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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. [ABSTRACT FROM AUTHOR]

Background: To evaluate the use of low-field magnetic resonance imaging (MRI) in active inflammatory bowel disease (IBD). Methods: MRI was executed in a consecutive cohort of 28 patients with Crohn disease (CD) and in 17 with ulcerative colitis (UC) prior to glucocorticoid treatment (1 mg prednisolone orally/kg body weight/day). MRI was repeated after 2-3 weeks (22 CD, 12 UC), and again after treatment completion or prior to surgery (18 CD, 6 UC). Five bowel segments were evaluated separately. MRIs were blindly evaluated by two observers, and findings compared with 39 leucocyte scintigraphies, 38 endoscopies, 15 double-contrast barium enemas, 66 small-bowel radiographic examinations and surgery in 23 patients. Results: In CD, blinded evaluation revealed a kappa (κ) of 0.84 (95% confidence interval (CI) 0.78-0.91). In UC, κ was 0.66 (95% CI 0.55-0.78). Agreements regarding disease extension between MRI and other modalities in CD were found in 345 bowel segments out of 391 (88.2%) at risk, and in UC in 209/235 (88.9%). Colonic disease activity gradings by radiography and endoscopy correlated significantly with T2-signal intensity (SIT2) and increments in T1-signal intensity (%SIT1) in both diseases. Significant correlations between MRI indices of disease activity and CDAI in CD (MRI-SIT2: P < 0.0001; MRI% SIT1: P = 0.0008) and the Powell-Tuck index in UC (MRI% SIT1: P = 0.008) were found. Conclusions: With low interobserver variation and high concordance of findings with other examinations, low-field MRI seems a valuable modality in active IBD. In addition, MRI expressions of disease activity correlate to clinical, radiographic and endoscopic disease activity. [ABSTRACT FROM AUTHOR]

OBJECTIVE: The aim of this study was to compare the treatment efficacies of subcutaneous interferon-α-2A (IFN-α- 2A) injections versus prednisolone enemas in active left-sided ulcerative colitis in an open-labeled, randomized study. METHODS: Sixteen ulcerative colitis patients received IFN-α- 2A subcutaneously (dosage: first wk, 9 MIU three times weekly [t.i.w.]; second wk. 6 MIU t.i.w.; wk 3-12, 3 MIU t.i.w.), and 16 received prednisolone enemas for 30 days (100 ml once daily, 0.25 mg of prednisolone/ml). The Powell-Tuck Index, Inflammatory Bowel Disease Questionnaire (IBDQ) score, and rectal histological activities were assessed before and after treatment. Thirteen patients in the IFN-α-2A group and all 16 in the prednisolone enema group completed the treatment. RESULTS: IFN-α-2A treatment showed significant improvements in the Powell-Tuck Index (p = 0.0002), IBDQ score (p = 0.002). and rectal histological activity scores (p = 0.02). In the enema group, significant improvements were found in the Powell-Tuck Index (p = 0.0009), whereas no significant improvements were detected in the IBDQ scores (p = 0.055) or rectal histological scores (p = 0.052). There were no differences between scores of the two groups either before or after treatment. Only moderate side effects from the IFN-α-2A treatment were seen during the first 2-4 wk of treatment. CONCLUSION: lFN-α-2A treatment resulted in significant depression of the disease activity as reflected by the Powell-Tuck Index. IBDQ score, and histological disease activity scoring. The preliminary trial thus suggests that IFN-α-2A may be effective in the treatment of active left-sided ulcerative colitis. Larger, randomized trials are, however, warranted to confirm this finding, owing to possible type II errors in group comparisons. [ABSTRACT FROM AUTHOR]

Background: Patients suspected of having perianal suppurative disease often undergo a combination of several potentially painful, invasive procedures to establish or rule out the diagnosis. To evaluate the accuracy of low-field magnetic resonance imaging (MRI) in distinguishing patients with active anal fistulae and patients with no active fistulation we performed a retrospective study. Methods: Fifty-six patients suspected of having anal fistulation were evaluated in the surgical outpatient clinic. Patients were examined with low-field MRI (0.1 T; gradient echo technique, TR/TE 1500/40 and TR/TE 115/25, +/- gadodiamide (0.1 mM/kg intravenously)) in both coronal and axial planes, using a body quadrature coil. Altogether 71 MRIs were performed. In selected patients evaluation included endoanal ultrasonography, fistulography, and/or surgery. On the basis of the combined results of all available follow-up data for 6 months after the MRI, patients were placed in groups either having active fistulation or not. Results: MRI findings agreed with the combined findings of other examinations in 54 patients. Active or possibly active fistulae were found in 36 cases, whereas 33 patients showed no active fistulae. The kappa value is 0.944 (95% confidence limits, 0.866-1.021). In two patients the MRI findings disagreed with the combined findings of the other modalities. Conclusion: The use of low-field MRI of the pelvic region in the investigation of suspected perianal fistulae is a feasible, reliable, and painless examination. MRI should be considered in patients with suspected complex anal fistulae. Future prospective studies are warranted. [ABSTRACT FROM AUTHOR]

A patient with active Crohn disease was evaluated by MRI at admission, clinical remission, and a new relapse. The MRI-estimated disease extension correlated with surgical findings, whereas ultrasonography underestimated and a small bowel series overestimated the extension. MRI disclosed the disappearance of intestinal edema at the time of clinical remission and, in contrast to ultrasonography, showed an abscess and a fistula, confirmed by surgery, at the new relapse. [ABSTRACT FROM AUTHOR]

Examines the use of low-field magnetic resonance imaging (MRI) in crohn disease and ulcerative colitis. Assessment of activity in inflammatory bowel disease; Comparison of disease with non-disease bowel segments with segments from healthy controls; Characteristics of chronic inflammatory bowel disease.

The article focuses on the characteristics of a Lactococcus lactis strain deficient in intracellular clpP and extracellular HtrA proteases. The strain, called clpP-htrA, could be construction through the conjugation between a clpP donor strain and an htrA recipient strain in the ZN9000 context. It allows heterologous gene expression under the control of nisin-controlled expression system.

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 (E eq ) 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 g 1 (r=-0.780, p=.005) decreased with increasing collagen ratio (amide III/CH 2 ) 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 E eq , g 2 and relaxation time τ 1 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., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background: To evaluate the use of low-field magnetic resonance imaging (MRI) in active inflammatory bowel disease (IBD)., Methods: MRI was executed in a consecutive cohort of 28 patients with Crohn disease (CD) and in 17 with ulcerative colitis (UC) prior to glucocorticoid treatment (1 mg prednisolone orally/kg body weight/day). MRI was repeated after 2-3 weeks (22 CD, 12 UC), and again after treatment completion or prior to surgery (18 CD, 6 UC). Five bowel segments were evaluated separately. MRIs were blindly evaluated by two observers, and findings compared with 39 leucocyte scintigraphies, 38 endoscopies, 15 double-contrast barium enemas, 66 small-bowel radiographic examinations and surgery in 23 patients., Results: In CD, blinded evaluation revealed a kappa (kappa) of 0.84 (95% confidence interval (CI) 0.78-0.91). In UC, kappa was 0.66 (95% CI 0.55-0.78). Agreements regarding disease extension between MRI and other modalities in CD were found in 345 bowel segments out of 391 (88.2%) at risk, and in UC in 209/235 (88.9%). Colonic disease activity gradings by radiography and endoscopy correlated significantly with T2-signal intensity (SI(T2)) and increments in T1-signal intensity (%SI(T1)) in both diseases. Significant correlations between MRI indices of disease activity and CDAI in CD (MRI-SI(T2): P <0.0001: MRI% SI(T1): P=0.0008) and the Powell-Tuck index in UC (MRI% SI(T1): P=0.008) were found., Conclusions: With low interobserver variation and high concordance of findings with other examinations. low-field MRI seems a valuable modality in active IBD. In addition, MRI expressions of disease activity correlate to clinical, radiographic and endoscopic disease activity.

Objective: The aim of this study was to compare the treatment efficacies of subcutaneous interferon-alpha-2A (IFN-alpha-2A) injections versus prednisolone enemas in active left-sided ulcerative colitis in an open-labeled, randomized study., Methods: Sixteen ulcerative colitis patients received IFN-alpha-2A subcutaneously (dosage: first wk, 9 MIU three times weekly [t.i.w.]; second wk, 6 MIU t.i.w.; wk 3-12, 3 MIU t.i.w.), and 16 received prednisolone enemas for 30 days (100 ml once daily, 0.25 mg of prednisolone/ml). The Powell-Tuck Index, Inflammatory Bowel Disease Questionnaire (IBDQ) score, and rectal histological activities were assessed before and after treatment. Thirteen patients in the IFN-alpha-2A group and all 16 in the prednisolone enema group completed the treatment., Results: IFN-alpha-2A treatment showed significant improvements in the Powell-Tuck Index (p = 0.0002), IBDQ score (p = 0.002), and rectal histological activity scores (p = 0.02). In the enema group, significant improvements were found in the Powell-Tuck Index (p = 0.0009), whereas no significant improvements were detected in the IBDQ scores (p = 0.055) or rectal histological scores (p = 0.052). There were no differences between scores of the two groups either before or after treatment. Only moderate side effects from the IFN-alpha-2A treatment were seen during the first 2-4 wk of treatment., Conclusion: IFN-alpha-2A treatment resulted in significant depression of the disease activity as reflected by the Powell-Tuck Index, IBDQ score, and histological disease activity scoring. The preliminary trial thus suggests that IFN-alpha-2A may be effective in the treatment of active left-sided ulcerative colitis. Larger, randomized trials are, however, warranted to confirm this finding, owing to possible type II errors in group comparisons.

Three beta-galactosidase genes from Bifidobacterium bifidum DSM20215 and one beta-galactosidase gene from Bifidobacterium infantis DSM20088 were isolated and characterized. The three B. bifidum beta-galactosidases exhibited a low degree of amino acid sequence similarity to each other and to previously published beta-galactosidases classified as family 2 glycosyl hydrolases. Likewise, the B. infantis beta-galactosidase was distantly related to enzymes classified as family 42 glycosyl hydrolases. One of the enzymes from B. bifidum, termed BIF3, is most probably an extracellular enzyme, since it contained a signal sequence which was cleaved off during heterologous expression of the enzyme in Escherichia coli. Other exceptional features of the BIF3 beta-galactosidase were (i) the monomeric structure of the active enzyme, comprising 1,752 amino acid residues (188 kDa) and (ii) the molecular organization into an N-terminal beta-galactosidase domain and a C-terminal galactose binding domain. The other two B. bifidum beta-galactosidases and the enzyme from B. infantis were multimeric, intracellular enzymes with molecular masses similar to typical family 2 and family 42 glycosyl hydrolases, respectively. Despite the differences in size, molecular composition, and amino acid sequence, all four beta-galactosidases were highly specific for hydrolysis of beta-D-galactosidic linkages, and all four enzymes were able to transgalactosylate with lactose as a substrate.

The DNA sequence of the replication module, part of the lysis module, and remnants of a lysogenic module from the lytic P335 species lactococcal bacteriophage phi31 was determined, and its regulatory elements were investigated. The identification of a characteristic genetic switch including two divergent promoters and two cognate repressor genes strongly indicates that phi31 was derived from a temperate bacteriophage. Regulation of the two early promoters was analyzed by primer extension and transcriptional promoter fusions to a lacLM reporter. The regulatory behavior of the promoter region differed significantly from the genetic responses of temperate Lactococcus lactis phages. The cro gene homologue regulates its own production and is an efficient repressor of cI gene expression. No detectable cI gene expression could be measured in the presence of cro. cI gene expression in the absence of cro exerted minor influences on the regulation of the two promoters within the genetic switch. Homology comparisons revealed a replication module which is most likely expressed from the promoter located upstream of the cro gene homologue. The replication module encoded genes with strong homology to helicases and primases found in several Streptococcus thermophilus phages. Downstream of the primase homologue, an AT-rich noncoding origin region was identified. The characteristics and location of this region and its ability to reduce the efficiency of plaquing of phi31 10(6)-fold when present at high copy number in trans provide evidence for identification of the phage origin of replication. Phage phi31 is an obligately lytic phage that was isolated from commercial dairy fermentation environments. Neither a phage attachment site nor an integrase gene, required to establish lysogeny, was identified, explaining its lytic lifestyle and suggesting its origin from a temperate phage ancestor. Several regions showing extensive DNA and protein homologies to different temperate phages of Lactococcus, Lactobacillus, and Streptococcus were also discovered, indicating the likely exchange of DNA cassettes through horizontal gene transfer in the dynamic ecological environment of dairy fermentations.

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.

Assessment of disease activity in inflammatory bowel disease (IBD), i.e., ulcerative colitis (UC) and Crohn's disease (CD), is done using clinical parameters and various biological disease markers. Ideally, a disease marker must: be able to identify individuals at risk of a given disorder, be disease specific, mirror the disease activity and, finally, be easily applicable for routine clinical purposes. However, no such disease markers have yet been identified for IBD. In this article, classical disease markers including erythrocyte sedimentation rate, acute phase proteins (especially orosomucoid and CRP), leukocyte and platelet counts, albumin, neopterin, and beta2-microglobulin will be reviewed together with emerging disease markers such as antibodies of the ANCA/ASCA type, cytokines (e.g., IL-1, IL-2Ralpha, IL-6, IL-8, TNF-alpha, and TNF-alpha receptors) and with various adhesion molecules. It is concluded that none of the pertinent laboratory surrogate markers of disease activity in IBD are specific or sensitive enough to replace basic clinical observation such as the number of daily bowel movements, general well-being, and other parameters in parallel. Further studies are highly warranted to identify and assess the clinical importance and applicability of new laboratory markers for the diagnosis or the disease activity of IBD.

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.

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.

In a previous study, we described the use of transposon Tn917-LTV1 for identification of environmentally regulated promoters in Lactococcus lactis. Here, we report the molecular analysis of one of these promoters, P170, that is upregulated at low pH during the transition to stationary phase. The minimal DNA region required for both promoter activity and pH regulation was mapped to a 51 bp fragment located 7 bp upstream of the transcriptional start site. This fragment lacked the consensus -35 promoter region, but it contained an 'extended' -10 promoter region. When a 28 bp segment, containing the consensus -35 region and 22 bp upstream of this in a constitutive promoter, was replaced with the corresponding sequence of P170, the hybrid promoter became regulated by pH and growth phase. This demonstrates that the P170 segment contains a cis-acting sequence involved in the control of promoter regulation. Transcriptional analysis showed that P170 is responsible for the transcription of a monocistronic gene orfX encoding a polypeptide homologous to a hypothetical protein from Bacillus subtilis. Analysis of total RNA from L. lactis grown at constant pH confirmed that transcription from P170 was induced between pH 6.5 and pH 6.0, but only when the culture entered stationary phase. Deletion analysis and chemical mutagenesis of P170 defined a specific region within the untranslated mRNA leader that is able to modulate the expression level directed by the P170 promoter. Deletion of a 72 bp HaeIII fragment from this leader region resulted in a 150- to 200-fold increase in the level of gene expression, without affecting the regulation. The functionality was confirmed by introducing this modulating element downstream of other lactococcal promoters.

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.

The Lactococcus lactis adhE gene, which encodes a multifunctional alcohol dehydrogenase, has been cloned and characterized. A DNA fragment encoding the putative alcohol dehydrogenase domain of the AdhE protein was cloned by screening an L. lactis genomic library in a fermentative mutant of Escherichia coli and selecting for the ability to grow anaerobically. Further analysis of the clone obtained allowed the cloning of the entire adhE gene sequence. Analysis of adhE expression in L. lactis during anaerobiosis showed induction at the transcriptional level, especially in medium containing glucose. Constructed mutant strains produced reduced amounts of ethanol under anaerobic conditions. With the L. lactis gene as a probe, adhE homologs were found in other industrially relevant lactic acid bacteria.

The Lactococcus lactis pfl gene, encoding pyruvate formate-lyase (PFL), has been cloned and characterized. The deduced amino acid sequence of the L. lactis PFL. protein showed high similarity to those of other bacterial PFL proteins and included the conserved glycine residue involved in posttranslational activation of PFL. The genetic organization of the chromosomal pfl region in L. lactis showed differences from other characterized pfl loci, with an upstream open reading frame independently transcribed in the same orientation as the pfl gene. The gene coding for PFL-activase (act), normally found downstream of pfl, was not identified in L. lactis. Analysis of pfl expression showed a strong induction under anaerobiosis at the transcriptional level independent of the growth medium used. During growth with galactose, pfl showed the highest levels of expression. Constructed L. lactis pfl strains were unable to produce formate under anaerobic growth. Higher levels of diacetyl and acetoin were produced anaerobically in the constructed Lactococcus lactis subsp. lactis biovar diacetylactis pfl strain.

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.

Two genes, hom and thrB, involved in threonine biosynthesis in Lactococcus lactis MG1614, were cloned and sequenced. These genes, which encode homoserine dehydrogenase and homoserine kinase, were initially identified by the homology of their gene products with known homoserine dehydrogenases and homoserine kinases from other organisms. The identification was supported by construction of a mutant containing a deletion in hom and thrB that was unable to grow in a defined medium lacking threonine. Transcriptional analysis showed that the two genes were located in a bicistronic operon with the order 5' hom-thrB 3' and that transcription started 66 bp upstream of the translational start codon of the hom gene. A putative -10 promoter region (TATAAT) was located 6 bp upstream of the transcriptional start point, but no putative -35 region was identified. A DNA fragment covering 155 bp upstream of the hom translational start site was functional in pAK80, an L. lactis promoter probe vector. In addition, transcriptional studies showed no threonine-dependent regulation of hom-thrB transcription.

Transposon Tn917-LTV1 was used to produce a collection of Lactococcus lactis strains with fusion of a promoterless lacZ gene to chromosomal loci. Screening 2,500 Tn917-LTV1 integrants revealed 222 that express beta-galactosidase on plates at 30 degrees C. Pulsed-field gel electrophoresis revealed Tn917-LTV1 insertions in at least 13 loci in 15 strains analyzed. Integrants in which beta-galactosidase expression was regulated by temperature or pH and/or arginine concentration were isolated. In most cases, the regulation observed on plates was reproducible in liquid medium. One integrant, PA170, produces beta-galactosidase at pH 5.2 but not at pH 7.0, produces more beta-galactosidase at 15 degrees C than at 30 degrees C, and has increased beta-galactosidase activity in the stationary phase. DNA fragments potentially carrying promoters from selected Lactococcus lactis integrants were cloned in Escherichia coli. A new promoter probe vector, pAK80, containing promoterless beta-galactosidase genes from Leuconostoc mesenteroides subsp. cremoris and the Lactococcus lactis subsp. lactis biovar diacetylactis citrate plasmid replication region was constructed, and the lactococcal fragments were inserted. Plasmid pAK80 was capable of detecting and discriminating even weak promoters in Lactococcus lactis. When inserted in pAK80, the promoter cloned from PA170 displayed a regulated expression of beta-galactosidase analogous to the regulation observed in PA170.

We have studied the formation of spontaneous mutations on plasmids present in the monomeric and dimeric states in a recF strain of Escherichia coli. Two test systems were employed: (i) the precise excision of Tn5 from the tetA gene of the plasmid pBR322 and (ii) operator constitutive (Oc) mutations on the pBR322-derived plasmid pPY97. The rate of Oc mutations was increased by a factor of three when this plasmid was present in the dimeric state compared to the monomeric state and the Oc phenotype was caused by small deletions in the operator sequence. No apparent mutational hot-spot was found. The rate of Tn5 excision was increased on dimeric compared to monomeric plasmids. Excision from a dimeric plasmid usually resulted in two types of mutant plasmids; a dimeric plasmid, where the Tn5 had excised from one of the plasmid units, and a monomeric parental pBR322. A mechanisms to account for this is suggested. Complementation tests revealed that the increased mutation rate on dimeric plasmids is the result of dimers being mutaphilic per se, rather than the result of a general, trans-acting increase in mutation rates of the host, induced by the presence of the dimeric plasmid. Furthermore, it was found that the rate of Tn5 excision from plasmids in the monomeric state was increased when the region carrying the inserted Tn5 was duplicated.

In order for us to evaluate the efficiency of perforator vein surgery and skin grafting in leg ulcer patients, 47 patients were randomized into 3 treatment groups (group A: surgery for incompetent perforators, group B: surgery for incompetent perforators and ulcer excision followed by grafting, group C: control group). All the patients were treated with a compression bandage. When cellulitis was observed, a systemic antibiotic was given; eczema was treated with a steroid ointment. Fourty patients were evaluated regularly during one year after entry. There were no differences between the 3 treatment groups considering base-line characteristics, median ulcer size at entry and after one year. According to a review of the initial phlebograms, the occurrence of post-thrombotic changes in the deep veins were recorded in the majority of the legs. Our results suggest that ligation of incompetent perforators and skin grafting, as used in the present study, may not offer an additional advantage for venous ulcer patients with insufficiency of the deep veins when compared to conservative treatment. However, the removal of insufficient superficial veins was not studied.

The complete nucleotide sequence of the plasmid pTX14-3 from Bacillus thuringiensis subsp. israelensis has been determined. The circular DNA molecule was 7649 bp and had a G + C content of 35.1%. Twenty-two open reading frames larger than 50 codons were identified. Ten of these open reading frames are suggested to be protein coding regions. The existence of the polypeptides encoded by the mob14-3 and rep14-3 genes were verified by maxi-cells analysis in Escherichia coli. Even though the rep14-3 gene was expressed in E. coli the plasmid pTX14-3 was unable to replicate in this bacterium. The minimal region of the plasmid pTX14-3 required for replication in B. thuringiensis was identified. Potential secondary structures upstream of the rep14-3 gene indicated regulation by antisense RNA and transcription attenuation. Extensive sequence homology with the B. thuringiensis subsp. thuringiensis plasmid pGI2 was found in the last part of the mob14-3 gene, downstream of the rep14-3 gene, and in the region containing the single-strand origin of replication (i.e., the minus origin) of pTX14-3. A sequence of 700 bp containing multiple direct repeats was found in an ORF encoding a glycine and proline rich protein of 35.9 kDa. 1.2 kbp upstream and 0.1 kbp downstream of this ORF was found a large direct repeat of 230 bp (87% identity). The region between this direct repeat was often spontaneously deleted from plasmid derivatives containing the entire pTX14-3.

pTX14-3 is a 7.5-kb cryptic plasmid isolated from a Bacillus thuringiensis subspecies israelensis strain. Like many other small plasmids in gram-positive bacteria, pTX14-3 replicates via a single-stranded DNA intermediate. The nucleotide sequence of the replication region was determined and an open reading frame of 636 base pairs encoding a protein necessary for plasmid replication was identified by deletion analysis. No significant homology was found between this open reading frame and those encoding replication proteins identified on other plasmids isolated from gram-positive bacteria, nor could we find any homology to plus origins from other single-stranded DNA plasmids. Consequently, it seems that the replicon of pTX14-3 belongs to a new family of replicons in the group of single-stranded DNA plasmids. The sequence of the single-strand origin (i.e., the minus origin) responsible for the conversion of single-stranded plasmid DNA to double-stranded plasmid DNA was also determined. A partial homology between the minus origin of pTX14-3 and the Bacillus subtilis plasmid pBAA1 was identified. A previously identified locus that suppresses formation of high molecular weight multimers was also minimized and sequenced.

Bacillus thuringiensis subspecies israliensis plasmids pTX14-1 and pTX14-3 were cloned and analyzed by Southern blot hybridization for their replication mechanism in Bacillus subtilis. The cloning of pTX14-1 into the replicon deficient vector pBOE335 showed the usual characteristics of single-stranded DNA plasmids, i.e., it generated circular single-stranded DNA and high molecular weight (HMW) multimers. The other plasmid, pTX14-3, behaved differently; it generated neither single-stranded DNA nor HMW multimers. Treatment with rifampicin did not result in the accumulation of single-stranded DNA. However, deletion of an EcoRI-PstI fragment resulted in the accumulation of both single-stranded DNA and HMW multimers. From various deletion derivatives, we have mapped the minus origin and the locus responsible for suppression of HMW multimer formation. Full activity of the minus origin and of the locus suppressing HMW formation was only observed on the native replicon, indicating a coupling to the plus strand synthesis.

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.

Following intravenous bolus injection of theophylline 2.37 . 10(-) mol kg-1 to guinea pigs (administered as aminophylline 52.0 mg kg-1), the elimination of the dose was more rapid in a group of 8 guinea pigs that had received theophylline 2.02 . 10(-4) mol kg-1 (aminophylline 44.3 mg kg-1 (aminophylline 44.3 mg kg-1) intraperitoneally twice daily for 12 days prior to the experiment than in a group of 10 non-pretreated guinea pigs of the same age. The difference was statistically significant in Student's t-test (P less than 0.02). The mean values in the group of pretreated animals were: kel 0.00457 min.-1, beta 0.00296 min.-1, clearance 2.04 ml kg-1 min.-1, Vd beta 693 ml kg-1. By way of comparison, the values obtained in non-pretreated guinea pigs were: kel 0.00293 min.-1 beta 0.00198 min.-1, clearance 1.50 ml kg-1 min.-1, Vd beta 757 ml kg-1. This result suggests enzyme induction to occur. The pharmacologically active theophylline metabolite 3-methylxanthine did not accumulate in the plasma during the long-term theophylline administration. The general plasma concentration level was 0-1.8 . 10(-8) mol ml-1 (0-3 microgram ml-1). In 5 per cent of the samples were detected concentrations in the range 1.8 . 10(-8) mol ml-1 (3-12 microgram ml-1), but the time of occurrence was sporadic.

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.

Aminophylline in the doses 6.31 . 10(-5) mol kg-1 and 2.37 . 10(-4) mol kg-1 (corresponding to 13.8 and 52.0 mg kg-1 respectively) and 3-methylxanthine 2.37 . 10(-4) mol kg-1 (39.4 mg kg-1) was administered to guinea pigs by intravenous bolus injection, n = 7, 10, and 6, respectively. The shape of semilogarithmic plots of all measured plasma theophylline concentrations versus time was compatible with the use of an open 2-compartment pharmacokinetic model assuming first-order distribution and elimination processes, but in the comparison of the mean values of the pharmacokinetic parameters obtained after the administration of the low and the high theophylline dose, statistical analysis by Student's t-test showed beta and V1 to be significantly altered (low dose: beta = 0.00338 min.-1, V1 = 392 ml kg-a; high dose: beta = 0.00198 min.-1, V1 = 528 ml kg-1; P less than 0.05). Following the administration of 3-methylxanthine, the observed plasma concentration time course of this pharmacologically active theophylline metabolite could be adequately described be means of the 2-compartment open model. The administered 3-methylxanthine was eliminated unchanged with a first-order rate constant ten times larger than the total elimination rate constant of theophylline itself, the latter being observed after the administration of the equimolar dose of aminophylline (kel 3MX = 0.029 min.-1, kel theophylline = 0.00293 min.-1). Clearance was calculated to 14.4 ml kg-1 min.-1 for 3-methylxanthine and 1.50 ml kg-1 min.-1 for theophylline. When aminophylline 2.37 . 10(-4) mol kg-1 had been administered, 3-methylxanthine was renally eliminated at a constant rate for the first hours after the injection, but it did not only constitute a few per cent of the theophylline-derived urine products.

The pharmacokinetics of the gamma-aminobutyric acid (GABA) agonist, THIP (Gaboxadol) has been studied following intramuscular administration of 10 or 20 mg THIP-monohydrate to six healthy human volunteers. Additional experiments with special reference to the possible occurrence of non-linear kinetics and to estimation of the efficiency of absorption were carried out in three beagle dogs. As estimated from the dogs, absorption of THIP from the site of injection was efficient (F = 0.94 +/- 0.16, mean +/- S.D., in 8 experiments). The time course of the THIP serum concentration was in all experiments adequately described by an open 1-compartment linear model. In most individual experiments a near maximum concentration was present at 15 or 10 min. after the administration to man and dog, respectively. In some subjects and dogs an uncertain relation was found between dose and the time integral of the THIP serum concentration, but the mode of variation did not suggest deviations from first-order kinetics at high doses or concentrations. The rate constant of elimination was estimated to 0.50 +/- 0.13 hr-1 and 0.52 +/- 0.13 hr-1 following injection of a 10 or 20 mg dose, respectively, to man (mean +/- S.D., n = 6), and similar values were obtained from the dogs.

Simultaneous administration of terbutaline and theophylline to guinea pigs did not cause significant alterations of the pharmacokinetic properties of any of the drugs. Terbutaline sulphate 24 microgram kg-1 and aminophylline 52 mg kg-1 (7.42 . 10(-8) and 2.37 . 10(-4) mol kg-1 respectively) were given by a bolus intravenous injection, producing plasma concentrations in the range 0-15 ng terbutaline sulphate per ml (0-46 nanomol 1(-1)) and 10-85 microgram theophylline per ml (50-429 mumol 1(-1)). Pharmacokinetic analyses of time courses of plasma concentrations of intact drugs and investigations of tissue distribution 1 hour after the administration were performed. The results showed a weak, statistically insignificant trend of the peripheral compartment of the 2-compartment model to sequester a larger fraction of the drugs when these were administered simultaneously.