Staphylococcus biofilm components as targets for vaccines and drugs.

Staphylococci have become the most common cause of nosocomial infections, especially in patients with predisposing factors such as indwelling or implanted foreign polymer bodies. The pathogenesis of foreign-body associated infections with S.aureus and S. epidermidis is mainly related to the ability of these bacteria to form thick, adherent multilayered biofilms. In a biofilm, staphylococci are protected against antibiotic treatment and attack from the immune system, thus making eradication of the infections problematic. This necessitates the discovery of novel prophylactic and therapeutic strategies to treat these infections. In this review, we provide an overview of staphylococcal biofilm components and discuss new possible approaches to controlling these persistent biofilm-dwelling bacteria.

Antibody response in patients with endocarditis caused by Staphylococcus aureus.

BACKGROUND: Staphylococcus aureus expresses a variety of adhesins involved in the colonization of host tissues. This study aimed to evaluate the role of staphylococcal surface proteins in the aetiology of infective endocarditis (IE) and the host immune response to infection. MATERIALS AND METHOD: The ELISA assays were used to assess the adherence of S. aureus isolates recovered from the blood cultures of 19 patients with IE (16 were drug abusers) to subendothelial matrix proteins. Anti-adhesin antibody titre was measured incubating surface-coated bacterial antigens with patients' IgG. S. aureus effects on platelet aggregation were evaluated with an aggregometer. RESULTS: Staphylococcus aureus isolates, from the patients with IE, exhibited a high expression of several surface components recognizing extracellular matrix proteins: clumping factors A and B (ClfA and ClfB) and fibronectin-binding proteins (FnbpA and FnbpB), whereas only four strains expressed the collagen-binding protein CNA. Bacteria also interacted with platelets both in the absence or presence of fibronectin or fibrinogen and some strongly supported platelet aggregation. Almost all patients presented significantly higher antibody reactivity to ClfA, ClfB, FnbpA, CNA and MAP (MHC class II analogous protein) than in sera from healthy individuals. On the contrary, the reactivity to CNA was remarkable only in three patients. The IgG preparations weakly inhibited the binding of bacteria to fibronectin, whereas they exhibited considerable blocking activity on staphylococcal attachment to fibrinogen or collagen. CONCLUSION: Adhesins ClfA, ClfB and FnbpA are produced in vivo and appear important factors both in valve colonization and in promoting host immune responses.

Monoclonal antibodies to CNA, a collagen-binding microbial surface component recognizing adhesive matrix molecules, detach Staphylococcus aureus from a collagen substrate.

Previous studies showed that Staphylococcus aureus expresses a collagen-binding MSCRAMM (Microbial Surface Component Recognizing Adhesive Matrix Molecules), CNA, that is necessary and sufficient for S. aureus cells to adhere to cartilage and is a virulence factor in experimental septic arthritis. We have now used a monoclonal antibody (mAb) approach to further analyze the structure and function of CNA. 22 mAbs raised against the minimal ligand binding domain, CNA-(151-318), were shown to bind to the MSCRAMM with similar affinity. All mAbs appear to recognize conformation-dependent epitopes that were mapped throughout the CNA-(151-318) domain using a chimeric strategy where segments of CNA are grafted on ACE, a structurally related MSCRAMM from Enterococcus faecalis. These mAbs were able to inhibit (125)I-collagen binding to CNA-(151-318) as well as to intact S. aureus cells. They also interfered with the attachment of bacteria to collagen substrates. Furthermore, some of the mAbs could effectively displace (125)I-collagen bound to the bacteria. These displacing mAbs were also able to detach bacteria that had adhered to a collagen substrate in a preincubation, raising the possibility that some of the mAbs may be used as therapeutic agents.

Routine immunofluorescence and light microscopy processing with a single renal biopsy specimen: 18 years' experience in a single centre.

To obtain a correct histological renal diagnosis, two adequate cortical biopsy cylinders are routinely taken for light microscopy and immunohistology in nephrology units. We describe our simple method for light microscopy (LM) study using the same frozen renal material used for immunofluorescence (IF). Of over 2,000 biopsies processed with this method, only three showed thawing artifacts and all occurred when the room temperature was above 30 degrees C. Our 18 years' experience and the large number of biopsies indicate that this method offers an interesting new way to do LM and IF with one biopsy core and could help stimulate etiopathogenic and diagnostic studies in nephrology. The other renal specimen can be used for histological investigation, applying recent molecular biology techniques (PCR, RT-PCR, in situ hybridization).

Heparin protection against Fe2+ -and Cu2+ -mediated oxidation of liposomes.

Heparin (HE) exhibited a protective effect on liposome peroxidation induced by Fe2+ and Cu2+, decreasing the formation of both conjugated dienes and thiobarbituric acid reactive substances (TBARS) in a dose-dependent manner. The antioxidant activity was more relevant in the oxidizing system employing Fe2+ and H2O2 and generating the highly reactive OH radical. The analysis of liposome size distribution by quasielastic laser light scattering showed that: (1) the native structure of the particles was completely lost after exposure to Fenton reagent; (2) the presence of HE in the reaction mixture completely prevented the peroxidative damage on liposomes. Thus, HE acts as an antioxidant factor on membrane lipid bilayer. This suggests that HE, released from mast-cell granules during inflammatory processes, might locally protect the cell membrane from the oxidative injuries.

The effect of cornea proteoglycans on liposome peroxidation.

Proteoglycans (PGs) from bovine cornea showed a protective effect on liposome peroxidation induced by Fe2+. Both chondroitin sulfate, dermatan sulfate-containing PG (CS,DS-PG) and keratan sulfate-containing PG (KS-PG) inhibited thiobarbituric acid-reactive substance formation when incubated with liposomes and Fe2+, CS,DS-PG being more effective than KS-PG. The native structure of PGs contributed markedly to antioxidant activity. Papain digestion of core protein reduced the protective effect of CS,DS-PG, whereas it abolished completely that of KS-PG. Apparently only hexuronate-containing glycosaminoglycan (GAG) chains may exert a significant antioxidant activity and this was confirmed using standard GAGs. Quasielastic laser light scattering was used to evaluate the structural consequence of peroxidative damage induced by Fenton reagent on liposomes. After exposure to the free-radical-generating system, a bimodal distribution of liposomes was observed, probably depending on the loss of native structure and fragmentation. Both CS,DS-PG and KS-PG prevented liposome breakdown. Again, free KS chains were ineffective against liposome damage, whereas DS and CS maintained the normal distribution of liposome size. These data support the hypothesis that PGs may represent part of the antioxidant mechanisms of organisms and suggest that modifications of PG content and/or composition might affect tissue sensitivity to oxidative stress.

The effect of heparin on Cu(2+)-mediated oxidation of human low-density lipoproteins.

The effect of heparin (HE) on the susceptibility of human low-density lipoprotein (LDL) to Cu(2+)-induced oxidation was investigated by monitoring conjugated diene formation. HE did not modify the maximum formation of conjugated diene, but increased markedly the lag phase. The plot of change in oxidation rate vs. time showed that the absolute value of Vmax was dependent on Cu2+ concentration and that HE increased the time necessary to reach Vmax. The value of constant K (the Cu2+ concentration producing a tlag of twice the minimum value) increased in the presence of HE, whereas the value of tmin (the time theoretically required for LDL oxidation at an infinite Cu2+ concentration) was not substantially affected. These results indicate that HE might play a protective antioxidant effect on LDL, probably affecting both the structural properties of the particle and the amount of Cu2+ available for the oxidation.

Collagenase-extractable proteoglycans from lesion-free areas of human aorta.

Different proteoglycan (PG) populations were isolated from normal human aorta by extraction of minced tissue with 4M GuHCl and by further digestion of the residue with collagenase. Dissociative extraction induced a complete disappearance of Alcian Blue positive material, which was demonstrable by transmission electron microscopy before the treatment around collagen fibrils and in pericellular areas. However, 4M GuHCl extraction solubilized only an average of 60% of aorta total hexuronate content. Collagenase treatment of the residue resulted in a complete loss of collagen fibril organization, which was coupled with a further hexuronate recovery, accounting for about one third of total tissue content. The bulk of PGs obtained in collagenase digest was retained by Sepharose CL-4B column. Their sulphated glycosaminoglycan (GAG) composition differed from PGs extracted with 4M GuHCl, containing only chondroitin sulphate (CS) and heparan sulphate (HS), without detectable traces of dermatan sulphate (DS). Moreover, they contained hyaluronic acid. The results obtained by agarose polyacrylamide gel electrophoresis (APGE) and Octyl-Sepharose chromatography, followed by further APGE and Sepharose CL-4B gel-filtration, carried out before and after treatment with Chondroitinase ABC and AC and Heparinase I and III, suggested that collagenase digest contained different PG populations, carrying mainly either CS or HS chains. Moreover, HS containing PGs showed higher hydrodynamic size and stronger properties of hydrophobic interactions than CS containing PGs.

Modifications of proteoglycans produced by human skin fibroblast cultures during replicative senescence.

The properties of proteoglycans (PGs) produced by normal human skin fibroblasts were investigated with increasing passage. The increase of subculture number was associated with a constant increase in PG molecular size, which was particularly evident in cell layer extracts. In the cell layer, the ratio of DS-PGs/HS-PGs was markedly higher in early passage cultures. Moreover, the cell layer from young cells contained lower amounts of radioactivity incorporated into the most hydrophobic PG populations, suggesting that the PG core protein might also undergo significant modification with increasing subcultures. There was no significant difference in energy charge value between early and late passage cultures, whereas the NAD/NADH ratio was found to decrease markedly in senescent cells.

Modifications of adhesion properties and proteoglycan structure in rat embryo fibroblast cultures with increasing passages.

Adhesion properties of rat embryo fibroblast cultures and proteoglycans (PGs) produced both in the growth medium and in the cell layer were investigated with increasing passages. Both cell-cell and cell-substrate adhesion increased with increasing subculture number. Cell adhesion properties were improved by cell treatment with chondroitinase ABC. The increase in subculture number was coupled with a constant increase of PG molecular size, which was particularly evident in cell layer extracts. The ratio HS-PGs/DS-PGs increased with increasing passages. PG modifications are likely to represent evidence of changes in extracellular matrix organization and could play a role in the increase of cell adhesion properties.

Effect of oxygen tension and lactate concentration on keratan sulphate and chondroitin sulphate biosynthesis in bovine cornea.

Calf cornea slices were incubated with [U-14C]glucose, in varying pO2 or lactate concentrations. Acid glycosaminoglycans were separated by ion-exchange chromatography after papain digestion. The percentage radioactivity incorporated into keratan sulphate increased markedly with decreased oxygen tension, whereas a concomitant relative decrease of the biosynthesis of glycosaminoglycuronans occurred. Similar results were obtained with increased lactate concentration. Our findings support the idea that keratan sulphate is a functional substitute for chondroitin sulphate in conditions of oxygen lack (Scott, J.E. and Haigh, M. (1988) J. Anat. 158, 95-108).

Biosynthesis of glycosaminoglycan precursors: evidences for different tissue specific forms of UDP-glucose dehydrogenase.

UDP-glucose dehydrogenase (UDPGDH) was extracted and partially purified from different rat tissues and the kinetic parameters and some properties of the enzyme were determined and compared. The pH optimum ranged between 8.6 and 9.4 for liver and kidney UDPGDH and between 8.4 and 8.6 for skin and lung UDPGDH. Liver and kidney enzymes showed a similar affinity for both UDPG and NAD. Lung and skin enzymes also showed similar affinity for both substrates, which differed however from that of liver and kidney UDPGDH. Both liver and kidney enzymes had a higher heat stability and a different electrophoretic mobility compared to skin and lung UDPGDH. These data suggest the existence of different tissue specific forms of the enzyme.

Pseudoxanthoma elasticum (PXE): ultrastructural and biochemical study on proteoglycan and proteoglycan-associated material produced by skin fibroblasts in vitro.

Pseudoxanthoma elasticum is a genetic disease characterized by progressive mineralization of elastic fibers. Previous studies suggested that other components, apart from elastin, might be involved in the alterations of this connective tissue disorder (Martinez-Hernandez and Huffer, 1974; Pasquali Ronchetti et al., 1981; 1986). Evidence is presented that proteoglycan metabolism is altered in PXE-affected patient. Urinary GAGs suggests an increased degradation of glucosamine-containing GAGs in the patient. Pulse and chase experiments on in vitro skin fibroblasts indicated a decreased rate of synthesis of [35SO4] containing GAGs or an increase of their turnover rate in PXE. Moreover, when PGs produced from skin fibroblasts were identified by ultracentrifugation and gel filtration in associative conditions, PXE fibroblasts produced a significantly higher amount of the high molecular weight fraction of sulfated PGs. This high molecular weight material was present both in the medium and in the matrix and disappeared under dissociative conditions or after treatment with hyaluronidase or with pancreas elastase. By electron microscopy, PXE fibroblasts appeared to produce and secrete an enormous amount of toluidine blue 0 positive material organized as filaments and amorphous masses. These data are in agreement with previous observations of the presence of abnormal masses of microfilaments, in the dermis of PXE patients, which were sensitive to hyaluronidase and partially to trypsin and elastase (Pasquali Ronchetti et al., 1986). The results seem to confirm that at least some of the alterations of connective tissues in PXE are due to abnormal PGs metabolism and to their tendency to form abnormal aggregates in the extracellular space.

Regulatory mechanisms of UDP-glucuronic acid biosynthesis in cultured human skin fibroblasts.

Kinetic parameters and regulatory properties of UDPGDH extracted from cultured human skin fibroblasts were determined and compared with those of UDPGDH from cornea and epiphysial-plate cartilage. Fibroblast enzyme showed an affinity for UDPG 7 times higher than cartilage enzyme and 42 times higher than cornea enzyme. UDP-xylose acted as a co-operative allosteric inhibitor, but under the same experimental conditions fibroblast enzyme was significantly less inhibited. These results were in agreement with the different GAG production of the cells we studied. Fibroblast UDPGDH activity was regulated by the NAD/NADH ratio and it was also affected by modifications of extracellular matrix composition. A significant increase of UDPGDH affinity for UDPG was observed after the treatment of the monolayers with Chase ABC.

The effect of extracellular matrix modifications on UDP-glucose dehydrogenase activity in cultured human skin fibroblasts.

The effect of modifications of the extracellular matrix on the biosynthesis of glycosaminoglycans was investigated in human skin fibroblast cultures by studying UDPGDH activity in order to evaluate: a), the histoenzymological and biochemical modifications induced by chondroitinase ABC treatment (new experimental conditions were developed in order to obtain minimum cell damage); b), the reversibility of these modifications; c), the effect of growing the cells in the presence of chondroitinsulfate; d), the specificity of the modifications induced. The results demonstrated that our experimental conditions specifically affected intracellular UDPGDH activity. Chondroitinase ABC treatment induced a reversible increase of UDP-glucuronic acid synthesis. On the contrary, the presence of chondroitinsulfate in the growth medium completely inhibited UDPGDH activity.

Proteoglycan modifications in cultured osteogenesis imperfecta skin fibroblasts.

The PGs produced in the growth medium by skin fibroblast cultures from two O.I. affected patients were investigated. After density gradient centrifugation, in the most dense fraction two main families of molecules appeared. The patient with the more severe clinical picture showed a lower content of the PGs with the highest molecular weight. The GAG composition of PGs was different in the two patients. The more severely affected one showed an increase of HS and a decrease of ChS content, in agreement with the lower value of galactosamine to glucosamine ratio in urinary GAGs.

Biochemical changes in the articular cartilage of the patella and femoral condyles in the lateral hyperpatellar syndrome.

The biochemical changes in the articular cartilage of the femoral condyles and the patella were studied during the earliest stage of patello-femoral arthrosis, with particular reference to chondromalacia of the patella in the "lateral hypertension syndrome", with the object of comparing them with the results obtained by Boni et al. (1977) in the initial phases of experimental arthrosis induced in rabbits by means of vitamin A. The biochemical determinations were done on samples of cartilage removed at operation from the medial and lateral femoral condyles and the medial and lateral patellar articular facets in ten patients. The biopsy samples were fixed in 80% alcohol and dried in the oven at 50 degrees C for twenty-four hours. The hexosamine and hydroxyproline content was then determined. This investigation demonstrated significant biochemical changes in this syndrome in the four areas examined. The data obtained indicate that only the mucopolysaccharide component of the joint cartilage is involved at this stage. The most interesting finding was marked diminution in the galactosamine/glucosamine ratio, which was markedly diminished - the opposite of what occurs in established arthrosis. These data appear to be identical with those found in the early phases of vitamin A induced arthrosis in experimental rabbits. The diminution of hexosamine content and the diminished galactosamine/glucosamine ratio were more marked in the femoral condyles, which appeared to have few lesions macroscopically. These changes were also present in both articular facets of patella, but were less marked, probably because the degeneration was more advanced in those areas, thus more closely resembling frank arthrosis.