependent on reduced TNF concentrations, since the relative attenuation of the production of these cytokines was similar in the presence or absence of a neutralizing anti-TNF antibody. The effect of P-ExA on cytokine production may offer a disadvantage to the host with respect to clearance of the infection.

ADP Ribose Transferases↗

PubMed2000

Broad-host-range shuttle vectors for screening of regulated promoter activity in viridans group streptococci: isolation of a pH-regulated promoter.

Viridans group streptococci are major constituents of the normal human oral flora and are also identified as the predominant pathogenic bacteria in native valve infective endocarditis. Little information is available regarding the regulation of gene expression in viridans group streptococci, either in response to changes in the oral environment or during development of endocarditis. We therefore constructed a set of broad-host-range vectors for the isolation of promoters from viridans group streptococci that are activated by specific environmental stimuli in vitro or in vivo. A genomic library of Streptococcus gordonii strain CH1 was constructed in one of the new vectors, and this library was introduced into a homologous bacterium by using an optimized electroporation protocol for viridans group streptococci. Because viridans group streptococci entering the bloodstream from the oral cavity encounter an increase in pH, we selected promoters upregulated by this specific stimulus. One of the selected promoter sequences showed homology to the promoter region of the hydA gene from Clostridium acetobutylicum, the expression of which is known to be regulated by the environmental pH. The isolation of this pH-regulated promoter shows that S. gordonii can sense an increase in the environmental pH, which serves as a signal for bacterial gene activation. Furthermore, this demonstrates the usefulness of these new selection vectors in research on adaptive gene expression of viridans group streptococci and possibly also of other gram-positive bacteria.

Base Sequence↗

PubMed2000

Interleukin-1 receptor type I gene-deficient mice are less susceptible to Staphylococcus epidermidis biomaterial-associated infection than are wild-type mice.

Elevated concentrations of interleukin-1 (IL-1) were found in tissue surrounding biomaterials infected with Staphylococcus epidermidis. To determine the role of IL-1 in biomaterial-associated infection (BAI), IL-1 receptor type I-deficient (IL-1R(-/-)) and wild-type mice received subcutaneous implants of silicon elastomer (SE) or polyvinylpyrrolidone-grafted SE (SEpvp), combined with an injection of 10(6) CFU of S. epidermidis or sterile saline. Neither mouse strain was susceptible to BAI around SE. IL-1R(-/-) mice with SEpvp implants had a no abscess formation and a reduced susceptibility to persistent S. epidermidis infection. The normal foreign body response, characterized by giant-cell formation and encapsulation, was delayed around SEpvp in wild-type mice but not in IL-1R(-/-) mice. This coincided with enhanced local IL-4 production in IL-1R(-/-) mice. These data suggest that inhibition of local IL-1 activity may be beneficial for the outcome of BAI.

Animals↗

PubMed2000

A shift from oral to blood pH is a stimulus for adaptive gene expression of Streptococcus gordonii CH1 and induces protection against oxidative stress and enhanced bacterial growth by expression of msrA.

Viridans group streptococci (VS) from the oral cavity entering the bloodstream may initiate infective endocarditis (IE). We aimed to identify genes expressed in response to a pH increase from slightly acidic (pH 6.2) to neutral (pH 7.3) as encountered by VS entering the bloodstream from the oral cavity. Using a recently developed promoter-screening vector, we isolated five promoter fragments from the genomic DNA of Streptococcus gordonii CH1 responding to this stimulus. No common regulatory sequences were identified in these promoter fragments that could account for the coordinate expression of the corresponding genes. One of the isolated fragments contained the promoter region and 5' end of a gene highly homologous to the methionine sulfoxide reductase gene (msrA) of various bacterial and eukaryotic species. This gene has been found to be activated in S. gordonii strain V288 in a rabbit model of IE (A. O. Kiliç, M. C. Herzberg, M. W. Meyer, X. Zhao, and L. Tao, Plasmid 42:67-72, 1999). We isolated and characterized the msrA gene of S. gordonii CH1 and constructed a chromosomal insertion mutant. This mutant was more sensitive to hydrogen peroxide, suggesting a role for the streptococcal MsrA in protecting against oxidative stress. Moreover, MsrA appeared to be important for the growth of S. gordonii CH1 under aerobic and anaerobic conditions. Both these properties of MsrA may contribute to the ability of S. gordonii to cause IE.

Amino Acid Sequence↗

PubMed2000

Enhanced susceptibility to subcutaneous abscess formation and persistent infection around catheters is associated with sustained interleukin-1beta levels.

A persistent Staphylococcus epidermidis infection in mice around a subcutaneous polyvinylpyrrolidone-grafted silicon elastomer catheter (SEpvp) but not around a conventional silicon elastomer catheter was observed. With SEpvp pericatheter tissue, protracted and exaggerated interleukin-1beta (IL-1beta) production was found. Apparently, sustained levels of IL-1beta are associated with enhanced susceptibility to biomaterial-associated S. epidermidis infection.

Abscess↗

PubMed2000

Altered gene expression in Staphylococcus aureus upon interaction with human endothelial cells.

Staphylococcus aureus is isolated from a substantial number of patients with infective endocarditis who are not known to have predisposing heart abnormalities. It has been suggested that the infection is initiated by the direct binding of S. aureus to human vascular endothelium. To determine the mutual response of the endothelial cells and the bacteria, we studied the interaction between S. aureus and human vascular endothelium. Scanning electron microscopic analyses showed that binding of S. aureus to human umbilical vein endothelial cells (HUVEC) mainly occurred via thread-like protrusions extending from the cell surface. Bound bacteria appeared to be internalized via retraction of the protrusions into newly formed invaginations of the endothelial cell surface. The growth phase of S. aureus had a major impact on the interaction with HUVEC. Logarithmically growing bacteria showed increased binding to, and were more readily internalized by, HUVEC compared to stationary-phase bacteria. To assess the bacterial response to the cellular environment, an expression library of S. aureus was used to identify genes whose expression was induced after 4 h of exposure to HUVEC. The identified genes could be divided into different categories based on the functions of the encoded proteins (transport, catabolism, biosynthesis, and DNA repair). Further analyses of five of the S. aureus transposon clones showed that HUVEC as well as human serum are stimuli for triggering gene expression in S. aureus.

Aspartic Acid↗

PubMed2000

Genotyping of Mycoplasma pneumoniae clinical isolates reveals eight P1 subtypes within two genomic groups.

Three methods for genotyping of Mycoplasma pneumoniae clinical isolates were applied to 2 reference strains and 21 clinical isolates. By a modified restriction fragment length polymorphism (RFLP) analysis of PCR products of the M. pneumoniae cytadhesin P1 gene, 5 subtypes were discriminated among 13 P1 type 1 strains and 3 subtypes were discriminated among 8 P1 type 2 strains. Sequence analysis of the 16S-23S rRNA gene spacer region and part of the 23S rRNA gene revealed one nucleotide difference in the intergenic spacer region in 3 of the 21 isolates. In the 23S rRNA gene sequence of the 8 P1 type 2 strains an extra adenosine was present, but it was absent from the 13 P1 type 1 strains. On the basis of M. pneumoniae genome sequence data, primers were designed to amplify large interrepeat fragments by long PCR, and these fragments were subsequently analyzed by RFLP analysis. Only two types, long PCR types 1 and 2, could be discriminated among the M. pneumoniae isolates. All P1 type 1 strains were assigned to long PCR type 1, and all P1 type 2 strains were assigned to long PCR type 2. These data obtained by three independent typing methods thus confirm the existence of two distinct M. pneumoniae genomic groups but expand the possibility of strain typing on the basis of variations within their P1 genes.

Adhesins, Bacterial↗

PubMed2000

Cross-linking and characterisation of gelatin matrices for biomedical applications.

Cross-linking of gelatin A and B with N,N-(3-dimethylaminopropyl)-N'-ethyl-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) was optimised by varying the NHS/EDC molar ratio at constant EDC concentration. Native and cross-linked gelatin gels were characterised using the degree of swelling, the number of free amine groups, the phase transition temperature, and titration of the carboxylic acid residues. The cross-linking reaction was most efficient at a NHS to EDC molar ratio of 0.2. At higher NHS/EDC molar ratios, the reaction of EDC with NHS becomes more pronounced, thereby reducing the effective amount of EDC for cross-linking. Swelling measurements of cross-linked gelatin gels gave deviating results when no NHS was used, which was explained by heterogeneous localisation of cross-links in the gelatin gel. The incorporation of undesired compounds into the gelatin gels during the cross-linking reaction was not observed. At optimal NHS to EDC molar ratio, gelatin A and B were cross-linked using increasing EDC/COOHgelatin molar ratios. A range of samples varying from very low cross-link density to very high cross-link density (at high EDC/COOHgelatin) was obtained. Stability of the gels is enhanced with increasing cross-link density, but a minimal cross-link density is required to obtain gelatin gels which are stable at 40 degrees C.

Animals↗