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.

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.

Prevention of bone mineral loss in postmenopausal women by norethisterone.

The effect of norethisterone on bone mineral metabolism was examined in 43 postmenopausal women. A significant decline in serum calcium, phosphate, and alkaline phosphatase in urinary calcium/creatinine ratio and in tubular maximum reabsorption of phosphate was demonstrated. There was no alteration in urinary hydroxyproline/creatinine ratio. The bone mineral content measured over two years by single photon absorptiometry was compared in 20 patients receiving norethisterone and a matched control group receiving placebo. There was significant protection against bone loss in the norethisterone group.

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.

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.

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.

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.

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.

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.

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.

Replication rate of Pseudomonas aeruginosa in the murine lung.

Using a method recently developed at our laboratory, we determined the initial rate of Pseudomonas aeruginosa replication in the lung under different experimental conditions. Mice were exposed to aerosols containing mixtures of a temperature-sensitive (ts) mutant of P. aeruginosa and its parental wild type (wt). The changes in the ratio of ts:wt were determined by quantitatively culturing homogenates of lungs from animals sacrificed over different time periods. The doubling time (DT) was calculated as the reciprocal of the slope of the linear portion of the curve generated by plotting n = (log [r0/rt])/log 2 against time where r is the ratio of ts:wt at a given time. The DTs measured in both outbred ICR mice and F1 hybrids (DBA/2J X B10.D2/nSnJ) were 32 and 30 min, respectively. These DTs were higher than that determined in the peritoneal cavities of ICR mice (20 min). The DT in the lungs of ICR mice rendered granulocytopenic by treatment with cyclophosphamide was 16 min. Experiments performed with inocula of different sizes showed that DTs tended to be higher in animals aerosolized with low doses of the ts-wt mixture.

Live attenuated bacterial vaccines: new approaches for safety and efficacy.

Problems arising from reversion to virulence in genetically attenuated bacterial vaccines can be overcome by the combination, in one strain, of multiple temperature-sensitive mutations of identical phenotype. Immunogenicity of attenuated strains may be enhanced by incorporation of mutations which permit limited replication in the vaccinee (thereby increasing antigen mass while minimising the possibility of vaccine reactions) and the expression of genes coding for antigens which are synthesised only during infection of the host.

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.

Differential growth characteristics and immunogenicity of tight and coasting temperature-sensitive mutants of Pseudomonas aeruginosa.

Genetically attenuated vaccines capable of limited replication in the vaccinate may elicit stronger, longer-lasting immunity than that induced by component, killed whole-cell, or nonreplicating live vaccines. We have isolated and partially characterized temperature-sensitive Pseudomonas aeruginosa mutants of two different phenotypes: a tight mutant, which ceases all growth immediately after its transfer to 36 degrees C, and a coaster, which continues to replicate for five generations at 36 degrees C. The growth profiles of the two temperature-sensitive phenotypes were compared both in vitro and in vivo; maintenance of the coasting phenotype in vivo was confirmed. The immunogenicity of the two phenotypes was compared in two models. In model 1, ICR mice were immunized intraperitoneally (i.p.) with graded doses of either mutant and challenged 3 weeks later i.p. with lethal doses of the wild-type strain. In model 2, DBA/2J mice were immunized intranasally with either mutant and subsequently challenged with an aerosolized inoculum of the wild-type strain, and lung clearance was measured over 4 h. In both models, the coaster demonstrated slightly higher immunogenic potential and, in addition, induced significantly higher levels of immunotype-specific serum immunoglobulin G after i.p. immunization.

Specific pulmonary defences against Pseudomonas aeruginosa after local immunization with temperature-sensitive mutants.

The specificity of the enhancement in lung defences after local immunization of mice with three temperature-sensitive (ts) mutants of Pseudomonas aeruginosa was investigated. The three selected mutants display altered growth characteristics when transferred from 29 degrees C to mammalian body temperature. Mice immunized with the live ts mutants by aerosol exposure or multiple intranasal inoculations were challenged with aerosols containing wild-type (wt) P. aeruginosa. Aerosol immunization with ts mutant A/10/25 significantly enhanced the lung clearance of the wt but did not enhance the clearance of either Klebsiella pneumoniae or Staphylococcus aureus. Aerosol immunization with ts mutants D/1/8 or E/9/9 enhanced the lung defences against the parental wt (of identical immunotype 1) but not against immunotype 4; similarly, intranasal immunization enhanced the lung defences against the parental wt but not against immunotypes 4 or 5. We conclude that local immunization with ts mutants of P. aeruginosa enhances lung defences against the wt in a genus- and immunotype-specific fashion. It is suggested that local immunity may play a central role in immunoprophylaxis against P. aeruginosa lung infection.

Effect of chemotactins released by Staphylococcus aureus and Pseudomonas aeruginosa on the murine respiratory tract.

Staphylococcus aureus, the Pseudomonas aeruginosa temperature-sensitive (ts) mutant A/10/25, and the P. aeruginosa parental wild type were aerosolized to C5-deficient mice, and the total number of polymorphonuclear leukocytes (PMN) recovered by lung lavage was determined 4 h after aerosol exposure. S. aureus induced a slight but significant recruitment of PMN, as compared with the effect of a saline aerosol. Both wild-type P. aeruginosa and the ts mutant induced a significant PMN recruitment of a magnitude ca. 180 times higher than that produced by S. aureus. Gentamicin-killed ts P. aeruginosa induced a PMN recruitment of a magnitude similar to that produced by live ts P. aeruginosa. Thorough washing of the bacteria, however, removed ca. 90% of the chemotactic activity. Exposure of the animals to a ts P. aeruginosa culture supernatant aerosol induced significant PMN recruitment into the lower airways. The same culture supernatants were chemotactic for mouse PMN in a dose-dependent fashion when tested in vitro in the absence of serum. Culture supernatants of S. aureus exhibited weak chemotactic activity in vitro and did not induce PMN recruitment in the lungs when aerosolized to DBA/2J mice. The results suggest that chemotactins released by P. aeruginosa may be an important virulence factor and play a significant role in lung tissue damage.

Quantitative determination of bacterial replication in vivo.

A new methodology which permits the quantitative measurement of absolute bacterial replication in vivo is proposed. Mice were inoculated with mixtures of temperature-sensitive mutants and parental wild types, and the changes in the ratios of the two strains were measured. The number of wild-type generations was calculated from the declining ratios over time with the formula n = log (r0/rt)/log 2; n is the number of generations, and r0 and rt are the ratio of temperature-sensitive mutants to the parental wild type at time zero and at the times sampled throughout the experiment. The replication rate was determined by regression analysis. A mathematical argument for the formula is presented. Using this technique, we determined the mean generation times of Escherichia coli (33 min) and Pseudomonas aeruginosa (20 min) in the peritoneal cavities of mice, in the face of host clearance mechanisms during the first stages of infection.