Cell-wall-associated proteins from Streptococcus equi subsp. equi, the causative agent of strangles, were analysed with a view to identifying a potential protective antigen. Preparations of these proteins, isolated from mutanolysin extracts of cell walls, were shown to contain one major high-M r protein species (apparent M r 220000 and 550000 when analysed by SDS-PAGE and gel-filtration chromatography, respectively). The high-M r protein bound horse fibrinogen and was purified under non-denaturing conditions using fibrinogen affinity chromatography. The fibrinogen-binding protein (FgBP) reacted with serum taken from horses recovering from strangles and protected mice against lethal challenge from S. equi subsp. equi. The sequence of the corresponding gene (fbp) was determined and shown to encode a mature protein (M r 54597) with predicted coiled-coil structure. An FgBP truncate, lacking the C-terminal cell wall/membrane anchor domain, was overexpressed in and purified from Escherichia coli and was shown to behave in an analogous fashion to the wild-type product in terms of M r estimation, fibrinogen binding and seroreactivity.

The sequence of the coagulase gene (coa) from Staphylococcus aureus strain 8325-4 is reported. The deduced amino acid sequence of the coagulase protein is compared with previously reported sequences of coagulases from strains 213 and BB. The secreted mature forms of coagulase proteins are composed of three distinct segments: (i) the N-terminal 150-270 residues, which are c. 50% identical, (ii) a central region with high (greater than 90%) residue identities, and (iii) a C-terminal region composed of repeated 27-amino-acid residue sequences. The variable N-terminal sequences are probably responsible for antigenic differences among coagulases of different serotype. The region of coagulase which binds to prothrombin and activates it to form staphylothrombin is also located in the N-terminal half of the protein. A site-specific substitution mutation in the coa gene, which abolished plasma clotting activity, was isolated by recombinational allele-replacement in strains 8325-4 and M60. The Coa- mutants did not show diminished virulence in subcutaneous and intramammary infections of mice. No evidence for a role for coagulase in virulence of toxigenic or nontoxigenic strains was obtained. This contradicts findings of several groups using Coa- mutants generated by chemical mutagenesis and suggests that the earlier results were obtained with strains that had suffered additional mutations in virulence-related genes.

The gene coding for protein A (spa) of Staphylococcus aureus 8325-4 has been inactivated by substituting part of the spa coding sequence for a DNA fragment specifying resistance to ethidium bromide. The in vitroconstructed spa::EtBrr substitution mutation was introduced into the S. aureus chromosome by recombinational allele replacement. Southern blot hybridization showed that the in vitro-constructed mutation was present in the chromosomal spa locus. We have previously reported the inactivation of the alpha-toxin gene (hly) by allele replacement with an in vitro-constructed hly::Emr (erythromycin resistance) mutation (M.

The effect of in-vivo administration of N-2-hydroxyethylpiperazine-N'-2- ethane sulphonic acid (HEPES) and taurine on rat paw oedema and reactive oxidant production was examined. Carrageenan-induced paw oedema was attenuated following intraperitoneal injection of HEPES. Chemiluminescence production by isolated peripheral blood mononuclear cells (PBMC) was reduced in HEPES-treated rats. Taurine-treated rats did not exhibit attenuation of paw oedema using subcutaneous or intraperitoneal administration but intracerebroventricular administration produced a significant reduction at a dosage of 4.0 mumol. No reduction in chemiluminescence production was observed by PBMC using subcutaneous or intraperitoneal administration of taurine, but intracerebroventricular administration produced a significant reduction at a dosage of both 0.4 and 4.0 mumol. Intravenous injection of [14C]HEPES or [3H]taurine demonstrated rapid clearance with a significantly longer half-life of HEPES compared with taurine. These results support previous reports of anti-inflammatory activity of taurine when administered centrally. The lack of anti-inflammatory effect when taurine was administered subcutaneously or intraperitoneally may be a consequence of rapid distribution or clearance. The greater anti-inflammatory effects of HEPES compared with taurine may be due to its slower distribution or clearance in-vivo.

Piezoelectric second generation lithotriptors are an established means of administering extracorporeal shockwave lithotripsy (ESWL) enabling treatment to be performed without anaesthesia or analgesia, but higher shockwave doses and multiple or staged treatment are frequently required. The bioeffects of this modality of ESWL, therefore, require further assessment. Seven experimental groups of adult male rabbits were treated using the EDAP LT.01 in order to determine the acute and chronic bioeffects of clinical dose, excess dose, divided excess dose, high frequency and multiple treatment (X10) piezoelectric shockwaves (PSW). Renal function was measured before and after treatment using mercaptoacetyltriglycine (MAG 3) scans. Gross and histological morphological changes were assessed at one and 30 days following application of PSW. Application of single clinical dose PSW was not associated with any significant functional or morphological renal injury. Excess dose PSW caused transient gross renal contusion, which resolved in the majority of animals with no persistent microscopic abnormality. Divided excess dose PSW resulted in no gross or microscopic damage. High frequency PSW was associated with mild histological abnormality. Multiple PSW treatments caused small discrete fibrotic lesions in all cases, without any change in renal function.

Decellularized porcine corneal scaffolds are a potential alternative to human cornea for keratoplasty. Although clinical trials have reported promising results, there can be corneal haze or scar tissue. Here, we examined if recellularizing the scaffolds with human keratocytes would result in a better outcome. Scaffolds were prepared that retained little DNA (14.89 ± 5.56 ng/mg) and demonstrated a lack of cytotoxicity by in vitro. The scaffolds were recellularized using human corneal stromal cells and cultured for between 14 in serum-supplemented media followed by a further 14 days in either serum free or serum-supplemented media. All groups showed full-depth cell penetration after 14 days. When serum was present, staining for ALDH3A1 remained weak but after serum-free culture, staining was brighter and the keratocytes adopted a native dendritic morphology with an increase (p < 0.05) of keratocan, decorin, lumican and CD34 gene expression. A rabbit anterior lamellar keratoplasty model was used to compare implanting a 250 μm thick decellularized lenticule against one that had been recellularized with human stromal cells after serum-free culture. In both groups, host rabbit epithelium covered the implants, but transparency was not restored after 3 months. Post-mortem histology showed under the epithelium, a less-compact collagen layer, which appeared to be a regenerating zone with some α-SMA staining, indicating fibrotic cells. In the posterior scaffold, ALDH1A1 staining was present in all the acellular scaffold, but in only one of the recellularized lenticules. Since there was little difference between acellular and cell-seeded scaffolds in our in vivo study, future scaffold development should use acellular controls to determine if cells are necessary.

No abstract

Decellularized porcine corneal scaffolds are a potential alternative to human cornea for keratoplasty. Although clinical trials have reported promising results, there can be corneal haze or scar tissue. Here, we examined if recellularizing the scaffolds with human keratocytes would result in a better outcome. Scaffolds were prepared that retained little DNA (14.89 ± 5.56 ng/mg) and demonstrated a lack of cytotoxicity by in vitro. The scaffolds were recellularized using human corneal stromal cells and cultured for between 14 in serum-supplemented media followed by a further 14 days in either serum free or serum-supplemented media. All groups showed full-depth cell penetration after 14 days. When serum was present, staining for ALDH3A1 remained weak but after serum-free culture, staining was brighter and the keratocytes adopted a native dendritic morphology with an increase (p < 0.05) of keratocan, decorin, lumican and CD34 gene expression. A rabbit anterior lamellar keratoplasty model was used to compare implanting a 250 µm thick decellularized lenticule against one that had been recellularized with human stromal cells. In both groups, host rabbit epithelium covered the implants, but transparency was not restored after 3 months. Post-mortem histology showed under the epithelium, a less-compact collagen layer, which appeared to be a regenerating zone with some α-SMA staining, indicating fibrotic cells. In the posterior scaffold, ALDH1A1 staining was present in all the acellular scaffold, but in only one of the recellularized lenticules. We conclude that recellularization with keratocytes alone may not be sufficiently beneficial to justify introducing allogeneic cells without concurrent treatment to further manage keratocyte phenotype.