Mechanical and strain behaviour of human Achilles tendon during in vitro testing to failure.

The Achilles tendon is the strongest tendon in the human body but its mechanical behaviour during failure has been little studied and the basis of its high tensile strength has not been elucidated in detail. In the present study, healthy, human, Achilles tendons were loaded to failure in an anatomically authentic fashion while the local deformation and strains were studied in real time, with very high precision, using digital image correlation (DIC). The values determined for the strength of the Achilles tendon were at the high end of those reported in the literature, consistent with the absence of a pre-existing tendinopathy in the samples, as determined by careful gross inspection and histology. Early in the loading cycle, the proximal region of the tendon accumulated high lateral strains while longitudinal strains remained low. However, immediately before rupture, the mid-substance of the Achilles tendon, its weakest part, started to show high longitudinal strains. These new insights advance the understanding of the mechanical behaviour of tendons as they are stretched to failure.

The role of the posterior bundle of the medial collateral ligament in posteromedial rotatory instability of the elbow.

Aims: The aim of this study was to evaluate two hypotheses. First, that disruption of posterior bundle of the medial collateral ligament (PMCL) has to occur for the elbow to subluxate in cases of posteromedial rotatory instability (PMRI) and second, that ulnohumeral contact pressures increase after disruption of the PMCL. Materials and Methods: Six human cadaveric elbows were prepared on a custom-designed apparatus which allowed muscle loading and passive elbow motion under gravitational varus. Joint contact pressures were measured sequentially in the intact elbow (INTACT), followed by an anteromedial subtype two coronoid fracture (COR), a lateral collateral ligament (LCL) tear (COR + LCL), and a PMCL tear (COR + LCL + PMCL). Results: There was no subluxation or joint incongruity in the INTACT, COR, and COR + LCL specimens. All specimens in the COR + LCL + PMCL group subluxated under gravity-varus loads. The mean articular contact pressure of the COR + LCL group was significantly higher than those in the INTACT and the COR groups. The mean articular contact pressure of the COR + LCL + PMCL group was significantly higher than that of the INTACT group, but not higher than that of the COR + LCL group. Conclusion: In the presence of an anteromedial fracture and disruption of the LCL, the posterior bundle of the MCL has to be disrupted for gross subluxation of the elbow to occur. However, elevated joint contact pressures are seen after an anteromedial fracture and LCL disruption even in the absence of such subluxation. Cite this article: Bone Joint J 2018;100-B:1060-5.

Toxicity of heavy metals for microorganisms isolated from slow sand filter schmutzdecke.

This paper describes the susceptibility of three species of environmental bacteria isolated from the schmutzdecke of biologically active slow sand filters to cadmium, chromium and lead. The microorganisms, all identified as members of the genus Pseudomonas, were grown in tryptic soy broth with various concentrations of the selected heavy metals. The mean generation times (MGT) of the bacteria were compared to evaluate the toxic effects of the heavy metals. All three species tolerated high doses of heavy metals and the MGTs increased exponentially as the heavy metal concentrations increased; 12 mg l(-1) was the highest dose tested and the bacteria continued to grow albeit at very slow rates. In dilute media, the toxic effects of heavy metals were enhanced, illustrating the protection effect of organic matter and heavy metal complex formation. Growth studies of the isolated microorganisms on half-strength tryptic soy agar containing 6 mg l(-1) heavy metals also proved useful in determining toxicity.

Biomass characterization of slow sand filtration schmutzdecke and its effects on filter performance.

A new approach of representing biomass by measuring the Total Kjeldahl Nitrogen (TKN) content of sand in the schmutzdecke of slow sand filtration (SSF) was established by correlating TKN, Volatile Solids (VS) and Total Plate Count, a better way for the representation of biomass against the usual method by measuring VS. Reasonably good correlations were found between TKN and Chemical Oxygen Demand (COD) and Biochemical Oxygen Demand (BOD) of the extract from the schmutzdecke sand. The effects of the concentration of TKN on the efficiency of the SSF used as a tertiary unit of a wastewater treatment plant in removing BOD, COD, Ammonia Nitrogen (NH(3)-N) and Total Microorganisms have been evaluated and better performances of the filter have been observed at higher TKN concentration. The removal of NH(3)-N was almost proportional to TKN concentration further proving the importance of measuring the NH(3)-N in the effluent to identify the degree of ripening of the filter.

Gender differences in Ayrshire teenagers' attitudes to sexual relationships, responsibility and unintended pregnancies.

Disadvantages and risks related to teenage pregnancy, together with a recent increase in Scottish conception rates among teenagers aged 13-15 years old, give cause for concern. Using an illustrated short story and questionnaire, attitudes of 129 Ayrshire teenagers aged 14-15 years old were examined to teenage pregnancy, and more generally to early sex. Seventy-three percent of girls advocated joint responsibility for contraceptive protection compared with only 46% of boys (p<0.01). Also, significantly more boys than girls saw nothing wrong with casual sex (21%vs. 5%, p<0.01), and significantly less boys than girls upheld the virtue of commitment in sexual relationships (27%vs. 54%, p<0.01). Sex education programmes should explore these gender-related disparities.

Regulation of expression of the nonhemolytic phospholipase C of Burkholderia cepacia.

Burkholderia cepacia is an opportunistic pathogen that causes serious pulmonary infections in cystic fibrosis patients. We have purified and partially characterized one potential virulence factor for the organism-a nonhemolytic phospholipase C-and we studied the effect of iron restriction and choline and phosphate concentrations on the expression of phospholipase C. Iron limitation did not affect expression, the effect of choline was variable, and high phosphate concentrations repressed expression. Experiments with heat-treated spent culture supernatants suggested that autoinducers affected the expression of the phospholipase and two other potential virulence factors, a protease and a lipase. We screened 26 B. cepacia isolates for autoinducer activity: 11 induced violacein production in the autoinducer-deficient mutant Chromobacterium violaceum CV026. Spent supernatants from two strains, one that was positive in the C. violaceum assay and one that was negative, were tested for inducing early expression of phospholipase C, protease, and lipase in homologous and heterologous cultures. Expression of all three enzymes was increased or induced at an earlier stage in the growth curve in every case, suggesting not only that autoinducers were involved in the regulation of the expression of these enzymes, but also that the autoinducers were of two different classes.

A nonhemolytic phospholipase C from Burkholderia cepacia.

Burkholderia cepacia is an opportunistic pathogen that causes serious pulmonary infections in cystic fibrosis patients. Although several potential virulence factors-a protease, lipase, and two phospholipases C (one hemolytic and one nonhemolytic)-have been identified, only two, the protease and the lipase, have been described in detail. The goal of this study was to purify and characterize a nonhemolytic phospholipase C secreted by B. cepacia strain Pc224c. The enzyme was concentrated from culture supernatants and purified by polyacrylamide gel electrophoresis. The 54-kDa protein was stable in the presence of sodium dodecyl sulfate (up to 10%) and at 4 degrees, 22 degrees, and 37 degrees C; it was, however, inactivated at 100 degrees C. The enzyme bound to glass, chromatography matrices, and polyvinylidene difluoride and cellulose membranes, suggesting that it is hydrophobic. In a genetic approach, primers based on conserved sequences of a B. cepacia Pc69 hemolytic phospholipase C and both the Pseudomonas aeruginosa hemolytic and nonhemolytic proteins were designed to identify the Pc224c nonhemolytic phospholipase C gene. One polymerase chain reaction product was identified; it was sequenced and the sequence compared with sequences in the BLAST database. The best match was the Pseudomonas aeruginosa hemolytic phospholipase C. Ten additional B. cepacia strains were screened for the gene by Southern hybridization; five had the 4-kb band, suggesting that these strains have a similar form of the PLC gene. Nine of the ten strains reacted with the probe, suggesting that similar sequences were present, but in another form.

Attenuated mutants of Staphylococcus aureus with two temperature-sensitive lesions: isolation and characterization.

The feasibility of constructing attenuated mutants of Staphylococcus aureus with two temperature-sensitive (ts) lesions for ultimate development of a live-attenuated strain was investigated. Temperature-sensitive S. aureus strain G/1/2, which grows well at 31 degrees C but does not replicate at 37 degrees C, was subjected to chemical mutagenesis. After two enrichment cycles, fifteen mutants able to grow at 25 degrees C but unable to grow at 31 degrees C, were identified. Growth curves with temperature shifts from 25 to 31 degrees C, and from 31 to 37 degrees C confirmed that these were mutants with two lesions (dts), each with a different cut-off temperature. The reversion frequency of mutant G/1/2 at 37 degrees C was 2 x 10(-6) whereas those of several dts mutants were much lower (dts7: 7 x 10(-9) and dts12: 1 x 10(-9)). There was no increase in ts mutation reversion rate in response to prolonged incubation at 37 degrees C. The data support the further development of these mutants for use as a stable attenuated vaccine.

Eligibility criteria for NHS long stay care: the relationship between clinical need, dependency, and staff perception.

A study was undertaken by a Scottish Health Authority to determine future provision of NHS long stay in-patient beds for young physically disabled people (aged < 65), and eligibility criteria for admission to such care. As part of the development of care in the community, only patients requiring specialist medical and nursing care should continue to be placed in NHS care. Resources freed from the resulting closure of NHS beds will be transferred to Social Services to develop alternative packages of care in the community, based on need rather than precedent. Achieving the balance, in terms of the correct level of continuance of NHS long stay care and redeployment of resources, requires careful planning. This study, involving all young physically disabled patients in NHS care in Argyll and Clyde Health Board, combined the assessment of dependency using validated scales (CAPE, FIM, and ERSS), with staff perception of dependency and with clinical criteria developed for a series of balance of care studies in this authority. These clinical criteria indicate the need for specialist medical and nursing care. By examining the relationship between dependency and staff perception, it has been possible to plan long stay provision on a population basis. The criteria for admission have been adopted for local clinical use and form the basis for appeals procedures for patients deemed appropriate for discharge.

Temperature-sensitive mutants of Actinobacillus pleuropneumoniae induce protection in mice.

Temperature-sensitive mutants of Actinobacillus pleuropneumoniae 4074, serotype 1, were isolated after treatment with nitrosoguanidine and enrichment with penicillin and D-cycloserine. Of the four temperature-sensitive mutants evaluated in mice, one (A-1) had a tight phenotype (i.e., it ceased replication immediately after transfer to the nonpermissive temperature [37 degrees C]) and three (1-2, 4-1, and 12-1) were coasters that continued replication for up to three generations after transfer to 37 degrees C. The reversion frequencies ranged from 10(-6) to 10(-9), and cutoff temperatures ranged from 33 to 35 degrees C. No major changes were detected in the biochemical profiles; agglutination reactions; electrophoretic profiles of the lipopolysaccharides, outer membrane proteins, and hemolysin proteins; hemolytic titers; or CAMP factor reactions of the mutants and the wild-type bacteria. Groups of 3- to 5-week-old, female ICR mice were immunized intranasally with three doses of 3.5 x 10(6) CFU of the mutants over 3 weeks and subsequently challenged intranasally with 5 50% lethal doses of the parental wild-type. Protection was induced by both the tight and the coaster mutants, with the 4-1 and 12-1 coasters eliciting greater protection (67 and 82%, respectively) than that induced by the A-1 tight mutant (57%). Intranasal immunization with both phenotypes induced serum antibody responses against the surface antigens and the hemolysin protein.

Immunization with temperature-sensitive mutants of Actinobacillus pleuropneumoniae induces protective hemolysin-neutralizing antibodies in mice.

The immunogenicity and protective potential of three temperature-sensitive mutants of Actinobacillus pleuropneumoniae were evaluated in mice with respect to antibodies against the capsular polysaccharide, lipopolysaccharide, outer membrane proteins, and hemolysin protein. Antibodies to the capsular polysaccharide and lipopolysaccharide could not be correlated with protection in the mice; there were no significant differences among the anti-capsular and anti-lipopolysaccharide antibody titers regardless of the severity of infection. Sera from mice immunized with the mutants and challenged with the wild type contained antibodies that reacted in immunoblots to four major outer membrane proteins(66, 39, 29, and 16 kDa) regardless of the severity of infection after challenge. Both the tight and coaster mutants synthesized and secreted the 105-kDa hemolysin protein exotoxin in vitro and in vivo; hemolysin protein neutralization titers and the blotting intensity of the sera, however, varied inversely with the severity of infection. Sera from mice surviving challenge with little to no lung involvement stained the hemolysin band more intensely and had significantly higher neutralization titers (P < 0.05) than sera from mice that either died or survived with severe pulmonary hemorrhage. These results confirm the importance of the hemolysin in pathogenesis and the need for including it in any vaccine preparation.

Temperature-sensitive mutants of bacterial pathogens: isolation and use to determine host clearance and in vivo replication rates.

Temperature-sensitive mutants of bacterial pathogens are relatively easy to obtain and characterize. We have used ts mutants of a number of bacterial pathogens: E. coli and Listeria monocytogenes to determine quantitatively the bacterial activities in vitro of macrophages and polymorphonuclear leukocytes; E. coli and P. cepacia to study clearance of the bacteria in vivo; and Salmonella enteritidis, Listeria monocytogenes, E. coli, P. aeruginosa, and P. cepacia to determine quantitatively the replication rates in the spleens, lungs, and peritoneal cavities of mice, and the lungs of chickens.

Genetically stable temperature-sensitive mutants of Salmonella typhi induce protection in mice.

Temperature-sensitive (ts) mutants of Salmonella typhi were isolated following treatment with nitrosoguanidine, and characterized with respect to cut-off temperature, ts phenotype and reversion frequency. Linkage of the ts mutations to selectable chromosomal markers was established by generalized transduction with bacteriophage phage Vi I, and the appropriate antibiotic resistances were transduced into the ts mutants. Multiple mutant S. typhi were then constructed by combining three independent ts mutations in one strain, utilizing linkage of three of the mutations to erythromycin-, streptomycin- and methylglyoxal-resistance. Several recombinants are genetically stable, with calculated reversion rates of less than 10(-22), and induce both protection from intraperitoneal challenge with the virulent parental wild-type S. typhi in mice and the formation of antibodies to the somatic O-9 and O-12, the flagellar H and the capsular Vi antigens.

Vi I typing phage for generalized transduction of Salmonella typhi.

Salmonella typhi Vi typing phages were used to transduce temperature-sensitive (Ts) mutants of Salmonella typhi. Antibiotic resistance and Ts+ markers were transduced at high frequency (> 10(-4) per virulent phage). Several markers were cotransduced by phage Vi I, suggesting that it may be useful for mapping studies of the S. typhi genome.

Studies of safety, infectivity and immunogenicity of a new temperature-sensitive (ts) 51-1 strain of Salmonella typhi as a new live oral typhoid fever vaccine candidate.

This report describes the results of a phase 1 study evaluating the safety, infectivity, and immunogenicity of a new live oral Salmonella typhi temperature-sensitive (ts) 51-1 typhoid fever vaccine in the human. Three normal male subjects aged 23-32 years received three oral doses of S. typhi ts 51-1, each dose containing 10(9) organisms. Prior to and following immunization each subject was carefully monitored by clinical and laboratory parameters over a 2 week period during which serial specimens of blood and stool were analysed for the presence of the organism. Blood specimens were also obtained for the determination of serum antibody and cell-mediated immune responses and stool filtrates were analysed for the development of coproantibody. The results of these studies indicate that: (1) the vaccine is well tolerated with no clinical or laboratory evidence of adverse reactions; (2) ts 51-1 was detected in only one stool specimen from one volunteer; the organism recovered displayed characteristics of the ts 51-1 vaccine strain; and (3) although no significant humoral or cell-mediated lymphocytotoxic immune responses were detected in the blood, coproantibody was detected in stool specimens from all of the three immunized subjects and IgA-armed ADCC activity was detected in two of three subjects. These studies indicate that S. typhi ts 51-1 may be a suitable strain for the development of an improved oral typhoid fever vaccine. Studies are in progress to determine optimal methods of vaccine delivery preparatory to large phase 2 studies of efficacy.

Temperature-sensitive mutants ofStaphylococcus aureus: Isolation and preliminary characterization.

Temperature-sensitive (ts) mutants ofStaphylococcus aureus were isolated after mutagenesis with nitrosoguanidine and two cycles of enrichment with Penicillin G and D-Cycloserine. The mutants expressed tight, coasting, and leaky phenotypes on solid media. In broth, however, most exhibited coasting for a limited number of generations. The reversion frequency of selected ts mutants was less than 10(-6). Intraperitoneal (i.p.) immunization with ts mutant G/1/2 conferred significant protection (0 dead/6 total vs. 7/7, immunized vs. control; p=0.0006) from lethal i.p. challenge with the parental wild-type (wt)S. aureus suspended in 5% porcine mucin, performed 28 days after i.p. administration of 10(8) colony-forming units. Protection induced by mutants of coasting phenotype was higher and lasted longer than that induced by mutants of the tight phenotype. The results of this study demonstrate that ts mutants ofS. aureus can be obtained and that ts mutants are able to induce protective immunity from subsequent challenge with the parental wt strain.

The effect of liposomal cefoperazone against Pseudomonas aeruginosa in a granulocytopenic mouse model of acute lung infection.

The therapeutic efficacy of liposomal cefoperazone against Pseudomonas aeruginosa was investigated in a granulocytopenic mouse model of acute lung infection. Granulocytopenia was induced in mice by intraperitoneal (i.p.) injection of 200 mg/kg cyclophosphamide. Mice were challenged by exposure to an aerosol containing P. aeruginosa and were treated i.p. with liposomal cefoperazone prepared by the dehydration-rehydration method. The half-life of free cefoperazone in the lungs following i.p. administration of the liposomal drug was significantly lengthened (13 min vs. 261 min), and the cefoperazone activity in the lungs remained above the MIC longer after administration of liposomal cefoperazone than after treatment with cefoperazone. Liposomal cefoperazone was more effective than cefoperazone alone in preventing death of granulocytopenic mice from lethal pulmonary challenge with P. aeruginosa (75% vs. 38% survival, p = 0.031). Finally, P. aeruginosa was cleared faster from the lungs of mice treated with liposomal cefoperazone when compared with those treated with cefoperazone. This study shows that incorporation of cefoperazone into liposomes enhances the activity of the antibiotic against P. aeruginosa in a granulocytopenic host.

Oral immunization of mice with temperature-sensitive Pseudomonas aeruginosa enhances pulmonary clearance of the wild-type.

DBA/2J mice were immunized daily for 3 days per os with 10(8)-10(9) colony forming units (c.f.u.) of two different temperature-sensitive (TS) mutants of Pseudomonas aeruginosa. At varying times after the final immunization the animals were exposed to aerosols of the parental immunotype 1, and the ability of the immunized and control mice to clear their lungs of the wild-type (WT) challenge was measured 4 h later. The number of c.f.u. remaining in the lungs of mice immunized with one mutant, D/1/8, was significantly less (p less than 0.01) than the number remaining in the lungs of control mice and mice immunized with a second TS mutant, E/9/9.

Immunization with temperature-sensitive mutants of Salmonella typhi induces protection in mice.

Temperature-sensitive (TS) mutants of Salmonella typhi were isolated following mutagenesis with nitrosoguanidine and two cycles of enrichment with penicillin and D-cycloserine. Several of the TS mutants were characterized with respect to growth profiles at permissive (29 degrees C) and non-permissive (36 degrees C) temperatures, reversion rates, and the potential for inducing protection against challenge in an animal model. All three TS mutants tested were immunogenic in mice; antibodies measured by enzyme-linked immunosorbent assay were produced following intraperitoneal (i.p.) immunization with three different doses of each of the mutants; i.p. immunization with the same mutants also induced highly significant protection (100%) from i.p. wild-type challenge; and oral immunization with one of the mutants significantly reduced shedding of the wild-type following oral challenge.