Tissue viscoelasticity is related to tissue composition but may not fully predict the apparent-level viscoelasticity in human trabecular bone - An experimental and finite element study.

Trabecular bone is viscoelastic under dynamic loading. However, it is unclear how tissue viscoelasticity controls viscoelasticity at the apparent-level. In this study, viscoelasticity of cylindrical human trabecular bone samples (n=11, male, age 18-78 years) from 11 proximal femurs were characterized using dynamic and stress-relaxation testing at the apparent-level and with creep nanoindentation at the tissue-level. In addition, bone tissue elasticity was determined using scanning acoustic microscope (SAM). Tissue composition and collagen crosslinks were assessed using Raman micro-spectroscopy and high performance liquid chromatography (HPLC), respectively. Values of material parameters were obtained from finite element (FE) models by optimizing tissue-level creep and apparent-level stress-relaxation to experimental nanoindentation and unconfined compression testing values, respectively, utilizing the second order Prony series to depict viscoelasticity. FE simulations showed that tissue-level equilibrium elastic modulus (Eeq) increased with increasing crystallinity (r=0.730, p=.011) while at the apparent-level it increased with increasing hydroxylysyl pyridinoline content (r=0.718, p=.019). In addition, the normalized shear modulus g1 (r=-0.780, p=.005) decreased with increasing collagen ratio (amide III/CH2) at the tissue-level, but increased (r=0.696, p=.025) with increasing collagen ratio at the apparent-level. No significant relations were found between the measured or simulated viscoelastic parameters at the tissue- and apparent-levels nor were the parameters related to tissue elasticity determined with SAM. However, only Eeq, g2 and relaxation time τ1 from simulated viscoelastic values were statistically different between tissue- and apparent-levels (p<.01). These findings indicate that bone tissue viscoelasticity is affected by tissue composition but may not fully predict the macroscale viscoelasticity in human trabecular bone.

The development of TnNuc and its use for the isolation of novel secretion signals in Lactococcus lactis.

We have previously used Tn917 for the identification and characterization of regulated promoters from Lactococcus lactis [Israelsen et al., Appl. Environ. Microbiol. 61 (1995) 2540-2547]. We describe here the construction of a new Tn917-transposon derivative, termed TnNuc, which includes the Staphylococcus aureus nuclease gene (nuc) as a reporter for secretion. Transposition of TnNuc into the L. lactis chromosome allows the generation of fusions in-frame with the nuc gene. TnNuc includes also lacZ, a reporter used for identification of relevant clones from the library, i.e. clones with Lac+ phenotype result from transposition of TnNuc into a functional gene on the L. lactis chromosome. The presence of a functional signal sequence at the upstream flanking region of the left repeat of the transposed element results in the detection of nuclease activity using a sensitive plate assay. TnNuc was used for the identification of novel secretion signals from L. lactis. The sequences identified included known and unknown lactococcal-secreted proteins containing either a signal peptidase-I or -II recognition sequence. In one case, the gene identified codes for a transmembrane protein. The sequences identified were used to study functionality when located in a plasmid under the control of the pH and growth phase-dependent promoter P170 [Madsen et al., Mol. Microbiol. 32 (1999) 75-87]. In all cases, concurrent secretion of nuclease was observed during induction of P170 in a fermentor.

Bacterial colonization and healing of venous leg ulcers.

UNLABELLED: The aim of the study was to evaluate a possible influence of selected bacterial species on healing of venous leg ulcers. Fifty-nine patients with venous leg ulcers were followed via frequent semiquantitative culture of bacteria from the ulcer surface and determination of the ulcer area over a period of 180 days. Occurrences of cellulitis were treated with systemic antibiotics. There was a significant difference in relative areas on days 90 and 180 when ulcers with growth of Pseudomonas aeruginosa were compared to those without (p = 0.0080 and 0.0133, respectively). Ulcers with P. aeruginosa were characterized to a great extent by enlargement in contrast to those without. Ulcers with growth of Staphylococcus aureus or haemolytic streptococci healed significantly more slowly than those without when relative areas were compared on day 180 (p = 0.0079 and 0.0492, respectively). Complete healing within the observation period of 180 days was observed in 10.5% of patients with P. aeruginosa and 35% of those without (p = 0.0631), in 21.6% of patients with S. aureus and 62.5% of those without (p = 0.0278), and in 10.5% of patients with haemolytic streptococci and 35% of those without (p = 0.0631). The initial areas of ulcers colonized with P. aeruginosa or S. aureus were significantly larger than those without, but no significant correlation between initial areas and ulcer healing was revealed. CONCLUSION: Our results suggest that P. aeruginosa in venous leg ulcers can induce ulcer enlargement and/or cause a healing delay. The results also suggest a healing delay caused by S. aureus and haemolytic streptococci. However, conclusions have to be treated with caution since P. aeruginosa was found in combination with haemolytic streptococci in 15.3% of the patients.

Ulcer bed infection. Report of a case of enlarging venous leg ulcer colonized by Pseudomonas aeruginosa.

We report a case of ulcer bed infection in an enlarging venous leg ulcer without clinical signs of cellulitis in the surrounding tissues. Signs of infection in the leg ulcer were: 1) cocci-like structures and bacteria-like rods around vessel walls in the viable ulcer bed, 2) vasculitis-like inflammation of deeply situated vessels of the viable tissue, 3) Pseudomonas aeruginosa-specific antibodies in the serum (other than against exotoxin A), 4) extensive epidermolysis of normal human skin by the wound exudate in vitro, and 5) P. aeruginosa exotoxin A in the wound exudate (23 ng/ml). In an in vitro cell assay, the wound exudate was cytotoxic and rabbit antibodies to exotoxin A, but not a serine proteinase inhibitor, inhibited this cytotoxicity. P. aeruginosa exotoxin A might contribute to the pathogenesis of the ulcer enlargement. The ulcer improved after the third skin graft, probably mainly due to effective treatment with a long-stretch compression bandage.

Biochemical and ultrastructural changes in rabbit sclera after treatment with 7-methylxanthine, theobromine, acetazolamide, or L-ornithine.

AIMS: To examine a possible effect of 7-methylxanthine, theobromine, acetazolamide, or L-ornithine on the ultrastructure and biochemical composition of rabbit sclera. METHODS: Groups of pigmented rabbits, six in each group, were dosed during 10 weeks with one of the substances under investigation, and one untreated group was the control. Samples of anterior and posterior sclera were taken for determination of hydroxyproline, hydroxylysine, proline, proteoglycans, uronic acids and dermatan sulphate, chondroitin sulphate, and hyaluronic acid. Sections were examined with electron microscopy, and the diameter of the individual collagen fibrils was measured. RESULTS: Treatment with theobromine produced a significant increase in the contents of hydroxylysine, hydroxyproline, and proline in both anterior and posterior sclera, while 7-methylxanthine increased the contents of hydroxyproline and proline selectively in posterior sclera. Acetazolamide, on the other hand, significantly decreased the contents of hydroxyproline and proline in samples from anterior sclera. Uronic acids in both anterior and posterior sclera were significantly reduced by treatment with 7-methylxanthine, and L-ornithine significantly reduced uronic acids in posterior sclera. An inverse correlation between contents of hydroxyproline and uronic acids was found. The mean diameter of collagen fibrils was significantly higher in the posterior sclera from rabbits treated with 7-methylxanthine or theobromine, and significantly lower in rabbits treated with acetazolamide or L-ornithine compared with controls. In the anterior sclera, fibril diameter was significantly reduced in all treatment groups compared with controls. A positive, significant correlation between fibril diameter and content of hydroxyproline and proline was found in posterior sclera. CONCLUSION: 7-Methylxanthine, a metabolite of caffeine, increases collagen concentration and the diameter of collagen fibrils in the posterior sclera, and may be useful for treatment or prevention of conditions associated with low level and/or inferior quality of scleral collagen, such as axial myopia, chronic open angle glaucoma, and possibly neovascular age related macular degeneration. The apparent loss of collagen induced by chronic treatment with acetazolamide should be taken into consideration as a potentially harmful side effect. These results may indicate that scleral biochemistry and ultrastructure are influenced by the retinal pigment epithelium. One possible explanation is that the scleral fibroblasts which produce the collagen are sensitive to changes in the physiological electric field created by the retinal pigment epithelium.

Care of pressure sores: a controlled study of the use of a hydrocolloid dressing compared with wet saline gauze compresses.

An occlusive hydrocolloid dressing (Comfeel Ulcus) was compared with a conventional wet saline gauze dressing regarding the effect on ulcer cleansing and healing processes, experience of pain and the consumption of nursing time, in a controlled, randomized and partially single-blind study with parallel groups of long-stay patients with pressure sores. After a few weeks' treatment the relative decrease in ulcer areas with time was larger in the group treated with the hydrocolloid dressing. The difference was almost statistically significant at week 5 (p = 0.054) and definite at week 6 (p = 0.006). At week 6 the median remaining ulcer area in per cent of the initial area was 0% in the hydrocolloid dressing group and 31% in the group treated with saline gauze (p = 0.016). Analysis of the healing distribution function showed the hydrocolloid dressing to be more effective, although the overall difference was non-significant (p = 0.15). Care of the pressure sore took significantly less time with hydrocolloid dressings.

[Colchicine treatment of recurrent steroid-dependent pericarditis in a patient with post-myocardial-infarction syndrome (Dressler's syndrome)]. / Kolkicinbehandling af recidiverende "steroidafhaengig" pericarditis hos en patient med postmyokardieinfarktsyndrom (Dressler's syndrom).

The usual treatment of pericarditis consists of non-steroid anti-inflammatory agents. In cases where the symptoms and/or the pericardial effusion persist or progress, the disease can be arrested in the majority of cases by employing steroids. In some patients, it may prove difficult to conclude steroid treatment as gradual withdrawal results in recurrence and it may, therefore, be necessary to continue with large doses of steroid for prolonged periods. There is, however, a possibility for another form of treatment. The present authors present the case history of a patient with pericarditis on the basis of the post-myocardial-infarction syndrome and in whom the symptoms recurred several times during attempts at gradual withdrawal of prednisolone. Treatment with colchicine was commenced. The patient rapidly became symptom free and has now been symptom free for 45 weeks without prednisolone. Colchicine was withdrawn after 33 weeks without recurrence. It is considered that trial of colchicine treatment can be recommended in cases of recurring pericarditis particularly when there are problems (recurrence) on attempted withdrawal of steroid treatment. Naturally, it is important to exclude specific causes requiring other forms of treatment as the cause of the pericarditis (e.g. malignant disease, tuberculosis, systemic disease etc.).

[How detailed information and how long consideration time do the potential trial subjects want?]. / Hvor udførlig information og hvor lang betaenkningstid ønsker potentielle forsøgspersoner?

Questionnaires were sent to 508 citizens in Copenhagen county and given to 200 patients attending two out-patient clinics. Questions concerned attitudes and preferences towards either a short or a more detailed information form about an invented trial, and preferences regarding required time for reflection before giving informed consent. One sixth of responders were satisfied with the short form, while about three quarters preferred the long form. Three quarters of the non-hospitalized citizens and the patients from one of the out-patient clinics required at least 24 hours before answering about consent. Among responders in the other outpatient clinic half of the patients only required 15 minutes, or stated that they could answer at once. We conclude that written information to trial patients should be detailed, as a majority of potential research participants prefer this. At least 24 hours should be given before an informed consent is obtained.

[Healing of venous leg ulcers. A randomized prospective study of a long-stretch versus short-stretch compression bandage]. / Heling af venøse bensår. En randomiseret prospektiv undersøgelse af en langstraek- versus en kortstraek-kompressionsbandage.

The aim of the study was to compare the efficacy of two types of compression bandage with different degrees of elasticity on healing of venous leg ulcers. Forty-three patients with venous leg ulcers were included and blindly randomized to treatment with one of the two types of bandage aiming at a sub-bandage pressure in the ankle area of 40 mmHg. Forty legs were evaluated. Healed ulcers after 12 months were observed in 71% of the long-stretch group and in 30% of the short-stretch group (p = 0.06). Using life-table analysis the predicted healing rate in the long-stretch group after 12 months was 81% and for the short-stretch group 31% (p = 0.03). The mean of relative ulcer areas at 12 months was 0.25 for the long-stretch group and 0.95 for the short-stretch group (p = 0.01). The present study appears to indicate a positive influence of the elasticity of a compression bandage applied with a sub-bandage pressure around 40 mmHg in the ankle area on venous ulcer healing.

[Increased incidence of anorexia nervosa in Denmark]. / Oget incidens af anorexia nervosa i Danmark.

Register information from the Danish Central Psychiatric Register about 915 anonymous patients, who during the period 1970-1989 had their first admission to a psychiatric institution in Denmark and had an eating disorder as their main diagnosis, ICD 8 306.50-59, were studied with regard to change in incidence. The incidence of these admissions increased from 0.42/100,000 in 1970 to a maximum of 1.36/100,000 in 1988. For women 15-24 years of age the incidence increased three times to 8.97/100,000 in 1989. During the time of our investigation, the number of discharges of patients with an eating disorder as their main diagnosis was constant from somatic hospitals, whereas discharges in general from psychiatric institutions increased.

[Anorexia nervosa in Denmark--changes in diagnosis]. / Anorexia nervosa i Danmark--diagnoseskift.

Using information retrieved from the Danish Central Psychiatric Register, 1017 anonymous patients who had been admitted to Danish psychiatric institutions during 1968-1986 under a diagnosis of eating disorder ICD 8,306.50-59 were traced throughout their altogether 3419 admissions over the same time period with the aim of examining the stability of the initial diagnosis. Fifty-nine percent maintained the diagnosis of eating disorder. Diagnostic change was found in 22%. Nineteen percent had eating disorder registered only as a subsidiary diagnosis. Two-thirds were readmitted. Diagnostic changes were mainly to and from those of personality disorder or neurosis. Only six percent ended up as being diagnosed with a psychosis. The high frequencies of diagnostic changes and subsidiary diagnoses support the concept of eating disorder as a clinical syndrome with fluctuating symptoms.

[Penicillin treatment of non-nosocomial pneumonia in Danish medical departments. A questionnaire]. / Penicillinbehandling af ekstranosokomielt erhvervet pneumoni på medicinske afdelinger i Danmark. En spørgeskemaundersøgelse.

This study describes the most common penicillin-regime for hospitalized patients with community-acquired pneumonia in Denmark. One hundred and seventy-one consultant physicians received an anonymous questionnaire, 85% were returned and 75% were evaluated. The most common regime is a treatment duration of six to ten days with 3-6 million IU of penicillin daily given in three doses. The most common route of administration is intravenously until the patient's body temperature drops. Then the same dose is given orally. Variations in strategy were revealed and call for further investigations to establish and maintain restrictive antibiotic regimes.

[Mortality of anorexia nervosa in Denmark 1970-1987]. / Dødeligheden af anorexia nervosa i Danmark 1970-1987.

Eight hundred and fifty-three patients were admitted to psychiatric institutions in Denmark with anorexia nervosa between 1970 and 1986. Based on register information, 50 deaths were recorded during a mean follow-up period of 7.8 years. Amongst these, five were males and 45 females. The standardized mortality ratio (SMR) was 9.1 in both sexes. The SMR was maximal during the first year after index admission. Suicide was the dominant cause of death amongst subjects who died from unnatural causes (18 of 22 cases). Among those who died from natural causes (24 subjects), 13 individuals died from anorexia nervosa, and 11 individuals died from other illnesses.

[Incidence of eating disorders in psychiatric health services in Denmark 1970-1993]. / Incidens af spiseforstyrrelser i psykiatrisk sygehusvaesen i Danmark 1970-1993.

By means of data from the nationwide Danish Psychiatric Case Register all first-ever diagnosed patients with eating disorders in Danish psychiatric in- and day-patient services were identified for the period 1970-1993, comprising 1529 persons in all, with 92% being female. Based on age-specific and age-standardized rates the regression lines were calculated for various definitions of eating disorders and definitions of periods. All were found to be increasing significantly. The maximum increase in rate was found among 15-24 year-old females: 0.40 per 100,000 per year. No changes in incidence were found in males.

The extent of written trial information: preferences among potential and actual trial subjects.

AIM: To investigate the preferred extent of written information in clinical trials among potential and actual trial participants. MATERIALS AND METHODS: Questionnaire survey among citizens of Copenhagen County (PUB, N=508), patients attending an out-patient clinic (OPC, N=200), and finally among participants in two clinical trials (ROC, N=32; MRCRUC, N=47--see Abbreviations). Questions concerned attitudes to and preferences towards a relatively short ("short form") and a more detailed information form ("long form") about a hypothetical, but realistic trial. RESULTS: Approximately 1/8 of the respondents in PUB were satisfied with the "short form", whereas this was the case for approximately 1/6 of outpatients and 1/5 of actual trial participants. Regarding the "long form" approximately three quarters of respondents in all groups were satisfied. Outpatients as a whole were satisfied to a larger extent that respondents from the PUB trial concerning the "short form" (p=0.04). The "long form" was preferred by a little less than 4/5 of respondents in all groups. CONCLUSION: Written information to trial subjects should be detailed, as a majority of both potential and actual research participants prefers this, given the choice between two information forms of different extent on the same trial.

Participating in a cancer clinical trial? The balancing of options in the loneliness of autonomy: a grounded theory interview study.

Cancer patients asked to participate in a randomised trial including chemotherapy at two university centres and a satellite centre were interviewed about perceptions and experiences (14 trial participating and 15 trial declining patients). The central phenomenon was a constant, cautious balancing of personal options searching for maximised effect, personal safety, trust, confidence and being cared for. Almost all developed a treatment preference and this was decisive for choices. Trial participants strongly wished to get the experimental treatment perceived as superior. They felt their freedom of choice being limited by randomisation. In contrast, trial decliners almost all focused on graver adverse effects related to the experimental treatment. A trusting and confident doctor-patient relationship was valued strongly. Yet, most breast cancer patients treated at the two large centres experienced a general lack of personal trust, confidence and being taken care of. The major reason was patients meeting too many physicians perceived as incompetent and unprepared. In contrast, the ovarian cancer patients treated at the satellite centre were content and satisfied with the main reason being the low number of physicians who were perceived as prepared, empathetic and knowledgeable. All patients expressed a feeling of "loneliness of autonomy" lacking sufficient knowledge and other resources to make educated choices.

Attitudes towards clinical research among cancer trial participants and non-participants: an interview study using a Grounded Theory approach.

The attitudes of women patients with cancer were explored when they were invited to participate in one of three randomised trials that included chemotherapy at two university centres and a satellite centre. Fourteen patients participating in and 15 patients declining trials were interviewed. Analysis was based on the constant comparative method. Most patients voiced positive attitudes towards clinical research, believing that trials are necessary for further medical development, and most spontaneously argued that participation is a moral obligation. Most trial decliners, however, described a radical change in focus as they faced the actual personal choice. Almost no one got an impression of clinical equipoise between treatments in the trials, and most patients expressed discomfort with randomisation. A patient's choice to participate was mainly determined by whether the primary focus was on treatment effect or on adverse effects. Both knowledge about and feelings towards trials originated mostly from the media, although paradoxically the media were largely seen as untrustworthy. Mistrust was shown towards the pharmaceutical industry, and although most patients originally trusted that doctors primarily pursued the interest of patients, they did not trust the adequacy of doctors or industry in maintaining self-regulation. Thus, public control measures were judged to be essential.

Construction and characterization of a Lactococcus lactis strain deficient in intracellular ClpP and extracellular HtrA proteases.

A Lactococcus lactis strain deficient in both its major proteases, intracellular (ClpP) and extracellular (HtrA), was constructed and characterized. This strain, hereafter called clpP-htrA, could be obtained only by conjugation between a clpP donor strain and an htrA recipient strain in the NZ9000 context, allowing heterologous gene expression under the control of the NICE (nisin-controlled expression) system. The clpP-htrA double mutant showed both higher stress tolerance (e.g. high temperature and ethanol resistance) and higher viability than single clpP or htrA mutant strains. In addition, the secretion rate of two heterologous proteins (staphylococcal nuclease Nuc and Nuc-E7) was also higher in clpP-htrA than in the wild-type strain. This strain should be a useful host for high-level production and quality of stable heterologous proteins.

Quantitative determination of the gamma-aminobutyric acid agonist, 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol, in serum by high-performance liquid chromatography.

A method was developed to determine pharmacologically relevant concentrations of the gamma-aminobutyric acid agonistic compound THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol) in serum. THIP is extracted from serum by a cation-exchange column, derivatized by dansyl chloride, and further separated and quantitated by reversed-phase high-performance liquid chromatography using measurement of ultraviolet absorption at the optimal wave-length (265 nm). The lower detection limit of the method is 0.07 mumol 1(-1) (10 ng ml-1) when 2 ml of serum are used for extraction. This sensitivity is sufficient for pharmacokinetic studies in man following administration of a presumably therapeutic dose of THIP. Blood samples collected from 28 patients treated with one or more of 26 drugs relevant for coadministration with THIP did not contain substances which would affect the reliable quantitation of THIP, if present.