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An integrated membrane system, producing high-quality industrial water from canal water, suffered severely from membrane biofouling. The full-scale plant (comprising: coagulation, moving bed filtration, ultrafiltration and reverse osmosis) has a capacity of 1750 m³/d. It is equipped with three reverse osmosis units and is located in Klazienaveen, the Netherlands. Fouling of the reverse osmosis membranes manifested as a frequent and rapid increase in pressure drop across the feed- brine channel of the first stage. Frequent chemical cleaning (CIP; cleaning in place) was therefore needed to prevent damage to the membranes. To reduce the cleaning frequency, a research project was conducted with the following objectives: controlling biofouling by dosing peracetic acid (PAA) and DBNPA and evaluating a newly developed in-line biofouling monitor, which monitors biofouling in an early stage. This in-line monitor continuously measures the increase in pressure drop across the feed-concentrate channel of the first element of the first stage in a unit. The in-line biofouling monitor demonstrated that the increase in head loss occurred almost exclusively in the first element and could be accurately measured. Biofouling was controlled by intermittently dosing PAA and DBNPA to two different units. The third unit was used as reference. The results of these efforts were: Intermittent dosing of PAA and DBNPA successfully controlled biofouling. It led to a reduction in the cleaning frequency from 12 times per year (and almost weekly in the summer) to once every three to four months; No increase in salt passage was observed, which could be attributed to the use of PAA or DBNPA. [ABSTRACT FROM AUTHOR]

Objective Although community-based studies reported an increased incidence of depression among demented persons compared with non-demented persons, it is not clear whether this relationship also exists among institutionalised elderly persons. The aim of this study was to compare the prevalence of diagnosed depressive disorders and mood symptoms between demented and non-demented residents living in Dutch homes for the elderly. Methods Cross-sectional analysis in 16 homes for the elderly of routine outcome measurements by trained nurse assistants using the Resident Assessment Instrument (RAI) between January 2007 and April 2008. Nurse assistants recorded all known medical diagnoses including dementia and depression, as well as a structured observation of the presence or absence of 11 mood symptoms over the last 3 days. Results 313 demented and 463 non-demented residents with complete data were included (99% of all residents, mean age 84 years). 24.6% of participants were diagnosed with a depressive disorder, with no statistically significant difference between demented and non-demented persons (p = 0.237). Mood symptoms were more prevalent in demented residents (p < 0.001, OR 2.14, 95%CI 1.56-2.93). Among residents with mood symptoms, demented residents were less likely to be diagnosed with a depressive disorder than non-demented residents (p = 0.039, OR 0.61, 95%CI 0.38-0.98). Conclusions The prevalence of diagnosed depressive disorders was comparable between demented and non-demented residents. However, demented residents suffered more from mood symptoms and may be at risk of under-diagnosis of depression. Copyright © 2009 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Propranolol, pindolol, practolol and metoprolol were investigated for β-adrenoceptor blocking activities in the guinea-pig right atrium and tracheal strip preparation, both in the absence and presence of 2.8% bovine serum albumin. Similarly, the influence of 2.8% albumin on the antagonism of ouabain-induced arrhythmias in the guinea-pig right atrium was studied. Local anesthetic activities were measured on the isolated, partially desheathed, frog sciatic nerve. Incubation with albumin decreased the in vitro atrial β-adrenoceptor blocking potency of propranolol against (-)-isoprenaline-induced positive chronotropism by a factor of 12 whereas the activity on tracheal receptors was 4.5 times lower. The activities of pindolol, practolol and metoprolol both on atrial and tracheal β-receptors were not significantly influenced by the presence of albumin. The results demonstrate the important role of albumin binding in causing differences in the in vivo and in vitro cardiac β-adrenoceptor blocking potency ratios of propranolol compared to the other β-receptor antagonists studied. The large difference between β-adrenoceptor blocking and minimal antiarrhythmic concentrations, the approximately 1000 times lower antiarrhythmic activity of practolol compared to propranolol and the very significant correlation between the antiarrhythmic and local anesthetic activities demonstrate that the antagonism of ouabain-induced arrhythmias in the isolated right atrium of the guinea pig is solely due to the 'membrane-stabilizing' properties of the β-receptor antagonists. The moderate decrease of the antiarrhythmic potency of propranolol by 2.8% albumin was concluded to have no relevance for the in vivo situation. Binding to 2.8% albumin was investigated using the ultracentrifugation technique. For propranolol a linear relationship was found between log concentration and percent binding which amounted to 88.2% at 10 M. In contrast, protein binding of pindolol, practolol and metoprolol was low and concentration independent. [ABSTRACT FROM AUTHOR]

The effect of increasing intravenous doses of theophylline and enprofylline, a new xanthine derivative, on bronchial responsiveness to methacholine was studied in eight asthmatic patients. Methacholine provocations were carried out on three days before and after increasing doses of theophylline, enprofylline, and placebo, a double blind study design being used. Methacholine responsiveness was determined as the provocative concentration of methacholine causing a fall of 20% in FEV1 (PC20). The patients were characterised pharmacokinetically before the main study to provide an individual dosage scheme for each patient that would provide rapid steady state plasma concentration plateaus of 5, 10, and 15 mg/l for theophylline and 1.25, 2.5, and 3.75 mg/l for enprofylline. Dose increments in the main study were given at 90 minute intervals. FEV1 showed a small progressive decrease after placebo; it remained high in relation to placebo after both drugs and this effect was dose related. Methacholine PC20 values decreased after placebo; mean values were higher after theophylline and enprofylline than after placebo (maximum difference 2.0 and 1.7 doubling doses of methacholine); the effect of both drugs were dose related. Thus enprofylline and theophylline when given intravenously cause a small dose related increase in FEV1 and methacholine PC20 when compared with placebo. [ABSTRACT FROM PUBLISHER]

Abstract Background The budesonide/formoterol combination is successfully used for fast relief of asthma symptoms in addition to its use as maintenance therapy. The temporarily increased corticosteroid dose during increasing inhaler use for symptom relief is likely to suppress any temporary increase in airway inflammation and may mitigate or prevent asthma exacerbations. The relative contribution of the budesonide and formoterol components to the improved asthma control is unclear. Methods The acute protective effect of inhaled budesonide was tested in a model of temporarily increased airway inflammation with repeated indirect airway challenges, mimicking an acute asthma exacerbation. A randomised, double-blind, cross-over study design was used. Asthmatic patients (n = 17, mean FEV1 95% of predicted) who previously demonstrated a ≥30% fall in forced expiratory volume in 1 second (FEV1) after inhaling adenosine 5'-monophosphate (AMP), were challenged on four consecutive test days, with the same dose of AMP (at 09:00, 12:00 and 16:00 hours). Within 1 minute of the maximal AMP-induced bronchoconstriction at 09:00 hours, the patients inhaled one dose of either budesonide/formoterol (160/4.5 μg), formoterol (4.5 μg), salbutamol (2 × 100 μg) or placebo. The protective effects of the randomised treatments were assessed by serial lung function measurements over the test day. Results In the AMP provocations at 3 and 7 hours after inhalation, the budesonide/formoterol combination provided a greater protective effect against AMP-induced bronchoconstriction compared with formoterol alone, salbutamol and placebo. In addition all three active treatments significantly increased FEV1 within 3 minutes of administration, at a time when inhaled AMP had induced the 30% fall in FEV1. Conclusions A single dose of budesonide/formoterol provided a greater protective effect against inhaled AMP-induced bronchoconstriction than formoterol alone, both at 3 and at 7 hours after inhalation. The acute protection against subsequent bronchoconstrictor stimuli such as inhaled AMP and the rapid reversal of airway obstruction supports the use of budesonide/formoterol for both relief and prevention in the treatment of asthma. Trial Registration ClinicalTrials.gov number NCT00272753

Abstract Background The objective of this article is to describe the design of a study to evaluate the clinical and economic effects of a Disease Management model on functional health, quality of care and quality of life of persons living in homes for the elderly. Methods This study concerns a cluster randomized controlled clinical trial among five intervention homes and five usual care homes in the North-West of the Netherlands with a total of over 500 residents. All persons who are not terminally ill, are able to be interviewed and sign informed consent are included. For cognitively impaired persons family proxies will be approached to provide outcome information. The Disease Management Model consists of several elements: (1) Trained staff carries out a multidimensional assessment of the patients functional health and care needs with the interRAI Long Term Care Facilities instrument (LTCF). Computerization of the LTCF produces immediate identification of problem areas and thereby guides individualized care planning. (2) The assessment outcomes are discussed in a Multidisciplinary Meeting (MM) with the nurse, primary care physician, nursing home physician and Psychotherapist and if necessary other members of the care team. The MM presents individualized care plans to manage or treat modifiable disabilities and risk factors. (3) Consultation by an nursing home physician and psychotherapist is offered to the frailest residents at risk for nursing home admission (according to the interRAI LTCF). Outcome measures are Quality of Care indicators (LTCF based), Quality Adjusted Life Years (Euroqol), Functional health (SF12, COOP-WONCA), Disability (GARS), Patients care satisfaction (QUOTE), hospital and nursing home days and mortality, health care utilization and costs. Discussion This design is unique because no earlier studies were performed to evaluate the effects and costs of this Disease Management Model for disabled persons in homes for the elderly on functional health and quality of care. Trail registration number ISRCTN11076857

Many initiatives integrating health and social care have been implemented in order to provide adequate care and support to older people living at home. Further development of existing initiatives requires iterative processes of developing, implementing and evaluating improvements to current practice. This case study provides insight into the process of improving an existing integrated care initiative in the Netherlands. Using a participatory approach, researchers and local stakeholders collaborated to develop and implement activities to further improve collaboration between health and social care professionals. Improvement activities included interprofessional meetings focussing on reflection and mutual learning and workplace visits. Researchers evaluated the improvement process, using data triangulation of multiple qualitative and quantitative data sources. According to participating professionals, the improvement activities improved their communication and collaboration by establishing mutual understanding and trust. Enabling factors included the safe and informal setting in which the meetings took place and the personal relationships they developed during the project. Different organisational cultures and interests and a lack of ownership and accountability among managers hindered the improvement process, whereas issues such as staff shortages, time constraints and privacy regulations made it difficult to implement improvements on a larger scale. Still, the participatory approach encouraged the development of partnerships and shared goals on the level of both managers and professionals. This case study highlights that improving communication between professionals is an important first step in improving integrated care. In addition, it shows that a participatory approach, in which improvements are co-created and tailored to local priorities and needs, can help in the development of shared goals and trust between stakeholders with different perspectives. However, stakeholders' willingness and ability to participate in such an improvement process is challenged by many factors., (© 2019 The Authors. Health and Social Care in the Community published by John Wiley & Sons Ltd.)

Background: Little is known about met and unmet needs of individuals in residential care, many of whom suffer from dementia. Unmet needs are associated with a decreased quality of life, worse mental health, dissatisfaction with services, and increased costs of care. The aim of this study was to compare the number and type of (unmet) needs of people with and without dementia in residential care in the Netherlands., Methods: 187 individuals in residents care or their relatives were interviewed to identify their care needs on 24 topics using the Camberwell Assessment of Needs for the Elderly (CANE) interview., Results: Individuals diagnosed with probable dementia reported more needs in total and more unmet needs in comparison with individuals without this diagnosis. More specifically, differences were found for the topics "accommodation", "money", "benefits", "medication management", "incontinence", "memory problems", "inadvertent self-harm", "company" and "daytime activities"., Conclusions: It seems that the differences in care needs between individuals with and without dementia can be attributed to actual differences in physical and cognitive functioning. Residents with dementia reported more often unmet needs which might imply that care for people with dementia can still be better attuned to their needs.

Background: Successfully introducing and maintaining care innovations may depend on the interplay between care setting, the intervention and specific circumstances. We studied the factors influencing the introduction and maintenance of a Multidisciplinary Integrated Care model in 10 Dutch residential care homes., Methods: Facilitating and impeding factors were studied and compared at the time of introduction of the interRAI-LTCF assessment method in residential care homes as well as three years later, by surveys and semi structured interviews among nurse staff, managers, and physicians., Results: Facilitating factors at introduction were positive opinions of staff and family physicians about the changes of the process of care and the anticipated improvement of quality of care. Staff was positive about the applicability of the software to support the interRAI-LTCF assessments. Impeding factors were time constraints to complete interRAI-LTCF assessments and insufficient computer equipment.In the maintenance phase, the positive attitude of the location manager and the perceived benefits of the care model and the interRAI-LTCF assessment method were most important. Impeding factors after 3 years remained the lack of time to complete the assessments and lack of sufficient computer equipment., Conclusions: Impeding and facilitating factors were comparable in the initial and maintenance phase. Adoption of the interRAI-LTCF assessment method depended on positive opinions of staff and management, continuing support of staff and the availability of sufficient computer equipment.

Objective: Although it is known that depression is highly prevalent in institutionalized older adults, little is known about its incidence and risk factors in nursing homes and residential care homes. The aim of this study was to investigate and compare the incidence and associated risk factors for depression in Dutch nursing homes and residential care homes., Design: Data on depression were extracted from the Vrije Universiteit naturalistic cohort on routine care monitoring with the Minimum Data Set of the Resident Assessment Instrument., Participants: A total of 1,324 residents in six nursing homes and 1,723 residents in 23 residential care homes with an average follow-up of 1.2 years., Measurements: Depression was defined as a clinical diagnosis according to Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, criteria and the use of antidepressants. Residents with prevalent depression at baseline were excluded., Results: The incidence rate was 13.6 per 100 person years in the nursing homes and 10.2 per 100 person years in the residential care homes. The independent risk factors for in-home depression for residents in nursing homes included dementia (OR: 1.7; 95% CI: 1.02-2.95) and a score of 3 or more on the Depression Rating Scale (odds ratio [OR]: 2.1; 95% confidence interval [CI]: 1.23-3.70). A protective effect was seen on the use of a hearing aid (OR: 0.3; 95% CI: 0.12-0.80). In the residential care homes, being male (OR: 2.1; 95% CI: 1.27-3.30), having cancer (OR: 2.9; 95% CI: 1.64-4.95), and a score of 2 or higher on the Cognitive Performance Scale (OR: 1.5; 95% CI: 1.05-2.22) increased the risk to develop depression. Age greater than 85 years (OR: 0.5; 95% CI: 0.31-0.67) and hearing impairment (OR: 0.8; 95% CI: 0.60-1.00) appeared to be protective., Conclusions: The incidence rate for depression in residents of Dutch nursing homes and residential care homes was high and the associated risk factors found may have important implications for staff., (2012 American Association for Geriatric Psychiatry)

Objective: To estimate and compare the prevalence and incidence of delirium and its risk factors in residents of Dutch nursing homes and residential care homes., Methods: Data were extracted from the Long-Term Care Facility (inter RAI-LTCF) version of the Resident Assessment Instrument, which was filled in a routine care cohort for a total of 3627 residents. 828 residents of six nursing homes and 1365 residents of 23 residential homes were included in the analyses. Delirium was defined as a positive score on the adjusted Nursing Home-CAM., Results: The prevalence of delirium was 8.9% in the nursing homes and 8.2% in the residential homes. The incidence was highest in the nursing homes with 20.7 versus 14.6 per 100 person-years. Multivariate tests of risk factors for developing delirium included chair restraints (OR 2.3; 95% CI 1.27-4.28), dementia (odds ratio (OR) 3.3; 95% confidence interval (CI) 2.03-5.24) and Parkinson's disease (OR 2.3; 95% CI 0.96-5.63) for residents in nursing homes, and dementia (OR 1.8; 95% CI 1.31-2.55) and fall incidents (OR 1.7; 95% CI 1.20-2.48) for residents in residential care homes., Conclusion: The prevalence and incidence of delirium was high in both nursing homes and residential care homes. More focus on modifiable risk factors such as the use of restraints in nursing homes and fall incidents in residential care homes may help to prevent delirium., (Copyright © 2011 John Wiley & Sons, Ltd.)

Objective: The objective of this study was to evaluate the cost-effectiveness of a Multidisciplinary Integrated Care (MIC) model compared to Usual Care (UC) in Dutch residential homes., Methods: The economic evaluation was conducted from a societal perspective alongside a 6 month, clustered, randomized controlled trial involving 10 Dutch residential homes. Outcome measures included a quality of care weighted sum score, functional health (COOP WONCA) and Quality Adjusted Life-Years (QALY). Missing cost and effect data were imputed using multiple imputation. Bootstrapping was used to analyze differences in costs and cost-effectiveness., Results: The quality of care sum score in MIC was significantly higher than in UC. The other primary outcomes showed no significant differences between the MIC and UC. The costs of providing MIC were approximately €225 per patient. Total costs were €2,061 in the MIC group and €1,656 for the UC group (mean difference €405, 95% -13; 826). The probability that the MIC was cost-effective in comparison with UC was 0.95 or more for ceiling ratios larger than €129 regarding patient related quality of care. Cost-effectiveness planes showed that the MIC model was not cost-effective compared to UC for the other outcomes., Interpretation: Clinical effect differences between the groups were small but quality of care was significantly improved in the MIC group. Short term costs for MIC were higher. Future studies should focus on longer term economic and clinical effects., Trial Registration: Controlled-Trials.com ISRCTN11076857.

Background: Sophisticated approaches are needed to improve the quality of care for elderly people living in residential care facilities. We determined the effects of multidisciplinary integrated care on the quality of care and quality of life for elderly people in residential care facilities., Methods: We performed a cluster randomized controlled trial involving 10 residential care facilities in the Netherlands that included 340 participating residents with physical or cognitive disabilities. Five of the facilities applied multidisciplinary integrated care, and five provided usual care. The intervention, inspired by the disease management model, consisted of a geriatric assessment of functional health every three months. The assessment included use of the Long-term Care Facility version of the Resident Assessment Instrument by trained nurse-assistants to guide the design of an individualized care plan; discussion of outcomes and care priorities with the family physician, the resident and his or her family; and monthly multidisciplinary meetings with the nurse-assistant, family physician, psychologist and geriatrician to discuss residents with complex needs. The primary outcome was the sum score of 32 risk-adjusted quality-of-care indicators., Results: Compared with the facilities that provided usual care, the intervention facilities had a significantly higher sum score of the 32 quality-of-care indicators (mean difference - 6.7, p = 0.009; a medium effect size of 0.72). They also had significantly higher scores for 11 of the 32 indicators of good care in the areas of communication, delirium, behaviour, continence, pain and use of antipsychotic agents., Interpretation: Multidisciplinary integrated care resulted in improved quality of care for elderly people in residential care facilities compared with usual care., Trial Registration: www.controlled-trials.com trial register no. ISRCTN11076857.

Background: The budesonide/formoterol combination is successfully used for fast relief of asthma symptoms in addition to its use as maintenance therapy. The temporarily increased corticosteroid dose during increasing inhaler use for symptom relief is likely to suppress any temporary increase in airway inflammation and may mitigate or prevent asthma exacerbations. The relative contribution of the budesonide and formoterol components to the improved asthma control is unclear., Methods: The acute protective effect of inhaled budesonide was tested in a model of temporarily increased airway inflammation with repeated indirect airway challenges, mimicking an acute asthma exacerbation. A randomised, double-blind, cross-over study design was used. Asthmatic patients (n = 17, mean FEV1 95% of predicted) who previously demonstrated a > or = 30% fall in forced expiratory volume in 1 second (FEV1) after inhaling adenosine 5'-monophosphate (AMP), were challenged on four consecutive test days, with the same dose of AMP (at 09:00, 12:00 and 16:00 hours). Within 1 minute of the maximal AMP-induced bronchoconstriction at 09:00 hours, the patients inhaled one dose of either budesonide/formoterol (160/4.5 microg), formoterol (4.5 microg), salbutamol (2 x 100 microg) or placebo. The protective effects of the randomised treatments were assessed by serial lung function measurements over the test day., Results: In the AMP provocations at 3 and 7 hours after inhalation, the budesonide/formoterol combination provided a greater protective effect against AMP-induced bronchoconstriction compared with formoterol alone, salbutamol and placebo. In addition all three active treatments significantly increased FEV1 within 3 minutes of administration, at a time when inhaled AMP had induced the 30% fall in FEV1., Conclusions: A single dose of budesonide/formoterol provided a greater protective effect against inhaled AMP-induced bronchoconstriction than formoterol alone, both at 3 and at 7 hours after inhalation. The acute protection against subsequent bronchoconstrictor stimuli such as inhaled AMP and the rapid reversal of airway obstruction supports the use of budesonide/formoterol for both relief and prevention in the treatment of asthma.

Systemic corticosteroids and additional short-acting beta2-agonists are commonly used in exacerbations of chronic obstructive pulmonary disease (COPD). In this double-blind study, the combination of a high-dose inhaled corticosteroid with a rapid-onset long-acting beta2-agonist was evaluated in the treatment of out-patient COPD exacerbations. The primary aim was to compare 14-day treatment effects of budesonide/formoterol to placebo on sputum eosinophils and, secondarily, on other indices of inflammation, forced expiratory flow in one second (FEV(1)), symptoms, health status, and adverse events. Forty-five patients not using steroids (37 male, 21/24 current/ex smoker, median packyears 38, age 65 years, FEV(1) 61% predicted), experiencing a COPD exacerbation, were treated at home with budesonide/formoterol (320/9 microg 4 times daily), prednisolone (30 mg daily), or placebo for 14 days. Sputum eosinophils were significantly reduced by budesonide/formoterol (-57%) compared to placebo (+24%) (p = 0.01). Budesonide/formoterol reduced total symptom scores significantly (p = 0.01) compared to placebo. The increase in FEV(1) by 2 weeks of treatment with budesonide/formoterol (125 ml) was not significantly different from that of placebo (43 ml) (p = 0.07). Budesonide/ formoterol treatment did not suppress morning serum cortisol compared to placebo (-16%; p = 0.50). In conclusion, budesonide/formoterol reduces sputum eosinophils and improves symptoms in the treatment of out-patient COPD exacerbations.

Background: The objective of this article is to describe the design of a study to evaluate the clinical and economic effects of a Disease Management model on functional health, quality of care and quality of life of persons living in homes for the elderly., Methods: This study concerns a cluster randomized controlled clinical trial among five intervention homes and five usual care homes in the North-West of the Netherlands with a total of over 500 residents. All persons who are not terminally ill, are able to be interviewed and sign informed consent are included. For cognitively impaired persons family proxies will be approached to provide outcome information. The Disease Management Model consists of several elements: (1) Trained staff carries out a multidimensional assessment of the patients functional health and care needs with the interRAI Long Term Care Facilities instrument (LTCF). Computerization of the LTCF produces immediate identification of problem areas and thereby guides individualized care planning. (2) The assessment outcomes are discussed in a Multidisciplinary Meeting (MM) with the nurse, primary care physician, nursing home physician and Psychotherapist and if necessary other members of the care team. The MM presents individualized care plans to manage or treat modifiable disabilities and risk factors. (3) Consultation by an nursing home physician and psychotherapist is offered to the frailest residents at risk for nursing home admission (according to the interRAI LTCF). Outcome measures are Quality of Care indicators (LTCF based), Quality Adjusted Life Years (Euroqol), Functional health (SF12, COOP-WONCA), Disability (GARS), Patients care satisfaction (QUOTE), hospital and nursing home days and mortality, health care utilization and costs., Discussion: This design is unique because no earlier studies were performed to evaluate the effects and costs of this Disease Management Model for disabled persons in homes for the elderly on functional health and quality of care. TRAIL REGISTRATION NUMBER: ISRCTN11076857.

Aims: Budesonide, unlike fluticasone propionate, undergoes fatty acid esterification in the lungs, and there is a need to characterize fully the distribution and fate of the two drugs after inhalation in humans., Methods: This open-label, randomized study was performed in adults undergoing whole lung or lobar resection resulting from lung cancer. Patients were given single 1000-mug doses of both budesonide and fluticasone propionate via dry powder inhalers before surgery. Tissue samples from peripheral and central lung, an ex vivo bronchial brush sample and intercostal muscle, together with plasma samples, were taken during surgery and analysed by liquid chromatography plus tandem mass spectrometry., Results: Lung tissue samples were obtained from 22 patients at surgery, 1-43 h after drug dosing. Budesonide was detectable from earliest sampling in central and peripheral lung tissue up to 10 h (in six of 22 samples), fluticasone propionate up to 22 h after inhalation (in 16 of 22 samples), and budesonide oleate up to 43 h after inhalation (in 21 of 22 samples). Budesonide, but not fluticasone propionate, was detected in intercostal muscle for up to 10 h after inhalation. Bronchial brush samples showed the presence of fluticasone propionate for up to 18 h, suggesting the presence of undissolved drug powder particles in the airway lumen., Conclusion: Sustained retention of esterified budesonide in the lungs supports the prolonged duration of action of budesonide and suitability for once-daily administration.

Rationale: Limited information is available on repeatability of inflammatory parameters in whole induced sputum samples from patients with COPD., Objectives: To study short-term and long-term repeatability in induced sputum samples in 22 patients with moderate to severe, stable COPD (mean age 64 yr, mean FEV(1) 1.91 L=65% of predicted). Samples were collected on 71 occasions twice within 1 to 7 days (mean 3.8 days) and on 12 occasions twice with an interval of 3 months while clinically stable. Cell differentials, markers of neutrophilic and eosinophilic inflammation, respiratory membrane permeability and size-selective permeation were assessed., Findings: Parameters of permeability and of size-selective permeation, % eosinophils and % neutrophils showed the best short-term repeatability with intra-class correlation coefficients (Ri) of 0.61 to 0.90, followed by total cell count (TCC) with Ri of 0.52. Repeatability of soluble cell activation markers was less satisfactory (Ri 0.34 to 0.52). Mean short-term within-patient variability for TCC and permeability was approximately 2-fold and for cell activation markers 3-fold; mean between-patients variability was twice as high. Inducing sputum slightly enhanced eosinophil numbers and % neutrophils and decreased % macrophages in successive IS samples. Long-term repeatability was comparable to short-term repeatability but variability increased., Conclusions: Repeatability of parameters assessed in whole sputum is similar as reported previously for sputum plugs. In COPD an induced sputum procedure has a minor pro-inflammatory effect. The current data facilitates power calculations but also indicates that studies using inflammatory markers in sputum may easily be underpowered.

Inhalers containing corticosteroids and long-acting beta2-agonists are becoming increasingly important in asthma management. A rapid effect is important to patients, particularly during exacerbations. We compared the onset of bronchodilation and patient-perceived relief from dyspnoea following single-inhaler budesonide/formoterol or salmeterol/fluticasone in a model of acute bronchoconstriction. A randomised, double-blind, double-dummy, single-dose, crossover study included 27 outpatients with asthma (mean age 35 years; mean FEV1 90% predicted normal). Immediately following methacholine-induced bronchoconstriction (fall in FEV1 > or = 30%), patients inhaled budesonide/formoterol (160/4.5 microg, 1 or 2 inhalations; Symbicort Turbuhaler), salmeterol/fluticasone (50/250 microg; Seretide Diskus) or placebo on 4 study days. Lung function and Borg score were assessed for 30 min. During methacholine-induced provocation (final mean FEV1 62.5% of baseline), mean Borg score increased 10-fold (from 0.3 to 3.0 units). Hereafter, mean FEV1 at 3 min improved significantly more after budesonide/formoterol 1 and 2 inhalations (37 and 38%, respectively) than after salmeterol/fluticasone (23%; P < 0.001) or placebo (10%; P < 0.001). Median recovery times to 85% of baseline FEV1 were shorter for budesonide/formoterol (1 or 2 inhalations: 3.3 and 2.8 min, respectively) than salmeterol/fluticasone (8.9 min; P < 0.001) and placebo (> 30 min). One min after budesonide/formoterol, dyspnoea was significantly reduced (Borg score -0.86 units, both doses) compared with salmeterol/fluticasone (-0.55 units; P < 0.05) and placebo (-0.23 units; P < 0.001). Budesonide/formoterol provides immediate bronchodilation, faster than salmeterol/fluticasone, which patients can feel during acute methacholine-induced bronchoconstriction.

The construction of layered DNA-RNA replicons has facilitated and expanded the use of alphavirus vectors to vaccine development, construction of packaging cell lines and long-term heterologous gene expression. In these vector systems, the alphavirus replicon is under the control of a strong RNA polymerase II promoter and replicon RNA is transcribed from DNA before transport to the cytoplasm. Efficient RNA amplification catalyzed by the viral replicase results in high levels of mRNA and the recombinant protein. Recently, we developed a temperature-regulated Sindbis replicon-based DNA expression system characterized by a linear increase of expression upon decrease of the temperature from 37 degrees C to 29 degrees C. Modifications known to affect transcription and nuclear export of RNA led to a 5-fold increase in expression in BHK cells and up to over 80-fold increase in CHO cells and BF fibroblasts in transient transfection experiments. Furthermore, reducing cell proliferation resulted in a further 2- to 3-fold higher expression. While increased expression per cell was responsible for some of the enhanced production, it was primarily the number of expressing cells that made the difference in most cell lines. Further experiments indicated that a threshold amount of replicon RNA had to reach the cytoplasm in order for replication to occur. Thus, alterations that improve transcription, nuclear export and stability of the RNA had a significant impact on protein production in the pCytTS expression system and probably in other layered DNA-based viral vectors. Furthermore the results indicate that RNA replication is differentially regulated in DNA layered RNA replicons versus viral infection., (Copyright 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 81: 553-562, 2003.)

The rat serotonin transporter (rSERT) is an N-glycosylated integral membrane protein with 12 transmembrane regions; the N-glycans improve the ability of the SERT polypeptide chain to fold into a functional transporter, but they are not required for the transmembrane transport of serotonin per se. In order to define the best system for the expression, purification and structural analysis of serotonin transporter (SERT), we expressed SERT in Escherichia coli, Pichia pastoris, the baculovirus expression system and in four different stable mammalian cell lines. Two stable cell lines that constitutively expressed SERT (Imi270 and Coca270) were constructed using episomal plasmids in HEK293 cells expressing the EBNA-1 antigen. SERT expression in the three different inducible stable mammalian cell lines was induced either by a decrease in temperature (cell line pCytTS-SERT), the addition of tetracycline to the growth medium (cell line T-REx-SERT) or by adding DMSO which caused the cells to differentiate (cell line MEL-SERT). All the mammalian cell lines expressed functional SERT, but SERT expressed in E. coli or P. pastoris was nonfunctional as assessed by 5-hydroxytryptamine uptake and inhibitor binding assays. Expression of functional SERT in the mammalian cell lines was assessed by an inhibitor binding assay; the cell lines pCytTS-SERT, Imi270 and Coca270 contained levels of functional SERT similar to that of the standard baculovirus expression system (250,000 copies per cell). The expression of SERT in induced T-REx-SERT cells was 400,000 copies per cell, but in MEL-SERT it was only 80,000 copies per cell. All the mammalian stable cell lines expressed SERT at the plasma membrane as assessed by [3H]-5-hydroxytryptamine uptake into whole cells, but the V(max) for the T-Rex-SERT cell line was 10-fold higher than any of the other cell lines. It was noticeable that the cell lines that constitutively expressed SERT grew extremely poorly, compared to the inducible cell lines whose growth rates were similar to the parental cell lines when not induced. In addition, the cell lines MEL-SERT, Imi270 and T-REx-SERT all expressed fully N-glycosylated SERT and no unglycosylated inactive protein, in contrast to the baculovirus expression system where the vast majority of expressed SERT was unglycosylated and nonfunctional., (Copyright 2003 Elsevier Science B.V.)

Alphaviruses are positive stranded RNA viruses that replicate to extremely high titers. Sindbis and Semliki Forest viral vectors are widely used tools for high-level production of recombinant proteins. Recent studies have broadened their scope to vaccine production, gene therapy, and analysis of cell function. Here we discuss the development of non-cytopathic and inducible expression vectors which can be applied to bioprocess development strategies. Furthermore, a Sindbis-based expression cloning system has been developed that allows for the rapid identification of genes encoding proteins with a selected functional activity.