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Intervertebral disc (IVD) degeneration causes low-back pain through disc compression, prolapse and herniation. Inflammation of the IVD and subsequent degeneration produce altered glycosylation profiles in several animal models of IVD injury and ageing, although the function of this altered glycosylation pattern in a human is unknown. Altered N-glycome, specifically sialylated and fucosylated N-glycosylation motif expression, might play a role in inflammation and disease progression. Healthy (foetal and adolescent idiopathic scoliosis) and degenerated (lumbar degeneration) human IVD glycosylation patterns were studied using lectin histochemistry. Small-molecule fluorinated sugar analogues (3Fax-Peracetyl Neu5Ac; 2F-Peracetyl-Fucose) were used to inhibit sialylation and fucosylation in an in vitro model of inflammation, to investigate their effects on the glycosignature, cell metabolism, extracellular matrix synthesis and cell migration. The effects of interleukin (IL)-1β, tumour necrosis factor (TNF)-α and IL-6 on glycosylation in human nucleus pulposus cells were investigated by lectin histochemistry, PCR and enzyme-linked immunosorbent assay (ELISA). In the in vitro model of IVD degeneration, cytokine-induced inflammation-induced hypersialylation was observed, as indicated by Sambucus nigra I binding. However, this modification was inhibited by the sialyltransferase inhibitor. Inhibition of sialylation and fucosylation modulates cell migration and protein translation of catabolic enzymes in response to inflammation. The altered patterns of glycosylation in human tissue in degeneration was consistent with previous IVD studies in murine, bovine and ovine models. The present study was the first functional investigation of glycosylation in human degenerated IVD, elucidating the role of the glycome in disease progression and identified potential therapeutic targets for future regenerative therapies.

Background Palliative care is becoming increasingly important because the number of patients with an incurable disease is growing and their survival is improving. Previous research tells us that early palliative care has the potential to improve quality of life (QoL) in patients with advanced cancer and their relatives. According to limited research on palliative care in the Netherlands, patients with advanced cancer and their relatives find current palliative care suboptimal. The aim of the eQuiPe study is to understand the experienced quality of care (QoC) and QoL of patients with advanced cancer and their relatives to further improve palliative care. Methods A prospective longitudinal observational cohort study is conducted among patients with advanced cancer and their relatives. Patients and relatives receive a questionnaire every 3 months regarding experienced QoC and QoL during the palliative trajectory. Bereaved relatives receive a final questionnaire 3 to 6 months after the patients’ death. Data from questionnaires are linked with detailed clinical data from the Netherlands Cancer Registry (NCR). By means of descriptive statistics we will examine the experienced QoC and QoL in our study population. Differences between subgroups and changes over time will be assessed while adjusting for confounding factors. Discussion This study will be the first to prospectively and longitudinally explore experienced QoC and QoL in patients with advanced cancer and their relatives simultaneously. This study will provide us with population-based information in patients with advanced cancer and their relatives including changes over time. Results from the study will inform us on how to further improve palliative care. Trial registration Trial NL6408 (NTR6584). Registered in Netherlands Trial Register on June 30, 2017.

Pro-inflammatory cytokines are considered to play a major role in osteoarthritis (OA), yet so far, the specific cytokines involved in the pathology of OA have not been identified. Oncostatin M (OSM) is a cytokine from the interleukin 6 (IL-6) family that has been shown to be elevated in synovial fluid of most rheumatoid arthritis (RA) patients, but only in a limited subset of OA patients. Little is known about OSM in the different joint tissues during OA and how its expression correlates with hallmarks of disease. Here, we mapped OSM expression in the joint tissues of two rat models of arthritis: an acute inflammatory model and an instability-induced osteoarthritic model. OSM expression was correlated with hallmarks of OA, namely cartilage damage, synovitis, and osteophyte formation. Reanalysis of an existing dataset on cytokine profiling of OA synovial fluid was performed to assess pattern differences between patients positive and negative for OSM. In the inflammatory model, OSM expression correlated with synovitis and osteophyte formation but not with cartilage damage. On the contrary, in the instability model of OA, an increase in synovitis, cartilage damage, and osteophyte formation was observed without changes in OSM expression. In line with these findings, synovial fluid of OA patients with detectable OSM contained higher levels of other inflammatory cytokines, namely interferon gamma (IFN-γ), IL-1α and tumor necrosis factor alpha (TNF-α), likely indicating a more inflammatory state. Taken together these data indicate OSM might play a prominent role in inflammatory phenotypes of OA.

Summary Background Antibiotics do not reduce mortality or short-term treatment non-response in patients receiving treatment for acute exacerbations of COPD in an outpatient setting. However, the long-term effects of antibiotics are unknown. The aim of this study was to investigate if the antibiotic doxycycline added to the oral corticosteroid prednisolone prolongs time to next exacerbation in patients with COPD receiving treatment for an exacerbation in the outpatient setting. Methods In this randomised double-blind placebo-controlled trial, we recruited a cohort of patients with COPD from outpatient clinics of nine teaching hospitals and three primary care centres in the Netherlands. Inclusion criteria were an age of at least 45 years, a smoking history of at least 10 pack-years, mild-to-severe COPD (Global Initiative of Chronic Obstructive Lung Disease [GOLD] stage 1–3), and at least one exacerbation during the past 3 years. Exclusion criteria were poor mastery of the Dutch language, poor cognitive functioning, known allergy to doxycycline, pregnancy, and a life expectancy of shorter than 1 month. If a participant had an exacerbation, we randomly assigned them (1:1; with permuted blocks of variable sizes [ranging from two to ten]; stratified by GOLD stage 1–2 vs 3) to a 7 day course of oral doxycycline 100 mg daily (200 mg on the first day) or placebo. Exclusion criteria for randomisation were fever, admission to hospital, and current use of antibiotics or use within the previous 3 weeks. Patients in both groups received a 10 day course of 30 mg oral prednisolone daily. Patients, investigators, and those assessing outcomes were masked to treatment assignment. The primary outcome was time to next exacerbation in all randomly allocated patients except for those incorrectly randomly allocated who did not meet the inclusion criteria or met the exclusion criteria. This trial is registered with the Netherlands Trial Register, number NTR2499. Findings Between Dec 22, 2010, and Aug 6, 2013, we randomly allocated 305 (34%) patients from the cohort of 887 patients to doxycycline (152 [50%]) or placebo (153 [50%]), excluding four (1%) patients (two [1%] from each group) who were incorrectly randomly allocated from the analysis. 257 (85%) of 301 patients had a next exacerbation (131 [87%] of 150 in the doxycycline group vs 126 [83%] of 151 in the placebo group). Median time to next exacerbation was 148 days (95% CI 95–200) in the doxycycline group compared with 161 days (118–211) in the placebo group (hazard ratio 1·01 [95% CI 0·79–1·31]; p=0·91). We did not note any significant differences between groups in the frequency of adverse events during the first 2 weeks after randomisation (47 [31%] of 150 in the doxycycline group vs 53 [35%] of 151 in the placebo group; p=0·54) or in serious adverse events during the 2 years of follow-up (42 [28%] vs 43 [29%]; p=1). Interpretation In patients with mild-to-severe COPD receiving treatment for an exacerbation in an outpatient setting, the antibiotic doxycycline added to the oral corticosteroid prednisolone did not prolong time to next exacerbation compared with prednisolone alone. These findings do not support prescription of antibiotics for COPD exacerbations in an outpatient setting. Funding Netherlands Organization for Health Research and Development.

Intervertebral disc (IVD) degeneration leading to chronic low back pain is a major problem in developed countries. Regeneration of the IVD can prevent pain and costs related to health care expenditure and work absenteeism, and improve quality of life. The IVD is the flexible structure between our vertebral bodies that allows for movement of our spinal column. Degeneration is thought to start in the nucleus pulposus (NP), the gelatinous core of the IVD. The osmotic value of a disc decreases during degeneration due to the characteristic loss of proteoglycans. To understand how the osmolality affects NP cells, they were cultured with medium of different osmolalities, adjusted with different osmolytes: NaCl, urea and sucrose. Gene expression for both the NP extracellular matrix (ECM) components type II collagen and aggrecan increased with increasing osmolality using NaCl or sucrose, but not urea. Protein production of these ECM components however, was not affected by increasing osmolality and was even decreased in the presence of urea or sucrose. Hydrogels are employed as NP cell carriers and can facilitate regeneration, either for clinical application or research into mechanisms of regeneration. We compared six different hydrogels based on natural polymers: alginate, agarose, fibrin, type II collagen, gelatin methacryloyl (gelMA), and hyaluronic acid–poly(ethylene glycol), and investigated the role of serum in the medium and of osmolarity during expansion or redifferentiation of NP cells. Agarose hydrogels seem to be the best option for in vitro culture of human NP cells, but for clinical application, type II collagen hydrogels may be better because they are more biocompatible. Culture in serum-free medium, necessary for clinical application, reduced the amount of proteoglycans produced during redifferentiation culture. Isolation and expansion of NP cells in high osmolarity medium increased regeneration, also in the absence of serum. High osmolality during redifferentiation culture again had no effect. One way hydrogels can influence regeneration is by their biomechanical properties. Stiffness sensing through the cell’s focal adhesions is believed to direct chondrogenesis, but the mechanisms involved are largely unknown. We compared focal adhesion formation and proteoglycan deposition by NP cells in a range of hydrogels. Using a focal adhesion kinase (FAK) inhibitor, we demonstrated that focal adhesion signaling is involved in the response of NP cells to stiffness of hydrogels that contain integrin binding sites (i.e. gelMA and type II collagen), but not to hydrogels deplete from integrin binding sites such as alginate and agarose, or CD44-binding hydrogels based on hyaluronic acid. NP cells can also be stimulated to regenerate by addition of growth factors or mesenchymal stromal cells (MSCs). Bone morphogenetic proteins (BMPs) are among the most promising growth factors for NP regeneration. It is not known which BMP, heterodimer or combination of homodimers would perform best for NP regeneration. Our results indicate that BMP4 might have the highest potential for regeneration of the intervertebral disc and can be applied together with MSCs. Moreover, the added value of BMP heterodimers over their respective homodimer BMP combinations depends on the BMP combination applied and the cells used.

Hydrogels can facilitate nucleus pulposus (NP) regeneration, either for clinical application or research into mechanisms of regeneration. However, many different hydrogels and culture conditions for human degenerated NP have been employed, making literature data difficult to compare. Therefore, we compared six different hydrogels of natural polymers and investigated the role of serum in the medium and of osmolarity during expansion or redifferentiation i n an attempt to provide comparators for future studies. Human NP cells of Thompson grade III discs were cultured in alginate, agarose, fibrin, type II collagen, gelatin methacryloyl (gelMA), and hyaluronic acid-poly(ethylene glycol) hydrogels. Medium containing fetal bovine serum and a serum-free (SF) medium were compared in agarose, gelMA, and type II collagen hydrogels. Isolation and expansion of NP cells in low compared to high osmolarity medium were performed before culture in agarose and type II collagen hydrogels in media of varying osmolarity. NP cells in agarose produced the highest amounts of proteoglycans, followed by cells in type II collagen hydrogels. The absence of serum reduced the total amount of proteoglycans produced by the cells, although incorporation efficiency was higher in type II collagen hydrogels in the absence than in the presence of serum. Isolation and expansion of NP cells in high osmolarity medium improved proteoglycan production during culture in hydrogels, but variation in osmolarity during redifferentiation did not have any effect. Agarose hydrogels seem to be the best option for in vitro culture of human NP cells, but for clinical application, type II collagen hydrogels may be better because, as opposed to agarose, it degrades in time. Although culture in SF medium reduces the amount of proteoglycans produced during redifferentiation culture, isolating and expanding the cells in high osmolarity medium can largely compensate for this loss.

During intervertebral disc (IVD) maturation, the main cell type shifts from notochordal cells (NCs) to chondrocyte-like cells (CLCs). NCs secrete factors with regenerative potential, making them an interesting focus for regenerative treatments. During initial development, these strategies preferably employ non-human donors due to easy availability of their NC-rich nucleus pulposus (NP) tissue. To increase the success of translating these strategies for clinical application, this study aimed to delineate whether NC-secreted factors of different species have a regenerative effect on human CLCs. Human, canine and porcine NC-rich NP tissue and NC-conditioned medium (NCCM) were analysed biochemically and histologically. Human CLC micro-aggregates from degenerated IVDs were cultured in human, canine or porcine NCCM. Collagen, glycosaminoglycan (GAG) and DNA content was determined and histology was performed. Canine and porcine NPs were richer in NCs than human NPs. Human NPs contained the highest collagen content, whereas the DNA and GAG content of canine NPs was significantly higher than that of human or porcine NPs. NCCM from all species significantly increased the DNA and GAG content of the human CLC micro-aggregates. Porcine and canine NCCM were significantly more potent than human NCCM in inducing GAG deposition, whereas only human NCCM induced collagen type II production. Secreted factors from human, canine and porcine NC-rich NPs exerted regenerative effects on human CLCs, indicating a cross-species effect. Bioactive compound(s) are present in NCCM of different species that may reverse human IVD degeneration, supporting further research into strategies based on NC-technology employing canine or porcine models for their translation into humans.

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Eosinophilic inflammation in chronic obstructive pulmonary disease (COPD) is predictive for responses to inhaled steroids. We hypothesised that the inflammatory subtype in mild and moderate COPD can be assessed by exhaled breath metabolomics. Exhaled compounds were analysed using gas chromatography and mass spectrometry (GC-MS) and electronic nose (eNose) in 28 COPD patients (12/16 Global Initiative for Chronic Obstructive Lung Disease (GOLD) stage I/II, respectively). Differential cell counts, eosinophil cationic protein (ECP) and myeloperoxidase (MPO) were measured in induced sputum. Relationships between exhaled compounds, eNose breathprints and sputum inflammatory markers were analysed and receiver operating characteristic (ROC) curves were constructed. Exhaled compounds were highly associated with sputum cell counts (eight compounds with eosinophils, 17 with neutrophils; pExhaled molecular profiles are closely associated with the type of inflammatory cell and their activation status in mild and moderate COPD. This suggests that breath analysis may be used for assessment and monitoring of airway inflammation in COPD.

Intervertebral disc (IVD) degeneration is associated with most cases of cervical and lumbar spine pathologies, amongst which chronic low back pain has become the number one cause of loss of quality-adjusted life years. In search of alternatives to the current less than optimal and usually highly invasive treatments, regenerative strategies are being devised, none of which has reached clinical practice as yet. Strategies include the use of stem cells, gene therapy, growth factors and biomaterial carriers. Biomaterial carriers are an important component in musculoskeletal regenerative medicine techniques. Several biomaterials, both from natural and synthetic origin, have been used for regeneration of the IVD in vitro and in vivo. Aspects such as ease of use, mechanical properties, regenerative capacity, and their applicability as carriers for regenerative and anti-degenerative factors determine their suitability for IVD regeneration. The current review provides an overview of the biomaterials used with respect to these properties, including their drawbacks. In addition, as biomaterial application until now appears to have been based on a mix of mere availability and intuition, a more rational design is proposed for future use of biomaterials for IVD regeneration. Ideally, high-throughput screening is used to identify optimally effective materials, or alternatively medium content comparative studies should be carried out to determine an appropriate reference material for future studies on novel materials.

High levels of histamine can be found in the airways of asthma patients. This study describes the effects of histamine on anti-CD3-induced production of IL-4, IL-5, and IFN-gamma by T cell clones from subjects with allergic asthma and healthy subjects. T cell clones were obtained from bronchoalveolar lavage (BAL) fluid and blood. The number of clones tested, and the percentage of clones in which histamine inhibited or enhanced cytokine production by more than 25%, were as follows: IL-4, 47, 8.5%, and 4.3%; IL-5, 43, 14%, and 30%; and IFN-gamma, 52, 40%, and 15%. Inhibition of IL-5 and IFN-gamma production was reversed by IL-2. The enhancement of IFN-gamma production was associated with an enhancement of both IL-2 production and proliferation. In 21% of the clones a combined effect consisting of inhibition of IFN-gamma production and enhancement of IL-5 production was found. This response was reversed by H2-receptor antagonists and was significantly associated with a histamine-induced increase in intracellular levels of cAMP. The role of cAMP in mediating the histamine effects was supported by the observations that the beta2-agonist salbutamol had effects similar to histamine and that high concentrations of PGE2 mimicked the inhibitory effects of histamine. Clones from BAL fluid and blood showed similar responses, as did clones from patients with asthma and from control subjects. The enhancement of IFN-gamma production by histamine, however, was found only in clones from healthy subjects. The results warrant further investigations on the role of cAMP in the regulation of cytokine production.

Rationale Adult-onset asthma differs from childhood-onset asthma in many respects. It is more heterogeneous, often severe and frequently associated with loss of lung function. To identify underlying mechanisms of adult-onset asthma and to capture predictors of disease progression, detailed characterization and phenotyping is necessary. Objectives To characterize adult-onset asthma and identify subphenotypes of adult-onset asthma. Methods A cohort of 200 patients with adult-onset (>18 year) asthma (age 54 (26–75) year) was recruited from one academic and three nonacademic pulmonary outpatient clinics in Amsterdam, the Netherlands. These patients were fully characterized with respect to clinical, functional and inflammatory markers. After data reduction, K-means nonhierarchical cluster analysis was performed to identify clusters of adult-onset asthma. Measurements and main results Patients with adult-onset asthma were predominately female (61%) and nonatopic (55%). Within this group of patients were identified three clusters of adult-onset asthma. Cluster 1 (n = 69) consisted of patients with severe eosinophilic inflammation-predominant asthma and persistent airflow limitation despite high-intensity anti-inflammatory treatment, with relatively low symptom scores. The second cluster was characterized by obese women with frequent symptoms, high healthcare utilization and low sputum eosinophils. The third cluster consisted of patients with mild-to-moderate, well-controlled asthma with normal lung function and low inflammatory markers. Repeatability accuracy was 98.2%. Conclusions Amongst patients with adult-onset asthma, three subphenotypes can be identified with distinct clinical and inflammatory characteristics. These subphenotypes help to understand the underlying pathobiology and provide clinicians with directions for personalized management.

Background Eosinophilic airway inflammation has successfully been used to tailor anti-inflammatory therapy in chronic obstructive pulmonary disease (COPD). Airway hyperresponsiveness (AHR) by indirect challenges is associated with airway inflammation. We hypothesized that AHR to inhaled mannitol captures eosinophilia in induced sputum in COPD. Methods Twenty-eight patients (age 58 ± 7.8 yr, packyears 40 ± 15.5, post-bronchodilator FEV1 77 ± 14.0%predicted, no inhaled steroids ≥4 wks) with mild-moderate COPD (GOLD I-II) completed two randomized visits with hypertonic saline-induced sputum and mannitol challenge (including sputum collection). AHR to mannitol was expressed as response-dose-ratio (RDR) and related to cell counts, ECP, MPO and IL-8 levels in sputum. Results There was a positive correlation between RDR to mannitol and eosinophil numbers (r = 0.47, p = 0.03) and level of IL-8 (r = 0.46, p = 0.04) in hypertonic saline-induced sputum. Furthermore, significant correlations were found between RDR and eosinophil numbers (r = 0.71, p = 0.001), level of ECP (r = 0.72, p = 0.001), IL-8 (r = 0.57, p = 0.015) and MPO (r = 0.64, p = 0.007) in sputum collected after mannitol challenge. ROC-curves showed 60% sensitivity and 100% specificity of RDR for >2.5% eosinophils in mannitol-induced sputum. Conclusions In mild-moderate COPD mannitol hyperresponsiveness is associated with biomarkers of airway inflammation. The high specificity of mannitol challenge suggests that the test is particularly suitable to exclude eosinophilic airways inflammation, which may facilitate individualized treatment in COPD. Trial registration Netherlands Trial Register (NTR): NTR1283

A 25 year old man with known chronic right sided heart failure and ascites due to a congenital heart defect presented with dyspnoea and a massive pleural effusion. Thoracoscopy revealed two diaphragmatic blebs. Changes in peritoneal and thoracic pressure during respiration resulted in periodic squirting of a ruptured bleb, illustrating preferential flow of peritoneal fluid into the thorax. The pleural effusion was successfully treated with drainage of ascitic fluid and chemical pleurodesis.

To the Editor: Thoracic masses due to extramedullary hematopoiesis are rare, occurring most often in patients with thalassemia or congenital hemolytic anemia.1 The masses usually cause no symptoms but can cause pleural effusion or hemothorax. Here, we describe a patient with respiratory insufficiency caused by large, hematopoietic thoracic masses. A 51-year-old woman with type III congenital dyserythropoietic anemia2 (diagnosed in 1971, with lifelong anemia) was referred to our clinic in December 1998 because of exertional dyspnea and respiratory insufficiency. In 1980, a routine radiograph of the chest had shown a small, right-sided thoracic mass; biopsy had revealed extramedullary hematopoiesis. In . . .

Cytokines produced by T-lymphocytes play an important regulatory role in inflammation in the airways of asthmatic patients. Our aim was to analyse the cytokine production by T-cell clones from bronchoalveolar lavage fluid (BAL) of patients with allergic asthma and the cytokine production of clones from the patients' peripheral blood (PB), as well as from BAL and blood from healthy controls. In 75 randomly selected CD4+ T-cell clones, we assessed the production of interleukin (IL)-2, IL-4 and interferon-gamma (IFN-gamma). After stimulation with anti-CD3, the clones from the asthmatic patients' BAL (A-BAL) produced significantly more IL-4 and IFN-gamma (median 0.32 and 4.17 ng.mL-1, respectively) than clones from A-PB (0.11 and 1.12 ng.mL-1, respectively). No evidence was found for a dominance of a type 1 or type 2 T-helper cell (Th1- or Th2)-cytokine profile in any of the groups. In three out of nine clones tested, the stimulation with anti-CD2/CD28/phorbol myristate acetate (PMA) induced a shift of the IFN-gamma/IL-4 ratio towards a Th2-type cytokine profile. Our results suggest that the clones from the asthmatic patients' bronchoalveolar lavage were derived from a more differentiated T-cell population. In several clones, the cytokine profile was still modulated by the stimulus applied. Similarly, local conditions in the airways may be involved in directing the cytokine production of T-cells.

The present results support a role for epithelial cells in the activation of T cells in an apparent antigen-independent manner. The transient expression of CD25 indicates a short acting T cells activation. Possibly, this event primes T cells to respond swiftly upon antigen-specific stimulation or to synthesize mediators that affect the local milieu. The molecular mechanism of interaction, although not well defined possibly involves LFA3-CD2 interactions. In T cell activation, via LFA3-CD2 interaction, the density of presented LFA3 molecules is critical. With the increase in the level of expression of LFA3 by epithelial cells this critical density may have been reached. However, based on what is known about T cell activation and CD25 expression in particular it is likely that additional signals such as soluble mediators are required for T cell activation by epithelial cells. Whether this mode of activation occurs in vivo remains to be established by studying ex vivo and in situ material. Not much is known about the expression of LFA3 by epithelial cells in vivo, nor about the stimuli that induce the upregulation of LFA3. In preliminary experiments with fluorescence microscopy we found that neither TNF-alpha nor IL-1 beta induce LFA3 in the same fashion as IFN-gamma. In conclusion, T cell activation by epithelial cells could be an important feature in inflammatory and immunological processes in mucosal systems such as the bronchi and deserves further research.

Both increased T cell numbers and their increased activation state have implicated an important role for T cells in chronic inflammatory reactions seen in the airways of (allergic) asthmatics. Airway epithelial cells are frequently exposed to stimuli that cause the release of mediators and the expression of cell adhesion molecules. We have examined whether human airway epithelial cells can activate lung-derived T cells. Clonal lung T cells showed an increased adherence to transformed airway epithelial cells that had been exposed previously for 2 h to human recombinant interferon-gamma (IFN-gamma; 100 U/ml). After an additional 16-24 h of culturing in the absence or presence of epithelial cells, T cells expressed increased levels of both the alpha-chain of the interleukin-2 receptor (IL-2R, CD25) and the transferrin receptor (CD71), both markers of T cell activation. T cells apparently activated by epithelial cells, however, did not produce IFN-gamma or IL-4 nor showed an increased proliferation on the addition of IL-2 (5-50 U/ml). The induced adherence to and the activation of T cells by epithelial cells is mediated largely by CD2 and its ligand lymphocyte functional antigen-3, a pathway known to up- and downregulate T cell functions

The effects of treatment with budesonide (200-mu-g twice daily) and terbutaline (500-mu-g four times daily) has been compared with the effects of placebo and terbutaline in 27 children with mild asthma, aged 7-14 years, in a double blind, randomised placebo controlled study over eight weeks. Bronchial responsiveness (PC20 histamine), lung function, the amplitude of diurnal variation in peak expiratory flow (PEF), and symptom scores were measured. Baseline FEV1 was over 70% predicted and PC20 histamine less than 8 mg/ml. Twelve children were treated with budesonide and terbutaline and 15 with placebo and terbutaline. After four and eight weeks of treatment the change in PC20 was significantly greater after terbutaline alone by 2.1 (95% CI 0.5-3.8) and 1.3 (95% CI 0.1-2.5) doubling doses respectively. Mean FEV1 did not change in either group. The change in afternoon and nocturnal PEF was significantly greater after budesonide and terbutaline than after terbutaline alone. The amplitude of diurnal variation in PEF did not change significantly in either group. Peak flow reversibility decreased in the budesonide group. There were no differences between treatments for cough and dyspnoea, but wheeze improved in the budesonide group. The children with mild asthma treated with budesonide and terbutaline showed improvement in bronchial responsiveness, afternoon and nocturnal PEF, and symptoms of wheeze and a fall in peak flow reversibility by comparison with those who received terbutaline alone.