Permeation of bioactive constituents from Arnica montana preparations through human skin in-vitro.

This study investigated and characterised transdermal permeation of bioactive agents from a topically applied Arnica montana tincture. Permeation experiments conducted over 48 h used polydimethylsiloxane (silastic) and human epidermal membranes mounted in Franz-type diffusion cells with a methanol-water (50:50 v/v) receptor fluid. A commercially available tincture of A. montana L. derived from dried Spanish flower heads was a donor solution. Further donor solutions prepared from this stock tincture concentrated the tincture constituents 1, 2 and 10 fold and its sesquiterpene lactones 10 fold. Permeants were assayed using a high-performance liquid chromatography method. Five components permeated through silastic membranes providing peaks with relative retention factors to an internal standard (santonin) of 0.28, 1.18, 1.45, 1.98 and 2.76, respectively. No permeant was detected within 12 h of applying the Arnica tincture onto human epidermal membranes. However, after 12 h, the first two of these components were detected. These were shown by Zimmermann reagent reaction to be sesquiterpene lactones and liquid chromatography/diode array detection/mass spectrometry indicated that these two permeants were 11,13-dihydrohelenalin (DH) analogues (methacrylate and tiglate esters). The same two components were also detected within 3 h of topical application of the 10-fold concentrated tincture and the concentrated sesquiterpene lactone extract.

Mechanical performance of polyurethane ureteral stents in vitro and ex vivo.

A serious problem associated with the use of ureteral stents is fracture in situ. Following clinical observations of fracture of polyurethane stents in vivo, this study examined the mechanical properties of 17 polyurethane stents (double-J containing drainage holes) retrieved from patients over a 24-week period of insertion. In addition, stents were immersed in human and artificial urine in an in vitro model at 37 degrees C to determine their general propensity to fracture. Mechanical properties of ureteral stents were examined using the standard ASTM D-412 tensile test and by the novel application of dynamic mechanical analysis (DMA). The ultimate tensile strength and elongation at break (but not the Young's modulus) of unused polyurethane stent sections containing side-drainage holes were greater than stent sections devoid of side-drainage holes. No correlations were observed between increased or decreased Young's modulus, ultimate tensile strength or elongation at break of polyurethane stents and their time of immersion in either human urine or artificial urine in simulated upper urinary tract conditions of 37 degrees C and 5% CO2. Similarly, no correlations were observed between Young's modulus, ultimate tensile strength or elongation of polyurethane stents and stent dwell time in situ. DMA of retrieved stents revealed that their tan delta value and storage modulus did not differ significantly from unused stents following dwell times in situ of up to 24 weeks. No changes in the glass transition temperatures were observed in retrieved stents. Although patient variation was observed, the results indicate that the polyurethane stents examined in vitro and following removal from patients did not exhibit any greater propensity to fracture than their unused counterparts. Fracture of retrieved polyurethane stents, arising in vivo and also during subsequent tensile testing, was observed to occur along the drainage holes, suggesting that elimination of these holes will reduce the incidence of polyurethane ureteral stent fracture in use.

Sequential polyurethane-poly(methylmethacrylate) interpenetrating polymer networks as ureteral biomaterials: mechanical properties and comparative resistance to urinary encrustation.

The mechanical properties and resistance to urinary encrustation of sequential-interpenetrating polymer networks (IPNs) composed of polyurethane (PU) and polymethylmethacrylate (PMMA), have been described. Mechanical properties were determined using tensile testing and dynamic mechanical analysis, whereas resistance to encrustation was examined using an in vitro model for encrustation simulating in vivo encrustation. Maximum and minimum tensile strength at break, Young's modulus, storage and loss moduli were associated with PMMA and PU, respectively. IPNs demonstrated intermediate mechanical properties which were dependent on the concentrations of the component polymers. Conversely, maximum elongation at break was observed for PU and this parameter decreased as the concentration of PMMA increased in the IPN. The dynamic mechanical damping parameter, tan delta, was similar for all IPNs at 37 degrees C. Increased advancing and decreased receding contact angles were observed for IPNs in comparison with the native PU. The rate and extent of encrustation, measured as the percentage surface coverage, was similar for PU, IPNs and PMMA. In contrast, encrustation on polyhydroxyethylmethacrylate, a model hydrogel, was greater than observed for the IPNs or component polymers. No apparent correlation was observed between the rate and/or extent of encrustation and polymer contact angle. It is concluded that these IPNs may be of clinical benefit in patients providing stent resistance to extrinsic compression of the ureter in comparison with native PU. The comparable resistance to encrustation between the IPNs and PU indicates that the use of IPNs should not be restricted in this regard.

Development of a model for assessment of biomaterial encrustation in the upper urinary tract.

A need exists for ureteral stent materials capable of preventing or reducing encrustation. The aim of this study, therefore, was to develop an in vitro model producing biomaterial encrustation similar to that on stents in vivo. Three models were designed and evaluated. Polyurethane stent sections were immersed in human urine (37 degrees C, 5% CO2): (1) with and (2) without crushed human kidney stone and (3) in an artificial urine (37 degrees C, 5% CO2). Encrustation of similar composition, as determined by infrared spectroscopy, X-ray diffraction and energy dispersive X-ray analysis, formed on stent materials in vivo, in artificial urine and in human urine with crushed kidney stone. Magnesium ammonium phosphate (struvite) and calcium phosphate (hydroxyapatite) predominated in all encrustations. The reproducibility and ease of use of the artificial urine model provided optimum encrustation assessment of materials presently used in ureteral stents and evaluation of novel biomaterials.

Characterization of biofilm and encrustation on ureteric stents in vivo.

OBJECTIVE: To examine the relationship between encrustation and microbial biofilm formation on indwelling ureteric stents. PATIENTS AND METHODS: Ureteric stents from 40 patients were examined for the presence of a microbial biofilm and encrustations. Bacteria in stent biofilms were isolated and identified. RESULTS: A profuse biofilm (> 10(4) c.f.u. cm-3) was identified on 11 (28%) stents. Enterococcus faecalis was the most common biofilm organism identified and Proteus spp. were not present. Encrustation was seen in 23 (58%) of stents and was not associated with the level of urinary calcium. The major risk factor for stent encrustation was the presence of urolithiasis. Importantly, there was no causative link between stent biofilm formation and encrustation. Both biofilm formation and encrustation increased with the duration of stenting. CONCLUSION: The results indicate that polyurethane is readily encrusted and colonized by bacteria in vivo despite antibiotic prophylaxis. Newer materials must be sought if effective long-term stenting is to be achieved.

Quaternary structure of the giant extracellular hemoglobin of the leech Macrobdella decora.

The molecular dimensions of the extracellular hemoglobin of the leech Macrobdella decora, determined by scanning transmission electron microscopy were 29.8 nm x 19.5 nm (diameter x height) for negatively stained specimens. Measurements of molecular mass (Mm) of unstained specimens with the microscope gave Mm = 3560 +/- 160 kDa. Small-angle X-ray scattering measurements gave a diameter of 28.0(+/- 0.5) nm, radius of gyration 10.5(+/- 0.2) nm and volume 7500(+/- 300) nm3. The hemoglobin had no carbohydrate and its iron content was found to be 0.23(+/- 0.02)% (w/w), corresponding to a minimum Mm of 24,000(+/- 1300) kDa. SDS/polyacrylamide gel electrophoresis of the unreduced hemoglobin showed that it consisted of three subunits, which have apparent Mm values of 12 (1), 25 (2) and 29 kDa (3). The reduced hemoglobin consisted of four subunits, I (12 kDa), II (14 kDa), III (26 kDa) and IV (30 kDa). Subunit 1 corresponded to subunit I, subunit 2 to subunits III and IV and subunit 3 to subunit II. Partial N-terminal sequences were obtained for subunit 1, the two chains of subunit 2 and one of the two chains of subunit 3, suggesting that the hemoglobin consists of at least five different polypeptide chains. The percentage fraction of the three unreduced subunits was determined by densitometry of SDS/polyacrylamide gel patterns and quantitative determination of Coomassie R-250 dye bound to the individual bands in reduced and unreduced patterns to be, monomer (subunit I) : non-reducible subunit (subunit 2) : reducible dimer (subunit 3) = 0.35 : 0.29 : 0.35 (S.D. = +/- 0.05). This corresponded to a stoichiometry of 74 +/- 11 : 37 +/- 5 : 38 +/- 6, assuming the molecular masses to be 17 kDa, 30 kDa and 34 kDa, taking into account the anomalously high mobility of annelid globins in SDS-containing gels. The stoichiometry calculated from the amino acid compositions of the hemoglobin and the three subunits was 82 +/- 12 : 29 +/- 4 : 40 +/- 8. Gel filtration of the hemoglobin at pH 9.8, at neutral pH subsequent to dissociation at pH 4 and at neutral pH in the presence of urea and Gu.HCl provided no evidence for the existence of a putative 1/12 of the whole molecule (Mm approx. 300 kDa). Furthermore, the largest subunits obtained had Mm of 60 to 100 kDa and had a much decreased content of subunit 2, suggesting that the hemoglobin was not a simple multimeric protein. Three-dimensional reconstruction from microscope images provided a model of Macrobdella hemoglobin that is very similar to the reconstruction of Lumbricus hemoglobin: the radial mass distribution curves are virtually superimposable.(ABSTRACT TRUNCATED AT 400 WORDS)

Meningococcal meningitis in children: clinical comparison of disease produced by the minor and major serologic groups of Neisseria meningitidis.

Since 1972, there has been an increased incidence of meningococcal disease due to the minor serologic groups, "serogroups," of Neisseria meningitidis. Few cases, however, have been reported in pediatric patients. We present 24 cases of meningococcal meningitis, ten of them (42%) due to serogroups X, Y, and Z N meningitidis. We believe these cases to be the largest group of pediatric patients with such meningitis thus far reported. The clinical disease produced by the minor serogroups was indistinguishable from that produced by the major serogroups. Our experience supports previous published reports that severe meningococcal disease does occur with the minor serogroups of N meningitidis, and this increasing incidence may be of major importance if vaccination programs are to be effective in controlling epidemic meningococcal disease.

Evaluation of the Cathra inoculating device for susceptibility testing of methicillin-susceptible and methicillin-resistant Staphylococcus aureus strains.

We compared the antimicrobial susceptibility results obtained with the Cathra replicator and reference methods when Staphylococcus aureus strains were tested by using agar dilution techniques. The Cathra replicator and the 0.001-ml calibrated loop gave results that fell within +/- 1 log2 dilution for greater than or equal to 95% of isolates when methicillin and cefamandole were tested.

Reliability of the MS-2 system in detecting methicillin-resistant Staphylococcus aureus.

The MS-2 system (Abbott Diagnostics, Division of Abbott Laboratories, Dallas, Tex.) is an automated system capable of rapid antimicrobial susceptibility testing. However, the short incubation periods used by the device may adversely affect its ability to detect slowly growing resistant organisms. Shortly after the introduction of the MS-2 system into the University of Mississippi Medical Center clinical microbiology laboratory, we noted discrepancies between the MS-2 and the disk diffusion susceptibility reports when methicillin-resistant Staphylococcus aureus isolates were tested. Subsequently, we determined the susceptibilities of 75 such isolates by the MS-2 and Kirby-Bauer disk diffusion methods and measured the minimum inhibitory concentrations of methicillin, oxacillin, and cephalothin for 33 of the 75 isolates by standardized agar dilution techniques. There was only 47% overall agreement between the MS-2 and disk diffusion methods when methicillin was tested and 15% agreement when cephalothin was the test drug. There was 93% or more overall agreement between the two methods when other antimicrobial agents were tested. The minimum inhibitory concentration of methicillin was greater than or equal to 16 micrograms/ml for all 33 isolates evaluated by the agar dilution method. A comparison of the MS-2 and agar dilution results revealed an overall agreement of 49% when the susceptibilities to methicillin were determined. The MS-2 system reported that multiple methicillin-resistant S. aureus isolates obtained from a single patient were either resistant, intermediate, or sensitive to methicillin. Inconsistent results were also obtained when a single isolate was tested simultaneously in 10 cuvette cartridges. We conclude that the MS-2 system does not reliably detect methicillin and cephalothin resistance among S. aureus.