Optimizing care for children with difficult-to-treat and severe asthma through specialist paediatric asthma centres: expert practical experience and advice.

Severe asthma in children carries an unacceptable treatment burden, yet its rarity means clinical experience in treating it is limited, even among specialists. Practical guidance is needed to support clinical decision-making to optimize treatment for children with this condition.This modified Delphi convened 16 paediatric pulmonologists and allergologists from northern Europe, all experienced in treating children with severe asthma. Informed by interviews with stakeholders involved in the care of children with severe asthma (including paediatricians, nurses and carers), and an analysis of European guidelines, the experts built a consensus focused on the gaps in existing guidance. Explored were considerations for optimizing care for patients needing biologic treatment, and for selecting home or hospital delivery of biologics. This consensus is aimed at clinicians in specialist centres, as well as general paediatricians, paediatric allergologists and paediatric pulmonologists who refer children with the most severe asthma to specialist care. Consensus is based on expert opinion and is intended for use alongside published guidelines.Our discussions revealed three key facets to optimizing care. Firstly, early asthma detection in children presenting with wheezing and/or dyspnoea is vital, with a low threshold for referral from primary to specialist care. Secondly, children who may need biologics should be referred to and managed by specialist paediatric asthma centres; we define principles for the specialist team members, tests, and expertise necessary at such centres, as well as guidance on when homecare biologics delivery is and is not appropriate. Thirdly, shared decision-making is essential at all stages of the patient's journey: clear, concise treatment plans are vital for patient/carer self-management, and structured processes for transition from paediatric to adult services are valuable. The experts identified the potential for specialist paediatric asthma nurses to play a significant role in facilitating multidisciplinary working.Through this project is agreed a framework of practical advice to optimize the care of children with severe asthma. We encourage clinicians and policymakers to implement this practical advice to enhance patient care.

Adulthood asthma after wheezing in infancy: a questionnaire study at 27 years of age.

BACKGROUND: Wheezing in early childhood is a heterogeneous condition, the longterm prognosis varying from total recovery to chronic asthma. Though short-term outcome has been actively studied, there is lack of data on long-term outcome until adulthood. The aim of the study was to evaluate the prevalence and risk factors of asthma at 26-29 years of age after early-life wheezing. METHODS: At the median age of 27.3 years (range 26.3-28.6), a questionnaire was sent to 78 study subjects hospitalized for wheezing at <24 months of age, and 59 (76%) answered. Asthma, allergy and weight status were compared with selected controls followed up from birth and with non-selected population controls recruited for this adulthood study. RESULTS: Doctor-diagnosed asthma was present in 20% of the former bronchiolitis patients, compared with 5% in the two control groups (OR 2.1, 95% CI 0.3-17.9 vs selected controls; OR 5.2, 95% CI 1.7-15.8 vs nonselected controls). The respective figures for current self-reported asthma were 41% and 7-10% (OR 11.4, 95% CI 2.3-56.1 vs selected controls; OR 12.2, 95% CI 4.4-33.7 vs nonselected controls). Current allergic rhinitis and current smoking were significantly associated with asthma, but current overweight or obesity was not. In multivariate analyses, early-life wheezing was an independent risk factor of adulthood asthma. CONCLUSION: An increased asthma risk in early-life wheezers continues, even after many symptom-free years at school age, at least until 27 years of age.

Differences in inflammatory responses and cytotoxicity in RAW264.7 macrophages induced by Streptomyces Anulatus grown on different building materials.

Streptomyces anulatus, an indicator microbe of mold in buildings, was grown on different building materials in order to study the impact of growth conditions on the ability of the spores of this microbe to induce toxicity and inflammatory responses. The microbes were grown for 2 months on sterilized and unsterilized wood, chipboard, concrete, plaster board and mineral wool in tight glass vessels under humid conditions. The highest microbial spore concentration was detected on the sterilized mineral wool followed by the sterilized plaster board and the unsterilized mineral wool. Mouse RAW264.7 macrophages were exposed in vitro for 24 h to the spores of S. anulatus and the production of the inflammatory mediators, nitric oxide (NO), tumor necrosis factor alpha (TNF alpha), interleukin-6 (IL-6), interleukin-10 (IL-10) and cytotoxicity, were measured. The dose equivalent to 5 x 10(5) spores/ml of medium was used to compare the different materials. The most intense production of NO (11.6 microM), TNF alpha (560 pg/ml) and IL-6 (2800 pg/ml) in macrophages was induced by the spores grown on sterilized plaster board. They also caused the greatest loss of cell viability (39%). The spores grown on sterilized concrete induced significant production of NO (1.5 microM) and decreased cell viability (22%), and the spores grown on unsterilized and sterilized mineral wool increased production of NO (4.1 microM and 0.8 microM, respectively). The spores did not stimulate production of the anti-inflammatory cytokine IL-10. These results indicate that the ability of S. anulatus to induce inflammatory responses and cytotoxicity in macrophages is dependent on the growth conditions provided by different building materials.

Pain behavior and response properties of spinal dorsal horn neurons following experimental diabetic neuropathy in the rat: modulation by nitecapone, a COMT inhibitor with antioxidant properties.

We attempted to characterize a spinal neuronal correlate of painful neuropathy induced by diabetes mellitus (DM). Pain behavior and response properties of spinal dorsal horn neurons were determined in rats with a streptozocin-induced DM. A catechol-O-methyltransferase inhibitor with potent antioxidant properties, nitecapone, was used in an attempt to attenuate neuropathic symptoms. Behaviorally DM induced mechanical hypersensitivity that was markedly attenuated by oral treatment with nitecapone. The antihyperalgesic effect of nitecapone was not reversed by naloxone, an opioid antagonist, or atipamezole, an alpha-2-adrenoceptor antagonist. Electrophysiological recordings performed in pentobarbitone-anesthetized animals revealed that the most distinct abnormality in response properties of spinal dorsal horn wide-dynamic range (WDR) neurons was the increase in their spontaneous activity observed in untreated but not in nitecapone-treated DM rats. Conditioning electrical stimulation and a lidocaine block of the rostroventromedial medulla (RVM) had a similar modulatory effect on evoked responses of spinal dorsal horn WDR neurons in all experimental groups. The response properties of spinal dorsal horn nociceptive-specific or low-threshold mechanoreceptive neurons were not markedly different between the experimental groups. The results indicate that increased spontaneous activity in spinal dorsal horn WDR neurons may be causally related to behaviorally observed mechanical hypersensitivity in DM. Attenuation of the increased spontaneous activity in WDR neurons may explain the antihyperalgesic effect by nitecapone, due to naloxone- and alpha-2-adrenoceptor-insensitive mechanisms. DM or nitecapone treatment did not produce significant changes in phasic or tonic descending pain regulation originating in the RVM.

Effect of growth medium on potential of Streptomyces Anulatus spores to induce inflammatory responses and cytotoxicity in RAW264.7 macrophages.

Epidemiological studies have shown an association between microbial growth in buildings and increased risk of respiratory symptoms and disease related to inflammatory reactions in the inhabitants96. The current study examined the affects of growth conditions of Streptomyces anulatus, isolated from indoor air of a moldy building, on the inflammatory potential of spores of this microbe. Spores were harvested from 15 growth media formulations, applied to RAW264.7 macrophages (10(5), 10(6), or 10(7) spores/million cells), and evaluated for the ability to stimulate production of inflammatory mediators and cytotoxicity in these cells 24 h after exposure. Streptomyces anulatus spores induced dose-dependent production of nitric oxide (NO) in macrophages, reaching a level from 4.2 microM to 39.2 microM depending on the composition of the growth medium of the microbe. Expression of inducible NO synthase (iNOS) was detected in macrophages after exposure to spores collected from all growth media. Production of reactive oxygen species (ROS) was significantly increased only by the highest dose of S. anulatus spores grown on glycerol-arginine agar. Furthermore production of cytokines was affected by growth medium; the highest dose-dependent levels of interleukin 6 (IL-6) ranged from 900 to 7800 pg/ml, and the levels of tumor necrosis factor alpha (TNFalpha) varied from 490 to 3200 pg/ml. The amount of dead macrophages after the exposure varied from 11% to 96%, depending also on the growth media of the microbe. Altogether, our results suggest that the growth medium of S. anulatus has a fundamental role in the ability of the spores to induce inflammatory responses and cytotoxicity in mammalian cells.

Inflammatory responses in RAW264.7 macrophages caused by mycobacteria isolated from moldy houses.

Mycobacterial strains (nonpathogenic Mycobacterium terrae, potentially pathogenic Mycobacterium avium-complex and Mycobacterium scrofulaceum), isolated from a moldy building, were studied with respect to their ability to stimulate macrophages (RAW264.7) to produce inflammatory mediators, and to cause cytotoxicity. Reactive oxygen species (ROS) were measured by chemiluminescence, cytokines (TNF-alpha, IL-6, IL-1, IL-10) immunochemically, nitric oxide (NO) by Griess-method, expression of inducible NO-synthase (iNOS) with Western Blot analysis and cytotoxicity with MTT-test. All the strains induced dose- and time-dependent production of NO, IL-6 and TNF-alpha in macrophages, whereas IL-1 or IL-10 production was not detected. The production of ROS and cytotoxicity was increased with the highest doses. Interestingly, different strains had significant differences in their ability to induce these responses, M. terrae being the most potent and M. avium-complex the weakest one. These results indicate that both non- and potentially pathogenic strains of mycobacteria present in moldy buildings are capable of activating inflammatory mechanisms in macrophages.

Effect of acute nicotine administration on striatal dopamine output and metabolism in rats kept at different ambient temperatures.

1. The effect of ambient temperature on the nicotine-induced (0.3, 0.5 or 0.8 mg kg(-1) s.c.) changes of the striatal concentrations of dopamine (DA) and its metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) was studied in freely-moving rats by in vivo microdialysis. 2. At the ambient temperature of 30 - 33 degrees C, but not at 20 - 23 degrees C, nicotine doses of 0.5 (P<0. 01) and 0.8 mg kg(-1) (P<0.05) significantly increased the extracellular DA concentration. The nicotine doses of 0.5 and 0.8 mg kg(-1) increased the DA metabolite levels similarly at both ambient temperatures studied (P

Nitric oxide and proinflammatory cytokines in nasal lavage fluid associated with symptoms and exposure to moldy building microbes.

Epidemiological data indicate that living or working in a moldy building is associated with increased risk of respiratory symptoms and disease related to inflammatory reactions, but biochemical evidence linking cause and effect is still scarce. The staff working in a mold-contaminated school, and a reference group without such exposure, were studied. Nasal lavage was performed and health data were collected with a questionnaire at the end of the spring term, after a 2.5-mo summer vacation, and at the end of the fall term. Here we show that concentrations of tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6), and nitric oxide (NO) in nasal lavage fluid were significantly higher in the exposed than in the control subjects at the end of the first exposure period. These inflammatory mediators decreased to reference group concentrations during the period when there was no exposure and the production of NO and IL-6 increased again during the reexposure in the fall term. Reports of cough, phlegm, rhinitis, eye irritation, and fatigue paralleled the changes in the measured inflammatory markers. These results point to an association between inflammatory markers in the nasal lavage fluid, the high prevalence of respiratory symptoms among the occupants, and chronic exposure to molds in the indoor environment.

Production of reactive oxygen species by man-made vitreous fibres in human polymorphonuclear leukocytes.

Human polymorphonuclear leukocytes (PMNL) or erythrocytes, isolated from human blood, were exposed to graded doses of asbestos (chrysotile), quartz, or man-made vitreous fibres (MMVF), i.e. refractory ceramic fibres (RCF), glasswool, or rockwool fibres. None of the MMVF affected either the viability of PMNL, as measured by trypan blue exclusion test, or induced haemolysis, whereas the positive controls, quartz and chrysotile, dose-dependently induced haemolysis in PMNL. MMVF did not increase the release of lactate dehydrogenase (LDH) from the PMNL, whereas the positive controls, chrysotile and quartz, induced a marked and dose-dependent release of LDH. When PMNL were exposed to MMVF, some of the fibre types slightly increased the levels of free intracellular calcium ([Ca2+]i) within the cells in a manner similar to that induced by chrysotile or quartz. All MMVF induced a dose-dependent production of reactive oxygen species (ROS) in PMNL, with RCF-induced production of ROS being the most marked. Production of ROS by MMVF seemed to depend on the availability of extracellular calcium because it could be attenuated with a Ca2+ channel blocker, verapamil, or a Ca2+ chelating agent, EGTA. Production of ROS may be a common pathway through which PMNL respond to MMVF-induced cell activation, but alterations of levels of free intracellular Ca2+ do not seem to be an absolute prerequisite for this effect. Fibre length seemed not to be an important factor in affecting the ability of MMVF to induce ROS production in PMNL. However, the balance between different elements in the fibre seemed importantly to affect the biological activity of a fibre.

Characterization of the decrease of extracellular striatal dopamine induced by intrastriatal morphine administration.

The effect of intrastriatally-administered morphine on striatal dopamine (DA) release was studied in freely moving rats. Morphine (1, 10 or 100 microM) was given into the striatum by reversed microdialysis, and concentrations of DA and its metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) were simultaneously measured from the striatal dialysates. Intrastriatally-administered morphine significantly and dose-dependently decreased the extracellular concentration of DA, the concentrations of the acidic DA metabolites were only slightly decreased. The effect of morphine was antagonized by naltrexone (2.25 mg kg(-1), s.c.). Pretreatment with a preferential kappa-opioid receptor antagonist, MR2266 [(-)-5,9 alpha-diethyl-2-(3-furylmethyl)-2'-hydroxy-6,7-benzomorphane; 1 mg kg(-1), s.c.], had no effect on the decrease of extracellular DA evoked by intrastriatal morphine (100 microM). Intrastriatal administration of the selective micro-opioid receptor agonist [D-Ala2,MePhe4,Gly-ol5] enkephalin (DAMGO; 1 microM), significantly decreased the extracellular concentration of DA in the striatum. When the rats were given morphine repeatedly in increasing doses (10-25 mg kg(-1), s.c.) twice daily for 7 days and withdrawn for 48 h, the decrease of extracellular DA induced by morphine (100 microM) was significantly less than that seen in saline-treated controls. Our results show that besides the well-known stimulatory effect there is a local inhibitory component in the action of morphine on striatal DA release in the terminal regions of nigrostriatal DA neurones. Tolerance develops to this inhibitory effect during repeated morphine treatment. Furthermore, our results suggest that the effect of intrastriatally-administered morphine is mediated by the micro-opioid receptors.

Diesel particles induce nitric oxide production in murine alveolar macrophages and rat airways.

The acute adverse health effects among respiratory and cardiovascular patients have been associated with particulate air pollution, containing diesel particles (DP). The mechanisms of these effects are unknown, but they may involve inflammation. We investigated the effects of DP (30-3000 µg/10(6) cells) on cell viability and production of nitric oxide (NO), interleukin-1 (IL-1), interleukin-6 (IL-6), interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) in murine RAW 264.7 macrophage cultures in vitro. DP caused a dose- and time-dependent NO-production and was cytotoxic in murine RAW 264.7 macrophages. Cytotoxicity preceded the increases in NO production. DP had minimal effects on cytokine production. A single intratracheal instillation of DP 1 and 5 mg/rat increased NO production and protein concentration in bronchoalveolar lavage fluid, and caused pulmonary edema and hemorrhage. The present results indicate that DP can induce both NO production and cytotoxicity in the lower respiratory tract, which may contribute to the short-term adverse respiratory effects of these particles.

Streptomyces anulatus from indoor air of moldy houses induce NO and IL-6 production in a human alveolar epithelial cell-line.

Moisture associated microbial growth in buildings may cause respiratory symptoms such as pulmonary inflammation. We studied the effects of spores of Streptomyces anulatus, commonly found in moldy buildings, on the production of nitric oxide (NO), interleukin-6 (IL-6), interleukin-5 (IL-5), interleukin-4 (IL-4), and tumour necrosis factor alpha (TNFα), as well as cell viability in human alveolar II type epithelial cell line (A549). Cells were exposed in vitro to S. anulatus spores with and without interferon-γ (IFNγ) in vitro. Lipopolysaccharide (LPS) was used as a reference substance. S. anulatus alone, and in combination with IFNγ induced NO and IL-6 production and decreased cell viability whereas IL-4, IL-5 or TNFα production were not affected. IFNγ alone had a weaker but otherwise similar effect as S. anulatus on NO and IL-6 production and it potentiated the effects of S. anulatus. LPS did not induce NO or cytokine production, or affect cell viability in A549 cells. These data indicate that spores of S. anulatus induce the excretion of inflammatory mediators in respiratory epithelial cells, which may partly explain the adverse respiratory health effects experienced by individuals exposed to the indoor air of moldy houses.

Locally infused taurine, GABA and homotaurine alter differently the striatal extracellular concentrations of dopamine and its metabolites in rats.

We studied in vivo the effects of locally infused taurine (50, 150, and 450 mM) on the striatal dopamine and its metabolites in comparison with those of GABA and homotaurine, a GABAA receptor agonist, in freely moving rats. The extracellular dopamine concentration was elevated maximally 2.5-, 2- and 4-fold by taurine, GABA and homotaurine, respectively. At 150 mM concentration, at which the maximum effects occurred, homotaurine increased the extracellular dopamine more than taurine or GABA. When taurine and GABA were infused simultaneously with tetrodotoxin the output of dopamine did not differ from that in the presence of tetrodotoxin alone. In comparison, tetrodotoxin did not inhibit the increase in extracellular dopamine caused by homotaurine. Furthermore, omission of calcium from the perfusion fluid inhibited the increase of extracellular dopamine caused by GABA. However, it did not block the increase of dopamine caused by taurine or homotaurine. The present study suggests that the effects of intrastriatal taurine, GABA and homotaurine on the striatal extracellular dopamine differ. Thus, these amino acids seem to affect the striatal dopaminergic neurons via more than one mechanism.

Cytotoxicity, production of reactive oxygen species and cytokines induced by different strains of Stachybotrys sp. from moldy buildings in RAW264.7 macrophages.

The ability of different strains of the fungus Stachybotrys, isolated from mold problem buildings, to induce cytotoxicity and production of important inflammatory mediators, i.e. nitric oxide (NO), reactive oxygen species (ROS), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in RAW264.7 macrophages were studied. Several strains of Stachybotrys sp. stimulated immediate increase in the ROS production and in 24-h exposure caused TNF-α and IL-6 release from these cells. However, none of the strains of Stachybotrys sp. was able to induce the expression of inducible nitric oxide synthase (iNOS) and subsequent production of NO in RAW264.7 cells. Moreover, there were significant differences in their ability to induce cytotoxicity in the macrophages. These results suggest that, in addition to direct cytotoxic effects of most Stachybotrys sp., some strains of Stachybotrys sp. stimulate production of inflammatory mediators, TNF-α and IL-6 which were associated with low cytotoxicity in RAW264.7 macrophages.

Optimization of aqueous-based film coating of tablets performed by a side-vented pan-coating system.

The purpose of this research was to characterize the aqueous-based hydroxypropylmethylcellulose (HPMC) film coating of tablets utilizing a laboratory-scale side-vented pan-coating apparatus (Thai coater). The process and apparatus parameters of potential importance with respect to the final film quality were evaluated by using fractional factorial design (2(6-2)IV) and the process was optimized using response surface methodology (central composite design). Rotating speed of the pan was identified as a major parameter with respect to film thickness (weight increase; p < 0.05) and breaking strength (p < 0.05) of the aqueous HPMC film-coated tablets. Increasing the rotating speed from 5 rpm to 10 rpm resulted in a mean relative change of -43.9% and 2.4% of film thickness (weight increase) and breaking strength, respectively. As expected, inlet air temperature significantly affected the moisture content of the final film-coated tablets (p < 0.01) and the film thickness (weight increase; p < 0.05), but the effects on the other responses studied were minimal or negligible. Pneumatic spraying pressure and position of the spray gun (excluding angle of the gun) did not affect the responses studied. The process parameters relevant to a side-vented pan-coating process can be identified (by fractional factorial design) and, consequently, optimized (by central composite design) by using the factorial design approach.

Effect of viability of actinomycete spores on their ability to stimulate production of nitric oxide and reactive oxygen species in RAW264.7 macrophages.

Spores of actinomycetes, mesophilic gram-positive bacteria, isolated from moldy houses, induced the expression of inducible NO-synthase (iNOS) with a subsequent NO-production in RAW264.7 macrophages. No differences were detected between production of nitric oxide (NO) by alive or irradiated spores of different strains of Actinomycetes sp. or Streptomyces sp. Moreover, a significant production of reactive oxygen species (ROS) occurred in the macrophages after their stimulation both by alive and irradiation killed spores of actinomycetes. However, ROS-responses in macrophage induced by dead spores were significantly lower compared to those induced by alive spores. The cytotoxicity of the spores of different actinomycetes differed widely. The production of NO and ROS did not depend directly on the viability of the spores, suggesting an important role for cell wall components in the activation of the cells.

Intrastriatal taurine increases striatal extracellular dopamine in a tetrodotoxin-sensitive manner in rats.

In vivo effects of locally administered taurine on striatal dopamine release and metabolism were studied by microdialysis in freely moving rats. Concentrations of dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in striatal dialysates were quantified by high pressure liquid chromatography (HPLC) using electrochemical detection. Infusion of 150 mM taurine into the striatum for 2 h induced a 2.5-fold increase in the extracellular dopamine concentration. Extracellular DOPAC concentration increased nearly 2-fold. Taurine infusion initially decreased HVA to 70% but afterwards increased it to 140% of the control. When taurine was infused simultaneously with 1 microM tetrodotoxin starting 60 min after tetrodotoxin, the output of dopamine did not differ from that in the presence of tetrodotoxin alone. Tetrodotoxin abolished the effects of taurine on dopamine metabolites as well. Tetrodotoxin-sensitivity of the effects of taurine on dopamine and its metabolites suggests that intrastriatal taurine elevates extracellular dopamine by releasing it from neuronal pool.

5-HT3 receptor antagonist MDL 72222 dose-dependently attenuates cocaine- and amphetamine-induced elevations of extracellular dopamine in the nucleus accumbens and the dorsal striatum.

The effects of a 5-HT3 receptor antagonist MDL 72222 on cocaine- and amphetamine-induced increases in extracellular dopamine in the nucleus accumbens and the dorsal striatum were studied with microdialysis technique using halothane anaesthesized rats. Dopamine and its metabolites were measured by HPLC with electrochemical detection. Cocaine elevated extracellular dopamine in the nucleus accumbens and to a lesser extent in the dorsal striatum, but it did not affect dopamine metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid. Pretreatment with MDL 72222 (25-100 micrograms/kg) dose-dependently attenuated cocaine-induced elevation of dopamine in both of the nuclei studied. Amphetamine elevated extracellular dopamine and reduced DOPAC and homovanillic acid equally in the nucleus accumbens and in the dorsal striatum. MDL 72222 also attenuated the amphetamine-induced elevation of extracellular dopamine concentration in both brain areas studied, but first at a dose of 100 micrograms/kg. The different potencies of the interactions of the 5-HT3 receptor antagonist with cocaine and amphetamine could be related to the different mechanisms by which these drugs primarily elevate extracellular dopamine.