Effect of Plasmid DNA Size on Chitosan or Polyethyleneimine Polyplexes Formulation.

Gene therapy could be simply defined as a strategy for the introduction of a functional copy of desired genes in patients, to correct some specific mutation and potentially treat the respective disorder. However, this straightforward definition hides very complex processes related to the design and preparation of the therapeutic genes, as well as the development of suitable gene delivery systems. Within non-viral vectors, polymeric nanocarriers have offered an ideal platform to be applied as gene delivery systems. Concerning this, the main goal of the study was to do a systematic evaluation on the formulation of pDNA delivery systems based on the complexation of different sized plasmids with chitosan (CH) or polyethyleneimine (PEI) polymers to search for the best option regarding encapsulation efficiency, surface charge, size, and delivery ability. The cytotoxicity and the transfection efficiency of these systems were accessed and, for the best p53 encoding pDNA nanosystems, the ability to promote protein expression was also evaluated. Overall, it was showed that CH polyplexes are more efficient on transfection when compared with the PEI polyplexes, resulting in higher P53 protein expression. Cells transfected with CH/p53-pDNA polyplexes presented an increase of around 54.2% on P53 expression, while the transfection with the PEI/p53-pDNA polyplexes resulted in a 32% increase.

Additive Manufacturing Tools to Improve the Performance of Chromatographic Approaches.

Chromatography is widely applied industrially. However, some limitations are associated with its common supports, and the impossibility to fully control their structural features is particularly restrictive. Additive manufacturing (AM) is emerging as a fast, highly precise, and reproducible technology for producing chromatographic supports that can improve its performance.

Dilemma on plasmid DNA purification: binding capacity vs selectivity.

Plasmid DNA chromatography is a powerful field in constant development and evolution. The use of this technique is considered mandatory in the production of an efficient and safe formulation to be applied for plasmid-mediated gene therapy. Concerning this, the search for an ideal chromatographic support/ligand combination motivated scientist to pursue a continuous improvement on the plasmid chromatography performance, looking for a progression on the ligands and supports used. The present review explores the different approaches used over time to purify plasmid DNA, ambitioning both high recovery and high purity levels. Overall, it is presented a critical discussion relying on the relevance of the binding capacity versus selectivity of the supports.

Purification of supercoiled p53-encoding plasmid using an arginine-modified macroporous support.

p53 is a tumour suppressor gene that has been explored for cancer gene therapy as a possible alternative to the common treatments. The use of plasmid DNA (pDNA) to carry the therapeutic gene has been considered, but it is requisite to preserve its supercoiled (sc) structure, for eliciting a more effective gene expression and therapeutic action. The purification of the sc pDNA using amino acids-based affinity chromatography has been successfully applied, exploring different amino acids and supports. From these studies, it stood out the selectivity of arginine for the recognition of sc pDNA. However, some limitation on the binding capacity was found in the arginine-agarose support, and in the case of monoliths, some fouling and clogging can limit sequential runs. By using macroporous support modified with arginine it was expected to take advantage of the selectivity of the ligand combined with the flow properties and binding capacity offered by the support. The arginine-modified macroporous support was characterized by SEM, EDX and FTIR also to verify the correct immobilization of arginine, and then used for pDNA purification. The support showed to be effective on the sc p53-pDNA isolation, and the robustness was also achieved by accomplishing the purification of plasmids with different sizes, only by slightly adjusting the experimental conditions. Regarding the dynamic binding capacity of the arginine-modified macroporous support, it was achieved an improvement of more than 50% in the pDNA binding capacity when compared with their homologous arginine-agarose commercial matrix, suggesting potential economic feasibility in case of scale-up.

Composite Central Face Design-An Approach to Achieve Efficient Alginate Microcarriers.

Microparticulated drug delivery systems have been used as promising encapsulation systems for protecting drugs for in vitro and in vivo applications, enhancing its stability, providing an increased surface to volume ratio, reducing adverse effects, and hence an improvement in bioavailability. Among the studied microparticles, there is a rising interest in the research of alginate microparticles for pharmaceutical and biomedical fields confirming its potential to be used as an effective matrix for drug and cell delivery. Moreover, calcium alginate has been one of the most extensively forming microparticles in the presence of divalent cations providing prolonged drug release and suitable mucoadhesive properties. Regarding the above mentioned, in this research work, we intended to produce Ca-alginate micro-vehicles through electrospraying, presenting high encapsulation efficiency (EE%), reduced protein release across the time, reduced swelling effect, and high sphericity coefficient. To quickly achieve these characteristics and to perform an optimal combination among the percentage of alginate and CaCl2, design of Experiments was applied. The obtained model presented to be statistically significant (p-value < 0.05), with a coefficient of determination of 0.9207, 0.9197, 0.9499, and 0.9637 for each output (EE%, release, swelling, and sphericity, respectively). Moreover, the optimal point (4% of alginate and 6.6% of CaCl2) was successfully validated.

Bioelectric impedance vector analysis (BIVA) in hospitalised children; predictors and associations with clinical outcomes.

BACKGROUND: Clinical use of bioelectric impedance is limited by variability in hydration. Analysis of raw bioelectric impedance vectors (BIVA), resistance (R), reactance (Xc) and phase angle (PA) may be an alternative for monitoring disease progression/treatment. Clinical experience of BIVA in children is limited. We investigated predictors of BIVA and their ability to predict clinical outcomes in children with complex diagnoses. METHODS: R, Xc and PA were measured (BODYSTAT Quadscan 4000) on admission in 108 patients (4.6-16.8 years, mean 10.0). R and Xc were indexed by height (H) and BIVA-SDS for age and sex calculated using data from healthy children. Potential predictors and clinical outcomes (greater-than-expected length-of-stay (LOS), complications) were recorded. RESULTS: Mean R/H-SDS was significantly higher (0.99 (SD 1.32)) and PA-SDS lower (-1.22 (1.68))) than expected, with a wide range for all parameters. In multivariate models, the Strongkids risk category predicted R/H-SDS (adjusted mean for low, medium and high risk = 0.49, 1.28, 2.17, p = 0.009) and PA-SDS (adjusted mean -0.52, -1.53, -2.36, p = 0.01). BIVA-SDS were not significantly different in patients with or without adverse outcomes. CONCLUSIONS: These complex patients had abnormal mean BIVA-SDS suggestive of reduced hydration and poor cellular health according to conventional interpretation. R/H-SDS was higher and PA-SDS lower in those classified as higher malnutrition risk by the StrongKids tool. Further investigation in specific patient groups, including those with acute fluid shifts and using disease-specific outcomes, may better define the clinical role of BIV.

DoE to improve supercoiled p53-pDNA purification by O-phospho-l-tyrosine chromatography.

P53 is implicated in various cellular functions and several studies have shown that transfection of cancer cells with wild-type p53-expressing plasmids could directly drive cells into growth arrest and/or apoptosis. In the present work, the 6.07 kbp pcDNA3-FLAG-p53 plasmid, which encodes the p53 tumor suppressor, was produced and recovered from a recombinant cell culture of Escherichia coli DH5α. Following plasmid biosynthesis, the O-phospho-l-tyrosine chromatographic matrix was explored to purify the supercoiled p53-encoding plasmid. In order to quickly determine the optimal chromatographic performance and to obtain the required purity degree, maximizing the recovery yield of the supercoiled plasmid DNA, the Composite Central Face design was applied. The model revealed to be statistically significant (p-value < 0.05), with coefficient of determination of 0.9434 for the recovery yield and 0.9581 for purity and the central point was successfully validated. After the chromatographic process optimization by using the design of experiments tool, 49.7% of the supercoiled p53-encoding plasmid was recovered with 98.2% of purity, when a decreasing ammonium sulphate gradient was applied. The dynamic binding capacity of the O-phospho-l-tyrosine agarose column was 0.35 ±â€¯0.02 mg pDNA/mL matrix at 50% of the breakthrough. Finally, the purified sample was analysed to assess the content of endotoxins, proteins and genomic DNA, showing that all these impurity levels were below the recommendations of the regulatory agencies.

Seroepidemiology of Neospora caninum among goats (Capra hircus) in the state of Paraíba, northeastern Brazil / Soroepidemiologia de Neospora caninum em caprinos (Capra hircus) do estado da Paraíba, Nordeste do Brasil

Although goat dairy farms in Brazil may have a higher risk of infection by Neospora caninum than beef farms, risk factor evaluation on a representative population remains to be fully established in Brazil. Accordingly, this study aimed to establish the occurrence of anti-N. caninum antibodies and factors associated with exposure in 406 blood samples from five dairy and three beef goat farms in the state of Paraíba, northeastern Brazil. Anti-N. caninum antibodies were detected by indirect immunofluorescence assay (IFA), with samples considered positive when reacting with dilution ≥ 1:50. A total of 106/406 goats (26.11%; 95% CI: 21.96-30.72%) were seroreactive comprising 2/61 (3.28%), 10/45 (22.22%), 13/50 (26.00%), 17/51 (33.33%) to 29/46 (63.04%) in dairy farms, and from 3/54 (5.56%), 12/50 (24.00%) to 20/49 (40.82%) on the beef farms. No significant associations were found in relation to age, gender, dairy versus beef farms, occurrence of abortions or mummified fetuses, and seroreactivity to N. caninum (P>0.05). In conclusion, goat farms in the state of Paraíba showed the highest occurrence of anti-N. caninum antibodies to date in Brazil.(AU)

Embora as criações caprinas de leite no Brasil possam ter maior probabilidade de risco de infecção por Neospora caninum do que as de carne, a avaliação dos fatores de risco em uma população representativa ainda não está totalmente estabelecida no Brasil. Dessa forma, este estudo teve por objetivo estabelecer a soroprevalência de N. caninum e seus fatores associados à exposição em 406 amostras de sangue de cinco fazendas de leite e três de corte provenientes do estado da Paraíba, região Nordeste do Brasil. A detecção de anticorpos anti-N. caninum foi realizada utilizando-se a reação de imunofluorescência indireta (RIFI), com as amostras consideradas positivas na diluição ≥ 1:50. No total, 106/406 (26,11%; IC 95%: 21,96-30,72%) caprinos foram sororreagentes, variando de 2/61 (3,28%), 10/45 (22,22%), 13/50 (26,00%), 17/51 (33,33%) a 29/46 (63,04%) em fazendas de leite, e de 3/54 (5,56%), 12/50 (24,00%) a 20/49 (40,82%) em fazendas de corte. Não foram observadas associações significativas entre idade, sexo, criação de leite e carne, ocorrência de abortamentos ou fetos mumificados e sororreatividade para N. caninum (P>0,05). Em conclusão, fazendas de caprinos da Paraíba mostraram as mais altas ocorrências de anticorpos anti-N. caninum até o momento no Brasil.(AU)

Role of TrkA signalling and mast cells in the initiation of osteoarthritis pain in the monoiodoacetate model.

OBJECTIVE: Aiming to delineate novel neuro-immune mechanisms for NGF/TrkA signalling in osteoarthritis (OA) pain, we evaluated inflammatory changes in the knee joints following injection of monoiodoacetate (MIA) in mice carrying a TrkA receptor mutation (P782S; TrkA KI mice). METHOD: In behavioural studies we monitored mechanical hypersensitivity following intra-articular MIA and oral prostaglandin D2 (PGD2) synthase inhibitor treatments. In immunohistochemical studies we quantified joint mast cell numbers, calcitonin gene-related peptide expression in synovia and dorsal root ganglia, spinal cord neuron activation and microgliosis. We quantified joint leukocyte infiltration by flow cytometry analysis, and PGD2 generation and cyclooxygenase-2 (COX-2) expression in mast cell lines by ELISA and Western blot. RESULTS: In TrkA KI mice we observed rapid development of mechanical hypersensitivity and amplification of dorsal horn neurons and microglia activation 7 days after MIA. In TrkA KI knee joints we detected significant leukocyte infiltration and mast cells located in the vicinity of synovial nociceptive fibres. We demonstrated that mast cells exposure to NGF results in up-regulation of COX-2 and increase of PGD2 production. Finally, we observed that a PGD2 synthase inhibitor prevented MIA-mechanical hypersensitivity in TrkA KI, at doses which were ineffective in wild type (WT) mice. CONCLUSION: Using the TrkA KI mouse model, we delineated a novel neuro-immune pathway and suggest that NGF-induced production of PGD2 in joint mast cells is critical for referred mechanical hypersensitivity in OA, probably through the activation of PGD2 receptor 1 in nociceptors: TrkA blockade in mast cells constitutes a potential target for OA pain.

In vitro activity of antifungals in combination with essential oils against the oomycete Pythium insidiosum.

AIMS: The aim of this study was to investigate the in vitro susceptibility of Pythium insidiosum to combinations of the antifungal drugs terbinafine or itraconazole with Melaleuca alternifolia, Mentha piperita and Origanum vulgare essential oils (EOs). METHODS AND RESULTS: In vitro combinations of antifungal drugs with EOs were evaluated by checkerboard microdilution method against 20 Brazilian isolates of P. insidiosum. The tests were performed according to protocol M38-A2, and the interpretation of each combination result was based on the values of the fractional inhibitory concentration index. The combinations of itraconazole with EOs presented prominent synergistic effects against P. insidiosum isolates, and no antagonism was observed with these combinations. However, the combinations of terbinafine with EOs resulted in indifferent or antagonistic effects. CONCLUSIONS: The combination of plant-derived bioactive compounds with antifungal drugs may be an alternative therapy for the control of infections caused by P. insidiosum. Studies of new therapeutic protocols involving these proposed combinations are needed. SIGNIFICANCE AND IMPACT OF THE STUDY: The antimicrobial combinations using EOs with terbinafine or itraconazole can be an attractive therapeutic option for controlling P. insidiosum infections.

In vitro susceptibility of Brazilian Pythium insidiosum isolates to essential oils of some Lamiaceae family species.

The present study aimed to evaluate the in vitro antimicrobial action of Origanum vulgare, Origanum majorana, Mentha piperita and Rosmarinus officinalis on Pythium insidiosum oomycete zoospores. The antimicrobial activity evaluation was performed by the broth microdilution method according to CSLI M38-A2 documentation adapted to phytopharmaceuticals. Twenty-two P. insidiosum isolates were evaluated, and the minimum inhibitory concentration was determined at 100% growth inhibition. All P. insidiosum isolates evaluated showed a minimum inhibitory concentration ranging from 0.05 to 1.75 mg/mL when O. vulgare oil was used and from 0.11 to 3.5 mg/mL for O. majorana, M. piperita and R. officinalis oils. The results obtained indicate that the essential oils tested showed antimicrobial activity on P. insidiosum, with O. vulgare essential oil showing the best performance. These findings emphasize the potential use of plant essential oils as control agents in P. insidiosum infections; further research, however, is needed so as the in vivo activity of these oils can also be evaluated.

Profile of depressive symptoms in women in the perinatal and outside the perinatal period: similar or not?

PURPOSES: To analyze which Beck Depression Inventory-II (BDI-II) and Postpartum Depression Screening Scores (PDSS) total and dimensional scores, as well as symptomatic answers proportions significantly differ between women in the perinatal period (pregnant/postpartum) without major depression, with major depression and women outside the perinatal period. METHODS: 572 pregnant women in the third trimester completed Beck Depression Inventory-II and Postpartum Depression Screening Scale and were assessed with the Diagnostic Interview for Genetic Studies. 417 of these were also assessed (with the same instruments) at three months postpartum. Ninety non-pregnant women or that did not have a child in the last year (mean age=29.42±7.159 years) also filled in the questionnaires. RESULTS: Non-depressed pregnant women showed lower scores than depressed pregnant women and higher scores than women outside the perinatal period in the BDI-II total score and in its Somatic-Anxiety dimension. Non-depressed postpartum women showed significantly higher scores than women outside the perinatal period only at Sleep/Eating Disturbances. Compared to women outside the perinatal period, pregnant women without depression presented higher scores only in the somatic items. Women with vs. without depression in the postpartum period did not significantly differ and both presented higher scores than women outside the perinatal period in the proportions of loss of energy and sleep changes. LIMITATIONS: Women outside the perinatal period were not diagnosed for the presence of a depressive disorder, but their BDI-II mean score was similar to the figures reported worldwide regarding women in childbearing age. CONCLUSION: In the perinatal period, most particularly at pregnancy, women experience significant somatic changes even if not clinically depressed. Cognitive-affective symptoms are more useful when assessing the presence of perinatal depression.

Selective purification of supercoiled p53-encoding pDNA with L-methionine-agarose matrix.

The p53 tumor suppressor gene has been widely explored for gene therapy as an alternative to the common treatments. Recently, the supercoiled conformation of a p53-encoding plasmid proved to be more efficient in cell transfection and protein expression than the open circular conformation. To successfully isolate this isoform, several chromatographic techniques have been used, namely affinity chromatography with amino acids as ligands. However, the study of new matrices and ligands with higher specificity and robustness for supercoiled plasmid purification is still required. The present work explores for the first time a new matrix of l-methionine-agarose to efficiently purify the supercoiled p53-encoding plasmid. The binding/elution conditions, such as salt concentration and temperature, were manipulated and combined to attain the best strategy. Therefore, the supercoiled plasmid isoform was purified from a clarified lysate by using a decreasing stepwise gradient comprising 2.35 and 1.7M ammonium sulfate in 10mM Tris-HCl, pH 8.0, and finally 10mM Tris-HCl, pH 8.0, at 5°C. After accomplishing the purification process, we performed several tests to assess the quality of the supercoiled plasmid, revealing that the amounts of proteins, gDNA, RNA, and endotoxins were significantly reduced or undetectable in the final formulation.

In vitro susceptibility of zoospores and hyphae of Pythium insidiosum to antifungals.

OBJECTIVES: The purpose of this study was to compare the in vitro susceptibilities of 22 Brazilian isolates of Pythium insidiosum to antifungals using a standardized inoculum of zoospores and a proposed novel inoculum prepared from cultured mycelia (hyphae) of P. insidiosum. METHODS: A zoospore suspension of P. insidiosum was obtained by the zoosporogenesis technique. The hyphal inoculum was prepared from a suspension of P. insidiosum mycelium. Susceptibility to each drug was evaluated using the CLSI M38-A2 method. RESULTS: Of the 88 MIC comparisons performed, 36 (41%) showed the same MIC value for the two inocula. The agreement (differences not greater than one dilution) between MICs obtained with both types of inocula was 39.8% (35/88). In other MIC comparisons analysed, 17 (19.3%) showed differences of two or three dilutions. CONCLUSIONS: We conclude that the use of hyphal inocula of P. insidiosum for in vitro susceptibility tests could be a suitable method for evaluating antimicrobial susceptibility, particularly when it is not possible to obtain a standardized zoospore inoculum.

A large community outbreak of Legionnaires disease in Vila Franca de Xira, Portugal, October to November 2014.

An outbreak of Legionnaires' disease with 334 confirmed cases was identified on 7 November 2014 in Vila Franca de Xira, Portugal and declared controlled by 21 November. Epidemiological, environmental and microbiological analysis identified industrial wet cooling systems to be the probable source of infection. Preliminary results from sequence-based typing of clinical specimens and environmental isolates confirmed this link. A series of meteorological phenomena are likely to have contributed to the scale of this outbreak.

Transient receptor potential ion channels in primary sensory neurons as targets for novel analgesics.

The last decade has witnessed an explosion in novel findings relating to the molecules involved in mediating the sensation of pain in humans. Transient receptor potential (TRP) ion channels emerged as the greatest group of molecules involved in the transduction of various physical stimuli into neuronal signals in primary sensory neurons, as well as, in the development of pain. Here, we review the role of TRP ion channels in primary sensory neurons in the development of pain associated with peripheral pathologies and possible strategies to translate preclinical data into the development of effective new analgesics. Based on available evidence, we argue that nociception-related TRP channels on primary sensory neurons provide highly valuable targets for the development of novel analgesics and that, in order to reduce possible undesirable side effects, novel analgesics should prevent the translocation from the cytoplasm to the cell membrane and the sensitization of the channels rather than blocking the channel pore or binding sites for exogenous or endogenous activators.

Antifungal, antioxidant and anti-inflammatory activities of Oenanthe crocata L. essential oil.

The present study reports the chemical composition, antifungal, antioxidant and anti-inflammatory properties as well as the cytotoxicity of Oenanthe crocata essential oil and one of its main compounds. The essential oil was obtained from the aerial parts of the plant by hydrodistillation and analysed by GC and GC/MS. The oil was predominantly composed of monoterpene hydrocarbons (85.8%), being the main compounds trans-ß-ocimene (31.3%), sabinene (29.0%) and cis-ß-ocimene (12.3%). For the antifungal activity, the minimal inhibitory and minimal lethal concentrations (MICs and MLCs) were determined. The oil was particularly active against dermatophytes and Cryptococcus neoformans, with MIC values ranging from 0.08 to 0.16 µL/mL. Regarding the anti-inflammatory activity, both the oil and sabinene demonstrated strong anti-inflammatory activity through nitric oxide (NO) production inhibition in lipopolysaccharide (LPS) plus interferon gamma (IFN-γ)-triggered macrophages. Furthermore, the essential oil showed a potent NO scavenging effect and inhibited inducible NO synthase expression. Interestingly, and although we detected a cytotoxic effect in macrophages and keratinocytes for the highest concentrations tested of the oil and sabinene, we also disclosed bioactive and safe concentrations to be further explored for therapeutic proposes. Taking together, these results support the use of the oil and sabinene for the management of dermatophytosis and/or inflammatory-related diseases.

Generating reference models for structurally complex data. Application to the stabilometry medical domain.

OBJECTIVES: We present a framework specially designed to deal with structurally complex data, where all individuals have the same structure, as is the case in many medical domains. A structurally complex individual may be composed of any type of single-valued or multivalued attributes, including time series, for example. These attributes are structured according to domain-dependent hierarchies. Our aim is to generate reference models of population groups. These models represent the population archetype and are very useful for supporting such important tasks as diagnosis, detecting fraud, analyzing patient evolution, identifying control groups, etc. METHODS: We have developed a conceptual model to represent structurally complex data hierarchically. Additionally, we have devised a method that uses the similarity tree concept to measure how similar two structurally complex individuals are, plus an outlier detection and filtering method. These methods provide the groundwork for the method that we have designed for generating reference models of a set of structurally complex individuals. A key idea of this method is to use event-based analysis for modeling time series. RESULTS: The proposed framework has been applied to the medical field of stabilometry. To validate the outlier detection method we used 142 individuals, and there was a match between the outlier ratings by the experts and by the system for 139 individuals (97.8%). To validate the reference model generation method, we applied k-fold cross validation (k = 5) with 60 athletes (basketball players and ice-skaters), and the system correctly classified 55 (91.7%). We then added 30 non-athletes as a control group, and the method output the correct result in a very high percentage of cases (96.6%). CONCLUSIONS: We have achieved very satisfactory results for the tests on data from such a complex domain as stabilometry and for the comparison of the reference model generation method with other methods. This supports the validity of this framework.

CFD modelling of the aerodynamic effect of trees on urban air pollution dispersion.

The current work evaluates the impact of urban trees over the dispersion of carbon monoxide (CO) emitted by road traffic, due to the induced modification of the wind flow characteristics. With this purpose, the standard flow equations with a kε closure for turbulence were extended with the capability to account for the aerodynamic effect of trees over the wind field. Two CFD models were used for testing this numerical approach. Air quality simulations were conducted for two periods of 31h in selected areas of Lisbon and Aveiro, in Portugal, for distinct relative wind directions: approximately 45° and nearly parallel to the main avenue, respectively. The statistical evaluation of modelling performance and uncertainty revealed a significant improvement of results with trees, as shown by the reduction of the NMSE from 0.14 to 0.10 in Lisbon, and from 0.14 to 0.04 in Aveiro, which is independent from the CFD model applied. The consideration of the plant canopy allowed to fulfil the data quality objectives for ambient air quality modelling established by the Directive 2008/50/EC, with an important decrease of the maximum deviation between site measurements and CFD results. In the non-aligned wind situation an average 12% increase of the CO concentrations in the domain was observed as a response to the aerodynamic action of trees over the vertical exchange rates of polluted air with the above roof-level atmosphere; while for the aligned configuration an average 16% decrease was registered due to the enhanced ventilation of the street canyon. These results show that urban air quality can be optimised based on knowledge-based planning of green spaces.

Airways changes related to air pollution exposure in wheezing children.

In this study, we aimed to evaluate the relationship between individual total exposure to air pollution and airway changes in a group of 51 wheezing children. Respiratory status was assessed four times (January 2006, June 2006, January 2007 and June 2007) during a 1-week period through a standardised questionnaire, spirometry, exhaled nitric oxide fraction and pH in exhaled breath condensate (EBC). Concentrations of particles with a 50% cut-off aerodynamic diameter of 10 µm (PM(10)), O(3), NO(2) and volatile organic compounds were estimated through direct measurements with an ad hoc device or air pollution modelling in the children's schools and at their homes in the same 4 weeks of the study. For each child, total exposure to the different air pollutants was estimated as a function of pollutant concentrations and daily activity patterns. Increasing total exposure to PM(10), NO(2), benzene, toluene and ethylbenzene was significantly associated with a decrease of forced expiratory volume in 1 s (FEV(1)) and with an increase of change in FEV(1). Increasing exposure to NO(2) and benzene was also related to a significant decrease of FEV(1)/forced vital capacity. Increasing exposure to PM(10), NO(2), benzene and ethylbenzene was associated with acidity of EBC. This study suggests an association in wheezing children between airway changes and total exposure to air pollutants, as estimated by taking into account the concentration in the various microenvironments attended by the children.