Physical learning environments' supportiveness to innovative pedagogies: students' and teachers' experiences.

Research into the relationship between innovative physical learning environments (PLEs) and innovative psychosocial learning environments (PSLEs) indicates that it must be understood as a network of relationships between multiple psychosocial and physical aspects. Actors shape this network by attaching meanings to these aspects and their relationships in a continuous process of gaining and exchanging experiences. This study used a psychosocial-physical, relational approach for exploring teachers' and students' experiences with six innovative PLEs in a higher educational institute, with the application of a psychosocial-physical relationship (PPR) framework. This framework, which brings together the multitude of PLE and PSLE aspects, was used to map and analyse teachers' and students' experiences that were gathered in focus group interviews. The PPR framework proved useful in analysing the results and comparing them with previous research. Previously-identified relationships were confirmed, clarified, and nuanced. The results underline the importance of the attunement of system aspects to pedagogical and spatial changes, and of a psychosocial-physical relational approach in designing and implementing new learning environments, including the involvement of actors in the discourse within and between the different system levels. Interventions can be less invasive, resistance to processes could be reduced, and innovative PLEs could be used more effectively.

High throughput transepithelial electrical resistance (TEER) measurements on perfused membrane-free epithelia.

Assessment of epithelial barrier function is critically important for studying healthy and diseased biological models. Here we introduce an instrument that measures transepithelial electrical resistance (TEER) of perfused epithelial tubes in the microfluidic OrganoPlate platform. The tubules are grown in microfluidic channels directly against an extracellular matrix, obviating the need for artificial filter membranes. We present TEER measurements on Caco-2 intestinal and renal proximal tubule epithelium. Forty tubules on one single plate were interrogated in less than a minute. We show that TEER measurement is significantly more sensitive than a fluorescent reporter leakage assay in response to staurosporine. We demonstrate a 40-channel time-lapse data acquisition over a 25 hour time period under flow conditions. We furthermore observed a 50% reduction in Caco-2 TEER values following exposure to a cocktail of inflammatory cytokines. To our best knowledge, this is the first instrument of its kind that allows routine TEER studies in perfused organ-on-a-chip systems without interference by artificial filter membranes. We believe the apparatus will contribute to accelerating routine adoption of perfused organ-on-a-chip systems in academic research and in industrial drug development.

Decontamination of the digestive tract and oropharynx in ICU patients.

BACKGROUND: Selective digestive tract decontamination (SDD) and selective oropharyngeal decontamination (SOD) are infection-prevention measures used in the treatment of some patients in intensive care, but reported effects on patient outcome are conflicting. METHODS: We evaluated the effectiveness of SDD and SOD in a crossover study using cluster randomization in 13 intensive care units (ICUs), all in The Netherlands. Patients with an expected duration of intubation of more than 48 hours or an expected ICU stay of more than 72 hours were eligible. In each ICU, three regimens (SDD, SOD, and standard care) were applied in random order over the course of 6 months. Mortality at day 28 was the primary end point. SDD consisted of 4 days of intravenous cefotaxime and topical application of tobramycin, colistin, and amphotericin B in the oropharynx and stomach. SOD consisted of oropharyngeal application only of the same antibiotics. Monthly point-prevalence studies were performed to analyze antibiotic resistance. RESULTS: A total of 5939 patients were enrolled in the study, with 1990 assigned to standard care, 1904 to SOD, and 2045 to SDD; crude mortality in the groups at day 28 was 27.5%, 26.6%, and 26.9%, respectively. In a random-effects logistic-regression model with age, sex, Acute Physiology and Chronic Health Evaluation (APACHE II) score, intubation status, and medical specialty used as covariates, odds ratios for death at day 28 in the SOD and SDD groups, as compared with the standard-care group, were 0.86 (95% confidence interval [CI], 0.74 to 0.99) and 0.83 (95% CI, 0.72 to 0.97), respectively. CONCLUSIONS: In an ICU population in which the mortality rate associated with standard care was 27.5% at day 28, the rate was reduced by an estimated 3.5 percentage points with SDD and by 2.9 percentage points with SOD. (Controlled Clinical Trials number, ISRCTN35176830.)

[Less ventilator-associated pneumonia after oral decontamination with chlorhexidine; a randomised trial]. / Minder beademingspneumonieën door orale decontaminatie met chloorhexidine; gerandomiseerde trial.

OBJECTIVE: To determine the effect of oral decontamination with either chlorhexidine (CHX, 2%) or the combination chlorhexidine-colistin (CHX-COL, 2%-2%) on the frequency and the time to onset of ventilator-associated pneumonia in Intensive Care patients. DESIGN: Double blind, placebo-controlled, multicentre, randomised trial. METHODS: Consecutive ICU patients needing at least 48 h of mechanical ventilation were enrolled in a randomized trial with 3 arms: CHX, CHX-COL, and placebo (PLAC). The trial medication was administered in the oral cavity every 6 h. Oropharyngeal swabs were obtained daily and analysed quantitatively for Gram-positive and Gram-negative microorganisms. Endotracheal colonisation was monitored twice weekly. Ventilator-associated pneumonia was diagnosed on the basis of a combination of clinical, radiological and microbiological criteria. RESULTS: Of 385 patients included, 130 received PLAC, 127 CHX and 128 CHX-COL. Baseline characteristics in the three groups were comparable. The daily risk of ventilator-associated pneumonia was reduced in both treatment groups compared to PLAC: 65% (HR= 0.352; 95% CI: 0.160-0.791; p = 0.012) for CHX and 55% (HR= 0.454; 95%/ CI: 0.224-0.925; p = 0.030) for CHX-COL. CHX-COL provided a significant reduction in oropharyngeal colonisation with both Gram-negative and Gram-positive microorganisms, whereas CHX significantly affected only colonisation with Gram-positive microorganisms. There were no differences in the duration of mechanical ventilation, ICU-stay or ICU-survival. CONCLUSION: Oral decontamination of the oropharyngeal cavity with chlorhexidine or the combination chlorhexidine-colistin reduced the incidence and the time to onset ofventilator-associated pneumonia.

Do cultures contribute to optimisation of antibiotic therapy in the intensive care unit?

Obtaining diagnostic microbiological cultures before initiating empirical antimicrobial therapy is part of the diagnostic work-up of intensive care patients with a clinical suspicion of infection. However, it is unknown to what extent these cultures provide a microbiological cause of infection and to what extent antimicrobial therapy is influenced. During a 6-month period, all episodes of suspected clinical infection were analysed and categorised as non-microbiologically proven infection (non-MPI) or MPI. Effects of culture results on antibiotic therapy were analysed for episodes of respiratory tract infection. Invasive diagnostic techniques were not routinely used for diagnosis of respiratory tract infections. Among 212 patients admitted, 147 episodes of clinical suspicion of infection were recorded (104 for respiratory tract infection) and 1147 microbiological cultures were obtained (0.64 culture per patient day). Antibiotics were administered on 1111 (62%) of 1803 patients days. Of the respiratory tract infections, 571 cultures resulted in 49 (47%) MPI. Cover with empirical antibiotics was inappropriate in 7 of 104 cases (8%) of respiratory infections. In 12 cases (11.5%) empirical therapy could have been changed based on culture results. Negative cultures were never followed by cessation of therapy, but the duration of treatment was significantly shorter for non-MPI. Forty-seven percent of respiratory tract infections were microbiologically confirmed and, based on culture results, empirical antimicrobial therapy could have been influenced in 11.5% of cases of respiratory tract infections. These findings provide aspects to evaluate and improve the diagnostic work-up of infections in the ICU.

[Intensive care medicine in the Netherlands, 1997-2001. II. Changes over time and differences between hospitals]. / Intensivecaregeneeskunde in Nederland, 1997-2001. II. Verandering in de tijd en verschillen tussen ziekenhuizen.

OBJECTIVE: To describe efficacy (mortality) and efficiency (length of admission) of intensive care (IC) treatment after admission due to a prior cardiothoracic operation or pneumonia, based on data from the Dutch National Intensive Care Evaluation (NICE) foundation. DESIGN: Descriptive. METHOD: Data for the period 1 January 1997-31 December 2001 were extracted from the NICE databank for patients admitted after cardiothoracic surgery and for patients admitted with pneumonia. The variables changes in time, risk factors for mortality, and differences between hospitals were analysed. RESULTS: There were 25,463 admissions to 5 hospitals following cardiothoracic surgery and 1408 admissions to 18 hospitals due to pneumonia. An increase in valve surgery was noted in the cardiothoracic surgery group: from about 10% to about 25%. In the group undergoing valve operations, there was an increase in the average age of the patients and in the number of patients with comorbidity. No significant differences in mortality between hospitals were detected. However, the length of ICU treatment differed. Hospital mortality in the pneumonia group was 33.9%. Differences between hospitals with respect to mortality (both crude mortality and severity-of-illness adjusted mortality) and length of ICU admission were found. CONCLUSION: With the NICE registration it is possible to detect differences and trends. This is a valuable tool for indicating where and how quality and efficiency in intensive care medicine can be improved.

Ectopic Wnt signal determines the eyeless phenotype of zebrafish masterblind mutant.

masterblind (mbl) is a zebrafish mutation characterised by the absence or reduction in size of the telencephalon, optic vesicles and olfactory placodes. We show that inhibition of Gsk3beta in zebrafish embryos either by overexpression of dominant negative dn gsk3beta mRNA or by lithium treatment after the midblastula transition phenocopies mbl. The loss of anterior neural tissue in mbl and lithium-treated embryos is preceded by posteriorization of presumptive anterior neuroectoderm during gastrulation, which is evident from the anterior shift of marker genes Otx2 and Wnt1. Heterozygous mbl embryos showed increased sensitivity to inhibition of GSK3beta by lithium or dn Xgsk3beta that led to the loss of eyes. Overexpression of gsk3beta mRNA rescued eyes and the wild-type fgf8 expression of homozygous mbl embryos. emx1 that delineates the telencephalon is expanded and shifted ventroanteriorly in mbl embryos. In contrast to fgf8, the emx1 expression domain was not restored upon overexpression of gsk3beta mRNA. These experiments place mbl as an antagonist of the Wnt pathway in parallel or upstream of the complex consisting of Axin, APC and Gsk3beta that binds and phosphorylates beta-catenin, thereby destabilising it. mbl maps on LG 3 close to a candidate gene axin1. In mbl we detected a point mutation in the conserved minimal Gsk3beta-binding domain of axin1 leading to a leucine to glutamine substitution at position 399. Overexpression of wild-type axin1 mRNA rescued mbl completely, demonstrating that mutant axin1 is responsible for the mutant phenotype. Overexpression of mutant L399Q axin1 in wild-type embryos resulted in a dose-dependent dominant negative activity as demonstrated by the loss of telencephalon and eyes. We suggest that the function of Axin1/Mbl protein is to antagonise the Wnt signal and in doing so to establish and maintain the most anterior CNS. Our findings provide new insights into the mechanisms by which the Wnt pathway generates anteroposterior polarity of the neural plate.

Guidelines for severe community-acquired pneumonia in the western world.

BACKGROUND: Recently, several guidelines (ATS 1993/IDSA 1998; ERS 1998; SWAB 1998) have been issued for the initial therapy of patients with community-acquired pneumonia. In patients who fulfil the criteria for severe community-acquired pneumonia (SCAP), it was advised to start with a macrolide (active against Legionella spp. and Mycoplasma pneumoniae) in combination with an agent active against both pneumococci and Pseudomonas aeruginosa by the ATS/IDSA guidelines, while the ERS suggested starting with a second or third generation cephalosporin, in combination with either a macrolide or second generation quinolon plus or minus rifampicin. In the SWAB guidelines, no recommendations for SCAP were made. METHODS: Sixty-two cases admitted to the intensive care units of a tertiary-care university hospital with SCAP between 1992 and 1996 were studied retrospectively. The causative pathogens, clinical and laboratory characteristics of severity, antibiotic therapy and mortality were analysed. Immunocompromised patients, patients using immunosuppressive agents and patients with a malignancy were excluded. RESULTS: Indices of severe illness were widely seen and 37% developed shock while 45% required vasoactive drugs. Bilobular or multilobular abnormalities were seen in 34% of the patients. Forty-five patients (73%) required artificial respiration and 54 (87%) had an underlying disease. The overall mortality was 42%. In 41 patients (66%), a pathogen was isolated. The most frequent causes of SCAP in this study were Streptococcus pneumoniae (22 cases or 35%), Haemophilus influenzae (seven cases or 11%), Pseudomonas aeruginosa (four cases or 7%), and other Enterobacteriaceae (twice in combination with pneumococci and once with H. influenzae). Legionella pneumophila was identified in three cases. In patients with severe chronic obstructive pulmonary disease (COPD), pneumococci were the most important pathogens six cases or 27%), followed by P. aeruginosa (14%) and H. influenzae (14%). CONCLUSIONS: The guidelines for the management of SCAP issued by the ATS and IDSA in 1993 are only partially adequate in the Dutch setting. Coverage of P. aeruginosa would seem useful, given the fact that isolation of this pathogen has been shown to be a predictor of mortality, but only in patients with severe COPD or structural disease of the lung, and especially in patients in whom the Gram stain reveals Gram-negative rods, as is also suggested in the revised IDSA guidelines (1998). Risk factors for P. aeruginosa could be added to the ERS guidelines. Including SCAP as a separate entity in the SWAB guidelines may be useful.

Hormone-inducible expression of secreted factors in zebrafish embryos.

The study of gene function at later stages of embryonic development by overexpression experiments is often complicated by genes exerting different functions at multiple stages of development, which renders analysis of stage-specific effects difficult. To address this problem an inducible expression system that supports timed expression of essentially any protein, including secreted proteins was designed. The system is based on a two step mechanism. A glucocorticoid inducible, Gal4-site binding chimeric transcription factor is expressed ubiquitously, whereas a gene of interest is placed under the control of a Gal4-site driven promoter. Treatment of zebrafish embryos injected with such constructs with the synthetic glucocorticoid dexamethasone results in readily detectable reporter activity within 3 h. The system was tested with induced expression of Xactivin(beta)B and X(wnt), which both were shown to induce morphological abnormalities, as well as alterations in the expression patterns of goosecoid and otx2, respectively. Coinjection of an inducible lacZ reporter vector served as an indicator for expressing cells in embryos. The present results demonstrate that this is a versatile inducible expression system for use in vertebrate embryos, that also supports expression of secreted proteins.

Protein kinase A is involved in the induction of early mesodermal marker genes by activin.

In this study we have investigated the role of cAMP-dependent protein kinase A (PKA) in the induction of the early mesodermal marker genes goosecoid and no tail by activin in zebrafish embryos. We show that upon treatment with activin, zebrafish blastula cells exhibit a rapid and transient increase in PKA activity. In these cells, activin rapidly induces the expression of the immediate early response genes goosecoid and no tail. Stimulation and inhibition of PKA by activin, respectively, enhances and reduces the induction of goosecoid and no tail mRNA expression. Similar effects of PKA stimulation and inhibition on the induction by activin of a 1.8 kb zebrafish goosecoid promoter construct were observed. The induction by activin of a fragment of the zebrafish goosecoid promoter that mediates an immediate early response to activin is blocked by inhibition of PKA. Activation of PKA alone has no effect in these experiments. Finally, inhibition of PKA in whole embryos by overexpression of a dominant negative regulatory subunit of PKA reduces the expression of no tail and goosecoid, whereas the expression of even-skippedl remains unaltered. Overexpression of the catalytic subunit of PKA in embryos does not affect expression of goosecoid, no tail or even-skippedl. These data show that in dissociated blastulae, PKA is required, but not sufficient for activin signalling towards induction of goosecoid and no tail. In intact zebrafish embryos, PKA contributes to induction of goosecoid and no tail, although it is not required or sufficient.

Domains of retinoid signalling and neurectodermal expression of zebrafish otx1 and goosecoid are mutually exclusive.

Retinoid signalling plays an important role in embryonic pattern formation. Excess of retinoic acid during gastrulation results in axial defects in vertebrate embryos, suggesting that retinoids are involved in early anteroposterior patterning. To study retinoid signalling in zebrafish embryos, we developed a novel method to detect endogenous retinoids in situ in embryos, using a fusion protein of the ligand inducible transactivation domain of a retinoic acid receptor and a heterologous DNA binding domain. Using this method, we show that retinoid signalling is localized in zebrafish embryos in the region of the embryonic shield, and towards the end of gastrulation in a posterior dorsal domain. To investigate the relationships between the spatial distribution of retinoid signalling and the regulation of retinoid target genes, we studied the downregulation by retinoic acid of two genes expressed in anterior regions of the embryo, goosecoid and otx1. These experiments show that expression of both genes is strongly downregulated in the anterior neurectoderm of zebrafish embryos treated with retinoic acid, whereas mesendodermal expression is only mildly affected. Interestingly, a significant downregulation of goosecoid expression by retinoic acid was observed only during midgastrulation but not in earlier stages. In agreement with these results, spatial expression of goosecoid and otx1 does not overlap with the region of retinoid signalling in the late gastrula. Our data support the hypothesis that a localized retinoid signal is involved in axial patterning during early development, at least in part through the repression of anterior genes in posterior regions of the embryo. Furthermore, our data suggest that the action of retinoids is spatially as well as temporally regulated in the developing embryo.

Regulation of the zebrafish goosecoid promoter by mesoderm inducing factors and Xwnt1.

Goosecoid is a homeobox gene that is expressed as an immediate early response to mesoderm induction by activin. We have investigated the induction of the zebrafish goosecoid promoter by the mesoderm inducing factors activin and basic fibroblast growth factor (bFGF) in dissociated zebrafish blastula cells, as well as by different wnts in intact embryos. Activin induces promoter activity, while bFGF shows a cooperative effect with activin. We have identified two enhancer elements that are functional in the induction of the goosecoid promoter. A distal element confers activin responsiveness to a heterologous promoter in the absence of de novo protein synthesis, whereas a proximal element responds only to a combination of activin and bFGF. Deletion experiments show that both elements are important for full induction by activin. Nuclear proteins that bind to these elements are expressed in blastula embryos, and competition experiments show that an octamer site in the activin responsive distal element is specifically bound, suggesting a role for an octamer binding factor in the regulation of goosecoid expression by activin. Experiments in intact embryos reveal that the proximal element contains sequences that respond to Xwnt1, but not to Xwnt5c. Furthermore, we show that the distal element is active in a confined dorsal domain in embryos and responds to overexpression of activin in vivo, as well as to dorsalization by lithium. The distal element is to our knowledge the first enhancer element identified that mediates the induction of a mesodermal gene by activin.

Active complex formation of type I and type II activin and TGF beta receptors in vivo as studied by overexpression in zebrafish embryos.

We have investigated the involvement of activin receptors and TGF beta type I receptor in zebrafish development. Overexpression of either full-length or a truncated form of mouse ActR-IIA interferes with the development. Different splice variants of mouse ActR-IIB have distinct effects; ActR-IIB4 induces abnormal embryos, whereas ActR-IIB2 does not. Activin and TGF beta type I receptors can induce axis duplications. Co-expression of ActR-IA or ActR-IB with the type II activin receptors results in a synergistic increase of the frequency of axis duplication. Moreover, ActR-IIB2 is synergistic with ActR-IA and ActR-IB, demonstrating that ActR-IIB2 can interact with the zebrafish ligand. Overexpression of TGF beta R-I with ActR-IIA or ActR IIB4 results in a synergistic increase in frequency of abnormal embryos, whereas in combination with ActR-IIB2 no such increase occurs.

Effects of retinoic acid on the expression of retinoic acid receptors during zebrafish embryogenesis.

The cDNAs encoding the zebrafish homologs of retinoic acid receptor alpha(zRAR alpha) and gamma (zRAR gamma) were isolated and their expression studied in normal and retinoic acid (RA) treated embryos. Expression boundaries in the central nervous system are clearly different from those observed in the mouse, which can only partly be explained by morphogenetic differences. Treatment of embryos with RA induces ectopic zRAR gamma expression in anterior brain structures and both zRAR alpha and zRAR gamma expression in the eyes. Furthermore, striking differences occur in the zRAR gamma expression pattern in pharyngeal arch mesenchyme. Since the development of all of these structures has been shown to be affected by exogenous RA, our data suggest a role for zRAR alpha and zRAR gamma in the establishment of the RA phenotype in zebrafish.

[Mild sequelae 12 years following massive aspiration of barium sulfate in the lungs]. / Geringe restverschijnselen 12 jaar na massale bariumsulfaat-aspiratie in de longen.

A twelve-year-old girl, who as a baby underwent an investigation of the oesophagus and stomach with barium roentgen contrast fluid, during which there was massive aspiration of barium contrast into the right lung, at follow-up showed no abnormalities, apart from slight signs of peribronchial granulomatosis or fibrosis. If there is an enhanced risk of aspiration or an oesophago-tracheal fistula is suspected it is better to use an isotonic water-soluble contrast fluid.

[The return of severe infections caused by Streptococcus pyogenes]. / De terugkeer van ernstige infecties door Streptococcus pyogenes.

Two patients with severe group A streptococcal infection associated with a toxic shock-like syndrome are described. Both isolates produced the pyrogenic exotoxin B. Since 1987 there have been many reports of these severe streptococcal infections. In order to know the incidence in the Netherlands, isolates from patients with severe streptococcal infection have to be serotyped (types of M-protein) and tested for streptococcal toxin production, and serum antibody levels have to be determined.

In vitro binding characteristics for cesium of two qualities of prussian blue, activated charcoal and Resonium-A.

The in vitro binding characteristics of radioactive 137Cs to two forms of Prussian blue [colloidally (soluble) K3Fe[Fe(CN)6] and insoluble Fe4[Fe(CN)6]3] and to activated charcoal and sodium polystyrene sulfonate (Resonium-A) were investigated by constructing Langmuir isotherms at pH = 1.0, 6.5 and 7.5 at 37 degrees C. At the three pHs investigated, 137Cs binding to activated charcoal and sodium polystyrene sulfonate was negligible. Binding of 137Cs to insoluble Prussian blue exceeded that for the soluble form and was pH dependent for both formulations. Maximum binding capacities were 87 mg/g (pH = 1.0), 194 mg/g (pH = 6.5) and 238 mg/g (pH = 7.5) for the insoluble form and 48 (pH = 1.0), 73 (pH = 6.5) and 78 (pH = 7.5) for the soluble form. As activated charcoal did not bind 137Cs, charcoal hemoperfusion is of no value. This has been confirmed by an in vitro experiment, using a Gambro Adsorbs 300 C cartridge.