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1.  Ca2+-Activated K+ Channel–3.1 Blocker TRAM-34 Attenuates Airway Remodeling and Eosinophilia in a Murine Asthma Model 
Key features of asthma include bronchial hyperresponsiveness (BHR), eosinophilic airway inflammation, and bronchial remodeling, characterized by subepithelial collagen deposition, airway fibrosis, and increased bronchial smooth muscle (BSM) mass. The calcium-activated K+ channel KCa3.1 is expressed by many cells implicated in the pathogenesis of asthma, and is involved in both inflammatory and remodeling responses in a number of tissues. The specific KCa3.1 blocker 5-[(2-chlorophenyl)(diphenyl)methyl]-1H-pyrazole (TRAM-34) attenuates BSM cell proliferation, and both mast cell and fibrocyte recruitment in vitro. We aimed to examine the effects of KCa3.1 blockade on BSM remodeling, airway inflammation, and BHR in a murine model of chronic asthma. BALB/c mice were sensitized with intraperitoneal ovalbumin (OVA) on Days 0 and 14, and then challenged with intranasal OVA during Days 14–75. OVA-sensitized/challenged mice received TRAM-34 (120 mg/kg/day, subcutaneous) from Days −7 to 75 (combined treatment), Days −7 to 20 (preventive treatment), or Days 21 to 75 (curative treatment). Untreated mice received daily injections of vehicle (n = 8 per group). Bronchial remodeling was assessed by histological and immunohistochemical analyses. Inflammation was evaluated using bronchoalveolar lavage and flow cytometry. We also determined BHR in both conscious and anesthetized mice via plethysmography. We demonstrated that curative treatment with TRAM-34 abolishes BSM remodeling and subbasement collagen deposition, and attenuates airway eosinophilia. Although curative treatment alone did not significantly reduce BHR, the combined treatment attenuated nonspecific BHR to methacholine. This study indicates that KCa3.1 blockade could provide a new therapeutic strategy in asthma.
doi:10.1165/rcmb.2012-0103OC
PMCID: PMC4035224  PMID: 23204391
asthma; KCa3.1; ion channel; remodeling; smooth muscle
2.  Bronchial hyperresponsiveness in lung transplant recipients: lack of correlation with airway inflammation 
Thorax  1997;52(6):551-556.
BACKGROUND: Bronchial hyperresponsiveness (BHR) to methacholine has been reported to occur in most lung transplant recipients. BHR to physical stimuli such as exercise and non-isotonic aerosols has not been as extensively studied in this subject population. This report aims to assess the presence and degree of BHR to methacholine and hypertonic saline in stable lung transplant recipients and to relate it to the presence of airway inflammation. METHODS: Ten patients undergoing bilateral sequential lung transplantation and six heart-lung transplant recipients, all with stable lung function, were recruited 66- 1167 days following transplantation. Subjects underwent a methacholine challenge and bronchoscopy for sampling of bronchoalveolar lavage fluid, transbronchial and endobronchial biopsy tissues. Hypertonic saline challenge was performed six days later. RESULTS: Nine of the 16 transplant recipients had positive methacholine challenges (geometric mean PD20 0.18 mg, interquartile range 0.058-0.509) and three of these subjects also had positive hypertonic saline challenges (PD15 = 2.3, 33.0, and 51.5 ml). No clear relationship was found between BHR to either methacholine or hypertonic saline and levels of mast cells, eosinophils or lymphocytes in samples of biopsy tissue or lavage fluid. CONCLUSIONS: Most of the lung transplant recipients studied were responsive to methacholine and unresponsive to hypertonic saline. BHR was not clearly related to airway inflammation, suggesting an alternative mechanism for BHR following lung transplantation from that usually assumed in asthma. 



PMCID: PMC1758572  PMID: 9227723
3.  Bacillus Calmette-Guérin Suppresses Asthmatic Responses via CD4+CD25+ Regulatory T Cells and Dendritic Cells 
Purpose
Bacillus Calmette-Guérin (BCG) is known to suppress the asthmatic responses in a murine model of asthma and to induce dendritic cells (DCs) maturation. Mature DCs play a crucial role in the differentiation of regulatory T cells (Tregs), which are known to regulate allergic inflammatory responses. To investigate whether BCG regulates Tregs in a DCs-mediated manner, we analyzed in a murine model of asthma.
Methods
BALB/c mice were injected intraperitoneally with BCG or intravenously with BCG-stimulated DCs and then sensitized and challenged with ovalbumin (OVA). Mice were analysed for bronchial hyperresponsiveness (BHR), the influx of inflammatory cells in the bronchoalveolar lavage (BAL) fluid, and histopathological changes in the lung. To identify the mechanisms, IgE, IgG1 and IgG2a in the serum were analysed and the CD25+ Tregs in the mice were depleted with anti-CD25 monoclonal antibody (mAb).
Results
BCG and the transfer of BCG-stimulated DCs both suppressed all aspects of the asthmatic responses, namely, BHR, the production of total IgE and OVA-specific IgE and IgGs, and pulmonary eosinophilic inflammation. Anti-CD25mAb treatment reversed these effects.
Conclusions
BCG can attenuate the allergic inflammation in a mouse model of asthma by a Tregs-related mechanism that is mediated by DCs.
doi:10.4168/aair.2014.6.3.201
PMCID: PMC4021237  PMID: 24843794
BCG; dendritic cells; T-lymphocytes, regulatory; asthma; allergic inflammation; mouse
4.  Crude Extracts of Caenorhabditis elegans Suppress Airway Inflammation in a Murine Model of Allergic Asthma 
PLoS ONE  2012;7(4):e35447.
Epidemiological studies suggest an inverse relationship between helminth infections and allergic disease, and several helminth-derived products have been shown to suppress allergic responses in animals. This study was undertaken to evaluate the effect of a crude extract of Caenorhabditis elegans on allergic airway inflammation in a murine model of asthma. Allergic airway inflammation was induced in BALB/c mice by sensitization with ovalbumin. The effect of the C. elegans crude extract on the development of asthma and on established asthma was evaluated by analyzing airway hyperresponsiveness, serum antibody titers, lung histology and cell counts and cytokine levels in the bronchoalveolar lavage fluid. The role of IFN-γ in the suppression of asthma by the C. elegans crude extract was investigated in IFN-γ knockout and wild-type mice. When mice were sensitized with ovalbumin together with the crude extract of C. elegans, cellular infiltration into the lung was dramatically reduced in comparison with the ovalbumin-treated group. Treatment of mice with the C. elegans crude extract significantly decreased methacholine-induced airway hyperresponsiveness and the total cell counts and levels of IL-4, IL-5 and IL-13 in the bronchoalveolar lavage fluid but increased the levels of IFN-γ and IL-12. Sensitization with the C. elegans crude extract significantly diminished the IgE and IgG1 responses but provoked elevated IgG2a levels. However, the suppressive effect of the C. elegans crude extract was abolished in IFN-γ knockout mice, and the Th2 responses in these mice were as strong as those in wild-type mice sensitized with ovalbumin. The crude extract of C. elegans also suppressed the airway inflammation associated with established asthma. This study provides new insights into immune modulation by the C. elegans crude extract, which suppressed airway inflammation in mice not only during the development of asthma but also after its establishment by skewing allergen-induced Th2 responses to Th1 responses.
doi:10.1371/journal.pone.0035447
PMCID: PMC3338843  PMID: 22558152
5.  320 Development of a Questionnaire for the Assessment of Bronchial Hyperresponsiveness in Korea 
Background
Bronchial hyperresponsiveness (BHR) is an important pathophysiological feature of asthma. In addition to the diagnostic significance, BHR is associated with the severity of airway inflammation and BHR- based treatment approaches has been shown to be effective. Nevertheless, challenge tests are time consuming, inconvenient to patients, and are not accessible in every primary care physicians. We aimed to develop a questionnaire for the assessment of BHR in Korean subjects.
Methods
From the 24 University-affiliated hospitals, we recruited 149 adults between age 20 and 40 years with more than one asthmatic symptom (cough, sputum or dyspnea) and who had bronchial provocation test. A list of 33 symptoms, past history of allergy or smoking and 10 provoking stimuli were selected for the BHR questionnaire. After a methacholine challenge test patients were asked to complete each questionnaire. For each item of questionnaire, diagnostic odds ratios for the presence of BHR were calculated and multiple logistic regression analysis was performed to select final questionnaire items. Receiver operating characteristic (ROC) curve analysis was used to evaluate the sensitivity and specificity of the selected questionnaire items.
Results
Methacholine challenge test was positive in 36 patients (24.2%). Eleven symptoms and 2 provoking stimuli items were statistically significant by the results of diagnostic odds ratio. According to the result of multiple logistic regression analysis, 4 items were finally selected for the significant BHR questionnaire: the presence of wheezing episode, past history of physician-diagnosed asthma, family history of asthma. The psychiatric stress was negatively associated provoking stimuli item for the presence of BHR. The area under the ROC curve was 0.80 (95% CI, 0.72-0.86). Sensitivity was 84.9% (95% CI, 68.1-94.9) and specificity was 65.5% (95% CI, 55.8-74.3).
Conclusions
Four BHR questionnaire items including wheezing episode, past history of physician-diagnosed asthma, family history of asthma and psyachiatric stress stimuli were able to assess the presence of BHR in Korean adults.
doi:10.1097/01.WOX.0000412083.06151.cc
PMCID: PMC3512762
6.  Influence of sensitization and allergen provocation procedures on the development of allergen-induced bronchial hyperreactivity in conscious, unrestrained guinea-pigs 
Mediators of Inflammation  1995;4(2):149-156.
The effects of different sensitization and allergen provocation regimens on the development of allergen-induced bronchial hyperreactivity (BHR) to histamine were investigated in conscious, unrestrained guinea-pigs. Similar early and late phase asthmatic reactions, BHR for inhaled histamine after the early (6 h) as well as after the late reaction (24 h), and airway inflammation were observed after a single allergen provocation in animals sensitized to produce mainly IgG or IgE antibodies, respectively. Repeating the allergen provocation in the IgE-sensitized animals after 7 days, using identical provocation conditions, resulted in a similar development of BHR to histamine inhalation. Repetition of the allergen provocation during 4 subsequent days resulted in a decreased development of BHR after each provocation, despite a significant increase in the allergen provocation dose necessary to obtain similar airway obstruction. The number of inflammatory cells in the bronchoalveolar lavage was not significantly changed after repeated provocation, when compared with a single allergen provocation. Finally, we investigated allergen-induced bronchial hyperreactivity by repetition of the sensitization procedure at day 7 and 14 (booster), followed by repeated allergen provocation twice a week for 5 weeks. Surprisingly, no BHR to histamine could be observed after either provocation, while the number of inflammatory cells in the bronchoalveolar lavage fluid after 5 weeks was enhanced compared with controls. These data indicate that both IgE and IgG sensitized guinea-pigs may develop bronchial hyperreactivity after a single allergen provocation. Repeated allergen exposure of IgE sensitized animals causes a gradual fading of the induced hyperreactivity despite the on-going presence of inflammatory cells in the airways, indicating a mechanism of reduced cellular activation.
doi:10.1155/S0962935195000263
PMCID: PMC2365622  PMID: 18475633
7.  A Novel Synthetic Mycolic Acid Inhibits Bronchial Hyperresponsiveness and Allergic Inflammation in a Mouse Model of Asthma 
Purpose
Recognition of microbes is important to trigger the innate immune system. Mycolic acid (MA) is a component of the cell walls of mycobacteria such as Mycobacterium bovis Bacillus Calmette-Guerin. MA has immunogenic properties, which may modulate the innate and adaptive immune response. This study aimed to investigate whether a novel synthetic MA (sMA) inhibits allergic inflammatory responses in a mouse model of asthma.
Methods
BALB/c mice were injected intraperitoneally with sMA followed by sensitization and challenge with ovalbumin (OVA). Mice were examined for bronchial hyperresponsiveness (BHR), the influx of inflammatory cells into the lung tissues, histopathological changes in the lungs and CD4+CD25+Foxp3+ T cells in the spleen, and examined the response after the depleting regulatory T cells (Tregs) with an anti-CD25mAb.
Results
Treatment of mice with sMA suppressed the asthmatic response, including BHR, bronchoalveolar inflammation, and pulmonary eosinophilic inflammation. Anti-CD25mAb treatment abrogated the suppressive effects of sMA in this mouse model of asthma and totally depleted CD4+CD25+Foxp3+ T cells in the spleen.
Conclusions
sMA attenuated allergic inflammation in a mouse model of asthma, which might be related with CD4+CD25+Foxp3+ T cell.
doi:10.4168/aair.2014.6.1.83
PMCID: PMC3881406  PMID: 24404398
Mycolic acid; asthma; allergic inflammation; regulatory T cells; mice
8.  IL-12-STAT4-IFN-γ axis is a key downstream pathway in the development of IL-13-mediated asthma phenotypes in a Th2 type asthma model 
Experimental & Molecular Medicine  2010;42(8):533-546.
IL-4 and IL-13 are closely related cytokines that are produced by Th2 cells. However, IL-4 and IL-13 have different effects on the development of asthma phenotypes. Here, we evaluated downstream molecular mechanisms involved in the development of Th2 type asthma phenotypes. A murine model of Th2 asthma was used that involved intraperitoneal sensitization with an allergen (ovalbumin) plus alum and then challenge with ovalbumin alone. Asthma phenotypes, including airway-hyperresponsiveness (AHR), lung inflammation, and immunologic parameters were evaluated after allergen challenge in mice deficient in candidate genes. The present study showed that methacholine AHR and lung inflammation developed in allergen-challenged IL-4-deficient mice but not in allergen-challenged IL-13-deficient mice. In addition, the production of OVA-specific IgG2a and IFN-γ-inducible protein (IP)-10 was also impaired in the absence of IL-13, but not of IL-4. Lung-targeted IFN-γ over-expression in the airways enhanced methacholine AHR and non-eosinophilic inflammation; in addition, these asthma phenotypes were impaired in allergen-challenged IFN-γ-deficient mice. Moreover, AHR, non-eosinophilic inflammation, and IFN-γ expression were impaired in allergen-challenged IL-12Rβ2- and STAT4-deficient mice; however, AHR and non-eosinophilic inflammation were not impaired in allergen-challenged IL-4Rα-deficient mice, and these phenomena were accompanied by the enhanced expression of IL-12 and IFN-γ. The present data suggest that IL-13-mediated asthma phenotypes, such as AHR and non-eosinophilic inflammation, in the Th2 type asthma are dependent on the IL-12-STAT4-IFN-γ axis, and that these asthma phenotypes are independent of IL-4Ralpha-mediated signaling.
doi:10.3858/emm.2010.42.8.054
PMCID: PMC2928926  PMID: 20592486
asthma; interferon-γ; interleukin-12; interleukin-13; respiratory hypersensitivity; Th2 cells
9.  Asymptomatic bronchial hyperreactivity and the development of asthma and other respiratory tract illnesses in children. 
Thorax  1994;49(8):757-761.
BACKGROUND--It is not clear whether asymptomatic bronchial hyperresponsiveness (BHR) in children is a risk factor for the subsequent development of asthma. A longitudinal study was conducted to determine the predictive value of BHR for the development of asthma in a primary care patient population. METHODS--A standard free running asthma screening test (FRAST) was applied to 956 schoolchildren aged between 4 and 11 years in 1985. Peak expiratory flow (PEF) rates were measured before hard running for six minutes and following a three minute rest period. Children with a fall in PEF of more than 15% were labelled as having a positive FRAST. Clinical data from the patients' notes and from symptom questionnaires were compared with age and sex matched controls for children known to have asthma, and for those with a positive FRAST but no asthma (BHR group). Over the ensuing six years to 1991 further clinical data were gathered to compare the development of asthma and other diseases of the airways in both the BHR groups and their controls. RESULTS--Of the 956 children exercised in 1985, 60 who were not known to have asthma had an abnormal test. Of the 55 of these studied in 1991, 32 (58%) had developed asthma. The sensitivity of a positive FRAST for the development of asthma was 58%, its specificity 97%, and positive predictive value 72%. Hay fever, eczema, otitis media, "bronchitis," and family history of atopy also occurred more commonly in this group. CONCLUSIONS--Asymptomatic BHR, as shown by exercise challenge, can predict the development of clinical asthma. This study has also shown a relation between BHR, asthma, and other diseases of the airways, notably upper respiratory tract infection, "bronchitis," and otitis media.
PMCID: PMC475119  PMID: 8091319
10.  Severity of Allergic Airway Disease Due to House Dust Mite Allergen Is Not Increased after Clinical Recovery of Lung Infection with Chlamydia pneumoniae in Mice 
Infection and Immunity  2013;81(9):3366-3374.
Chlamydia pneumoniae is associated with chronic inflammatory lung diseases like bronchial asthma and chronic obstructive pulmonary disease. The existence of a causal link between allergic airway disease and C. pneumoniae is controversial. A mouse model was used to address the question of whether preceding C. pneumoniae lung infection and recovery modifies the outcome of experimental allergic asthma after subsequent sensitization with house dust mite (HDM) allergen. After intranasal infection, BALB/c mice suffered from pneumonia characterized by an increased clinical score, reduction of body weight, histopathology, and a bacterial load in the lungs. After 4 weeks, when infection had almost resolved clinically, HDM allergen sensitization was performed for another 4 weeks. Subsequently, mice were subjected to a methacholine hyperresponsiveness test and sacrificed for further analyses. As expected, after 8 weeks, C. pneumoniae-specific antibodies were detectable only in infected mice and the titer was significantly higher in the C. pneumoniae/HDM allergen-treated group than in the C. pneumoniae/NaCl group. Intriguingly, airway hyperresponsiveness and eosinophilia in bronchoalveolar lavage fluid were significantly lower in the C. pneumoniae/HDM allergen-treated group than in the mock/HDM allergen-treated group. We did observe a relationship between experimental asthma and chlamydial infection. Our results demonstrate an influence of sensitization to HDM allergen on the development of a humoral antibacterial response. However, our model demonstrates no increase in the severity of experimental asthma to HDM allergen as a physiological allergen after clinically resolved severe chlamydial lung infection. Our results rather suggest that allergic airway disease and concomitant cellular changes in mice are decreased following C. pneumoniae lung infection in this setting.
doi:10.1128/IAI.00334-13
PMCID: PMC3754214  PMID: 23817611
11.  Effects of nebulized ketamine on allergen-induced airway hyperresponsiveness and inflammation in actively sensitized Brown-Norway rats 
Since airway hyperresponsiveness (AHR) and allergic inflammatory changes are regarded as the primary manifestations of asthma, the main goals of asthma treatment are to decrease inflammation and maximize bronchodilation. These goals can be achieved with aerosol therapy. Intravenous administration of the anesthetic, ketamine, has been shown to trigger bronchial smooth muscle relaxation. Furthermore, increasing evidence suggests that the anti-inflammatory properties of ketamine may protect against lung injury. However, ketamine inhalation might yield the same or better results at higher airway and lower ketamine plasma concentrations for the treatment of asthma. Here, we studied the effect of ketamine inhalation on bronchial hyperresponsiveness and airway inflammation in a Brown-Norway rat model of ovalbumin(OVA)-induced allergic asthma. Animals were actively sensitized by subcutaneous injection of OVA and challenged by repeated intermittent (thrice weekly) exposure to aerosolized OVA for two weeks. Before challenge, the sensitizened rats received inhalation of aerosol of phosphate-buffered saline (PBS) or aerosol of ketamine or injection of ketamine respectivity. Airway reactivity to acetylcholine (Ach) was measured in vivo, and various inflammatory markers, including Th2 cytokines in bronchoalveolar lavage fluid (BALF), as well as induciable nitric oxide synthase (iNOS) and nitric oxide (NO) in lungs were examined. Our results revealed that delivery of aerosolized ketamine using an ultrasonic nebulizer markedly suppressed allergen-mediated airway hyperreactivity, airway inflammation and airway inflammatory cell infiltration into the BALF, and significantly decreased the levels of interleukin-4 (IL-4) in the BALF and expression of iNOS and the concentration of NO in the inflamed airways from OVA-treated rats. These findings collectively indicate that nebulized ketamine attenuated many of the central components of inflammatory changes and AHR in OVA-provoked experimental asthma, potentially providing a new therapeutic approach against asthma.
doi:10.1186/1476-9255-4-10
PMCID: PMC1876456  PMID: 17480224
12.  Exaggerated IL-17 response to epicutaneous sensitization mediates airway inflammation in the absence of IL-4 and IL-13 
Background
Atopic dermatitis (AD) is characterized by local and systemic Th2 responses to cutaneously introduced allergens and is a risk factor for asthma. Blockade of Th2 cytokines has been suggested as therapy for AD.
Objectives
To examine the effect of the absence of IL-4 and IL-13 on the Th-17 response to epicutaneous (EC) sensitization in a mouse model of allergic skin inflammation with features of AD.
Methods
Wild-type (WT), IL-4KO, IL-13KO and IL-4/13 double KO (DKO) mice were subjected to EC sensitization with ovalbumin (OVA) or saline and airway challenged with OVA. Systemic immune responses to OVA, skin and airway inflammation, and airway hyperresponsiveness (AHR) were examined.
Results
OVA sensitized DKO mice exhibited impaired Th2 driven responses with undetectable OVA specific IgE and severely diminished eosinophil infiltration at sensitized skin sites, but intact dermal infiltration with CD4+ cells. DKO mice mounted an exaggerated IL-17A, but normal IFN-γ and IL-5 systemic responses. Airway challenge of these mice with OVA caused marked upregulation of IL-17 mRNA expression in the lungs, increased neutrophilia in bronchoalveolar lavage fluid (BALF), airway inflammation characterized by mononuclear cell infiltration with no detectable eosinophils, and bronchial hyperresponsiveness to methacholine that were reversed by IL-17 blockade. IL-4, but not IL-13, was identified as the major Th2 cytokine that downregulates the IL-17 response in EC sensitized mice.
Conclusion
EC sensitization in the absence of IL-4/IL-13 induces an exaggerated Th17 response systemically, and in lungs following antigen challenge that results in airway inflammation and AHR.
Clinical implications
Blockade of IL-4 may promote IL-17-mediated airway inflammation in AD.
doi:10.1016/j.jaci.2009.07.040
PMCID: PMC2895457  PMID: 19815118
IL-17; Th2 cytokines; atopic dermatitis; asthma
13.  ISO-1, a Macrophage Migration Inhibitory Factor Antagonist, Inhibits Airway Remodeling in a Murine Model of Chronic Asthma 
Molecular Medicine  2010;16(9-10):400-408.
Airway remodeling is the process of airway structural change that occurs in patients with asthma in response to persistent inflammation and leads to increasing disease severity. Drugs that decrease this persistent inflammation play a crucial role in managing asthma episodes. Mice sensitized (by intraperitoneal administration) and then challenged (by inhalation) with ovalbumin (OVA) develop an extensive eosinophilic inflammatory response, goblet cell hyperplasia, collagen deposition, airway smooth muscle thickening, and airway wall area increase, similar to pathologies observed in human asthma. We used OVA-sensitized/challenged mice as a murine model of chronic allergic airway inflammation with subepithelial fibrosis (i.e., asthma). In this OVA mouse model, mRNA and protein of macrophage migration inhibitory factor (MIF) are upregulated, a response similar to what has been observed in the pathogenesis of acute inflammation in human asthma. We hypothesized that MIF induces transforming growth factor-β1 (TGF-β1) synthesis, which has been shown to play an important role in asthma and airway remodeling. To explore the role of MIF in the development of airway remodeling, we evaluated the effects of an MIF small-molecule antagonist, (S,R)3-(4-hy-droxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester (ISO-1), on pathologies associated with the airway-remodeling process in the OVA mouse model. We found that administration of ISO-1 significantly mitigated all symptoms caused by OVA treatment. In addition, the treatment of OVA-sensitized mice with the MIF antagonist ISO-1 significantly reduced TGF-β1 mRNA levels in pulmonary tissue and its protein level in bronchial alveolar lavage fluid supernatants. We believe the repression of MIF in the ISO-1 treatment group led to the significant suppression observed in the inflammatory responses associated with the allergen-induced lung inflammation and fibrosis in our murine asthma (OVA) model. Our results implicate a possible function of MIF in the pathogenesis of chronic asthma and suggest that MIF might be an important therapeutic target for airway remodeling.
doi:10.2119/molmed.2009.00128
PMCID: PMC2935952  PMID: 20485865
14.  Sustained reduction in bronchial hyperresponsiveness with inhaled fluticasone propionate within three days in mild asthma: time course after onset and cessation of treatment 
Thorax  2003;58(6):500-504.
Background: Bronchial hyperresponsiveness (BHR) is characteristic of asthmatic airways, is induced by airway inflammation, and is reduced by inhaled corticosteroids (ICS). The time course for the onset and cessation of the effect of ICS on BHR is unclear. The effect of inhaled fluticasone propionate (FP) on BHR in patients with mild persistent asthma was assessed using time intervals of hours, days and weeks.
Methods: Twenty six asthmatic patients aged 21–59 years were selected for this randomised, double blind, parallel group study. The effect of 250 µg inhaled FP (MDI) administered twice daily was compared with that of placebo on BHR assessed using a dosimetric histamine challenge method. The dose of histamine inducing a decrease in forced expiratory volume in 1 second (FEV1) by 15% (PD15FEV1) was measured before and 6, 12, 24 and 72 hours, and 2, 4 and 6 weeks after starting treatment, and 48 hours, 1 week and 2 weeks after cessation of treatment. Doubling doses of changes in PD15FEV1 were calculated and area under the curve (AUC) statistics were used to summarise the information from individual response curves.
Results: The increase in PD15FEV1 from baseline was greater in the FP group than in the placebo group; the difference achieved significance within 72 hours and remained significant until the end of treatment. In the FP group PD15FEV1 was 1.85–2.07 doubling doses above baseline between 72 hours and 6 weeks after starting treatment. BHR increased significantly within 2 weeks after cessation of FP treatment.
Conclusions: A sustained reduction in BHR to histamine in patients with mild asthma was achieved within 3 days of starting treatment with FP at a daily dose of 500 µg. The effect tapered within 2 weeks of cessation of treatment.
doi:10.1136/thorax.58.6.500
PMCID: PMC1746689  PMID: 12775860
15.  Block Copolymer/DNA Vaccination Induces a Strong Allergen-Specific Local Response in a Mouse Model of House Dust Mite Asthma 
PLoS ONE  2014;9(1):e85976.
Background
Allergic asthma is caused by abnormal immunoreactivity against allergens such as house dust mites among which Dermatophagoides farinae (Der f) is a common species. Currently, immunotherapy is based on allergen administration, which has variable effect from patient to patient and may cause serious side effects, principally the sustained risk of anaphylaxis. DNA vaccination is a promising approach by triggering a specific immune response with reduced allergenicity.
Objective
The aim of the study is to evaluate the effects of DNA immunization with Der f1 allergen specific DNA on allergic sensitization, inflammation and respiratory function in mice.
Methods
Mice were vaccinated 28 and 7 days before allergen exposure with a Der f1-encoding plasmid formulated with a block copolymer. Asthma was induced by skin sensitization followed by intra-nasal challenges with Der f extract. Total lung, broncho-alveolar lavage (BAL) and spleen cells were analyzed by flow cytometry for their surface antigen and cytokine expression. Splenocytes and lung cell IFN-γ production by CD8+ cells in response to Der f CMH1-restricted peptides was assessed by ELISPOT. IgE, IgG1 and IgG2a were measured in serum by ELISA. Specific bronchial hyperresponsiveness was assessed by direct resistance measurements.
Results
Compared to animals vaccinated with an irrelevant plasmid, pVAX-Der f1 vaccination induced an increase of B cells in BAL, and an elevation of IL-10 and IFN-γ but also of IL-4, IL-13 and IL-17 producing CD4+ lymphocytes in lungs and of IL-4 and IL-5 in spleen. In response to CD8-restricted peptides an increase of IFN-γ was observed among lung cells. IgG2a levels non-specifically increased following block copolymer/DNA vaccination although IgE, IgG1 levels and airways resistances were not impacted.
Conclusions & Clinical Relevance
DNA vaccination using a plasmid coding for Der f1 formulated with the block copolymer 704 induces a specific immune response in the model of asthma used herein.
doi:10.1371/journal.pone.0085976
PMCID: PMC3908923  PMID: 24497934
16.  Airway inflammation, basement membrane thickening and bronchial hyperresponsiveness in asthma 
Thorax  2002;57(4):309-316.
Background: There are few data in asthma relating airway physiology, inflammation and remodelling and the relative effects of inhaled corticosteroid (ICS) treatment on these parameters. A study of the relationships between spirometric indices, airway inflammation, airway remodelling, and bronchial hyperreactivity (BHR) before and after treatment with high dose inhaled fluticasone propionate (FP 750 µg bd) was performed in a group of patients with relatively mild but symptomatic asthma.
Methods: A double blind, randomised, placebo controlled, parallel group study of inhaled FP was performed in 35 asthmatic patients. Bronchoalveolar lavage (BAL) and airway biopsy studies were carried out at baseline and after 3 and 12 months of treatment. Twenty two normal healthy non-asthmatic subjects acted as controls.
Results: BAL fluid eosinophils, mast cells, and epithelial cells were significantly higher in asthmatic patients than in controls at baseline (p<0.01). Subepithelial reticular basement membrane (rbm) thickness was variable, but overall was increased in asthmatic patients compared with controls (p<0.01). Multiple regression analysis explained 40% of the variability in BHR, 21% related to rbm thickness, 11% to BAL epithelial cells, and 8% to BAL eosinophils. The longitudinal data corroborated the cross sectional model. Forced expiratory volume in 1 second improved after 3 months of treatment with FP with no further improvement at 12 months. PD20 improved throughout the study. BAL inflammatory cells decreased following 3 months of treatment with no further improvement at 12 months (p<0.05 v placebo). Rbm thickness decreased in the FP group, but only after 12 months of treatment (mean change –1.9, 95% CI –3 to –0.7 µm; p<0.01 v baseline, p<0.05 v placebo). A third of the improvement in BHR with FP was associated with early changes in inflammation, but the more progressive and larger improvement was associated with the later improvement in airway remodelling.
Conclusion: Physiology, airway inflammation and remodelling in asthma are interrelated and improve with ICS. Changes are not temporally concordant, with prolonged treatment necessary for maximal benefit in remodelling and PD20. Determining the appropriate dose of inhaled steroids only by reference to symptoms and lung function, as specified in current international guidelines, and even against indices of inflammation may be over simplistic. The results of this study support the need for early and long term intervention with ICS, even in patients with relatively mild asthma.
doi:10.1136/thorax.57.4.309
PMCID: PMC1746305  PMID: 11923548
17.  Mast Cells Protect against Airway Mycoplasma pneumoniae under Allergic Conditions 
Background
Infection with Mycoplasma pneumoniae in asthma can occur both acutely and chronically with an associated Th2 inflammatory response and/or increased numbers of bronchial mast cells. Mast cells have previously been shown to promote mycoplasma clearance in mice; however, it is unknown whether mast cells would aid M. pneumoniae clearance under allergic conditions.
Objective
Our aim was to determine the impact of allergic inflammation on mast cell-mediated lung M. pneumoniae clearance. Furthermore, as we have previously demonstrated an essential role for IL-6 in lung M. pneumoniae clearance we also investigated the role of mast cell-derived IL-6.
Methods
Mast cell deficient WBB6F1/J-KitW/KitW-v mice were challenged with ovalbumin to induce airway inflammation prior to M. pneumoniae infection. The role of mast cell-derived IL-6 in bacterial clearance was further investigated by reconstitution of mast cell deficient mice with IL-6-/- mast cells.
Results
Allergic mast cell deficient mice exhibited increased lung M. pneumoniae burden compared to control littermates. Intravenous adoptive transfer of wild type and IL-6-/- mast cells significantly improved M. pneumoniae clearance in mast cell deficient mice. Acutely after M. pneumoniae infection, allergen-challenged mast cell deficient mice had increased levels of the pro-inflammatory cytokines IL-6 and TNF-α in the BAL fluid. The total number of neutrophils was also increased in mast cell deficient mice.
Conclusions
Our results establish that mast cells aid host defense against M. pneumoniae in an allergic setting and that while IL-6 is necessary for lung M. pneumoniae clearance, mast cell-derived IL-6 is not required.
doi:10.1111/j.1365-2222.2010.03488.x
PMCID: PMC2895012  PMID: 20345998
Asthma; host defense; innate immunity; mast cells; Mycoplasma pneumoniae
18.  Antiasthmatic Effects of Herbal Complex MA and Its Fermented Product MA128 
This study was conducted to determine if oral administration of the novel herbal medicine, MA, and its Lactobacillus acidophilus fermented product, MA128, have therapeutic properties for the treatment of asthma. Asthma was induced in BALB/c mice by systemic sensitization to ovalbumin (OVA) followed by intratracheal, intraperitoneal, and aerosol allergen challenges. MA and MA128 were orally administered 6 times a week for 4 weeks. At 1 day after the last ovalbumin exposure, airway hyperresponsiveness was assessed and samples of bronchoalveolar lavage fluid, lung cells, and serum were collected for further analysis. We investigated the effect of MA and MA128 on airway hyperresponsiveness, pulmonary eosinophilic infiltration, various immune cell phenotypes, Th2 cytokine production, OVA-specific IgE production, and Th1/Th2 cytokine production in this mouse model of asthma. In BALB/c mice, we found that MA and MA128 treatment suppressed eosinophil infiltration into airways and blood, allergic airway inflammation and AHR by suppressing the production of IL-5, IL-13, IL-17, Eotaxin, and OVA-specific IgE, by upregulating the production of OVA-specific Th1 cytokine (IFN-γ), and by downregulating OVA-specific Th2 cytokine (IL-4) in the culture supernatant of spleen cells. The effectiveness of MA was increased by fermentation with Lactobacillus acidophilus.
doi:10.1155/2012/769508
PMCID: PMC3235914  PMID: 22203879
19.  Low dose of Mycoplasma pneumoniae (Mp) infection enhances an established allergic inflammation in mice: Role of prostaglandin E2 (PGE2) pathway 
Summary
Background
Over 40% of chronic stable asthma patients have evidence of respiratory Mycoplasma pneumoniae (Mp) infection as detected by polymerase chain reaction (PCR), but not by serology and culture, suggesting a low-level Mp involved in chronic asthma. However, the role of such a low-level Mp infection in regulation of allergic inflammation remains unknown.
Objective
To determine the impact of a low-level Mp infection in mice with established airway allergic inflammation on allergic responses such as eosinophilia and chemokine eotaxin-2, and the underlying mechanisms (i.e., prostaglandin E2 [PGE2] pathway) since PGE2 inhalation before allergen challenge suppressed eosinophil infiltration in human airways.
Methods
BALB/c mouse models of ovalbumin (OVA)-induced allergic asthma with an ensuing low-dose or high-dose Mp were used to assess IL-4 expression, BAL eosinophil, eotaxin-2 and PGE2 levels, and lung mRNA levels of microsomal prostaglandin E synthase-1 (mPGES-1). Primary alveolar macrophages (pAMs) from naïve BALB/c mice were cultured to determine if Mp-induced PGE2 or exogenous PGE2 down-regulates IL-4/IL-13-induced eotaxin-2.
Results
Low-dose Mp in allergic mice significantly enhanced IL-4 and eotaxin-2, and moderately promoted lung eosinophilia, whereas high-dose Mp significantly reduced lung eosinophilia and tended to decrease IL-4 and eotaxin-2. Moreover, in both OVA-naïve and allergic mice, lung mPGES-1 mRNA and BAL PGE2 levels were elevated in mice infected with high-dose, but not low-dose Mp. In pAMs, IL-4/IL-13 significantly increased eotaxin-2, which was reduced by Mp infection accompanied by dose-dependent PGE2 induction. Exogenous PGE2 inhibited IL-4/IL-13-induced eotaxin-2 in a dose-dependent manner.
Conclusions
This study highlights a novel concept on how differing bacterial loads in the lung modify the established allergic airway inflammation, and thus interact with an allergen to further induce Th2 responses. That is: Unlike high-level Mp, low-level Mp fails to effectively induce PGE2 to down-regulate allergic responses (e.g., eotaxin-2), thus maintaining or even worsening allergic inflammation in asthmatic airways.
doi:10.1111/j.1365-2222.2009.03309.x
PMCID: PMC2784117  PMID: 19552640
asthma; Mycoplasma pneumoniae; eotaxin-2; PGE2; alveolar macrophages
20.  Phosphodiesterase 4B is essential for TH2-cell function and development of airway hyperresponsiveness in allergic asthma 
Background
Cyclic AMP (cAMP) signaling modulates functions of inflammatory cells involved in the pathogenesis of asthma, and type 4 cAMP-specific phosphodiesterases (PDE4s) are essential components of this pathway. Induction of the PDE4 isoform PDE4B is necessary for Toll-like receptor signaling in monocytes and macrophages and is associated with T cell receptor/CD3 in T cells; however, its exact physiological function in the development of allergic asthma remains undefined.
Objectives
We investigated the role of PDE4B in the development of allergen-induced airway hyperresponsiveness (AHR) and TH2-driven inflammatory responses.
Methods
Wild-type and PDE4B−/− mice were sensitized and challenged with ovalbumin and AHR measured in response to inhaled methacholine. Airway inflammation was characterized by analyzing leukocyte infiltration and cytokine accumulation in the airways. Ovalbumin-stimulated cell proliferation and TH2 cytokine production were determined in cultured bronchial lymph node cells.
Results
Mice deficient in PDE4B do not develop AHR. This protective effect was associated with a significant decrease in eosinophils recruitment to the lungs and decreased TH2 cytokine levels in the bronchoalveolar lavage fluid. Defects in T-cell replication, TH2 cytokine production, and dendritic cell migration were evident in cells from the airway-draining lymph nodes. Conversely, accumulation of the TH1 cytokine IFN-γ was not affected in PDE4B−/− mice. Ablation of the orthologous PDE4 gene PDE4A has no impact on airway inflammation.
Conclusion
By relieving a cAMP-negative constraint, PDE4B plays an essential role in TH2-cell activation and dendritic cell recruitment during airway inflammation. These findings provide proof of concept that PDE4 inhibitors with PDE4B selectivity may have efficacy in asthma treatment.
doi:10.1016/j.jaci.2010.08.014
PMCID: PMC3002752  PMID: 21047676
Asthma; PDE4B; TH2 cytokines; airway hyperresponsiveness; airway inflammation; cAMP signaling
21.  Effect of aging on pulmonary inflammation, airway hyperresponsiveness and T and B cell responses in antigen sensitized and challenged mice 
Background
The effect of aging on several pathologic features of allergic-asthma (pulmonary inflammation, eosinophilia, mucus-hypersecretion), and their relationship with airway hyperresponsiveness (AHR) is not well characterized.
Objective
To evaluate lung inflammation, mucus-metaplasia and AHR in relationship to age in murine models of allergic-asthma comparing young and older mice.
Methods
Young (6-week) and older (6-, 12- 18-month) BALB/c mice were sensitized and challenged with ovalbumin (OVA). AHR and bronchoalveolar fluid (BALF) total inflammatory cell count and differential were measured. To evaluate mucus-metaplasia, quantitative PCR for the major airway mucin-associated gene, MUC-5AC, from lung tissue was measured, and lung tissue sections stained with periodic acid-Schiff (PAS) for goblet-cell enumeration. Lung tissue cytokine gene expression was determined by qPCR, and systemic cytokine protein levels by ELISA from spleen-cell cultures. Antigen-specific serum IgE was determined by ELISA.
Results
AHR developed in both aged and young OVA-sensitized/challenged mice (OVA-mice), and was more significantly increased in young OVA-mice than in aged OVA-mice. However, BALF eosinophil numbers were significantly higher, and lung histology showed greater inflammation in aged OVA-mice than in young OVA-mice. MUC-5AC expression and numbers of PAS+ staining bronchial epithelial cells were significantly increased in the aged OVA-mice. All aged OVA-mice had increased IL-5 and IFN-γ mRNA expression in the lung and IL-5 and IFN-γ protein levels from spleen cell cultures compared to young OVA-mice. OVA-IgE was elevated to a greater extent in aged OVA-mice.
Conclusions
Although pulmonary inflammation and mucus-metaplasia after antigen sensitization/challenge occurred to a greater degree in older mice, the increase in AHR was significantly less compared with younger OVA-mice. Antigen treatment produced a unique cytokine profile in older mice (elevated IFN-γ and IL-5) compared with young mice (elevated IL-4 and IL-13). Thus, the airway response to inflammation is lessened in aging animals, and may represent age-associated events leading to different phenotypes in response to antigen provocation.
doi:10.1111/j.1365-2222.2007.02775.x
PMCID: PMC2818115  PMID: 17845421
Aging; murine; asthma; airway hyperresponsiveness; eosinophil; inflammation
22.  Haemophilus influenzae Infection Drives IL-17-Mediated Neutrophilic Allergic Airways Disease 
PLoS Pathogens  2011;7(10):e1002244.
A subset of patients with stable asthma has prominent neutrophilic and reduced eosinophilic inflammation, which is associated with attenuated airways hyper-responsiveness (AHR). Haemophilus influenzae has been isolated from the airways of neutrophilic asthmatics; however, the nature of the association between infection and the development of neutrophilic asthma is not understood. Our aim was to investigate the effects of H. influenzae respiratory infection on the development of hallmark features of asthma in a mouse model of allergic airways disease (AAD). BALB/c mice were intraperitoneally sensitized to ovalbumin (OVA) and intranasally challenged with OVA 12–15 days later to induce AAD. Mice were infected with non-typeable H. influenzae during or 10 days after sensitization, and the effects of infection on the development of key features of AAD were assessed on day 16. T-helper 17 cells were enumerated by fluorescent-activated cell sorting and depleted with anti-IL-17 neutralizing antibody. We show that infection in AAD significantly reduced eosinophilic inflammation, OVA-induced IL-5, IL-13 and IFN-γ responses and AHR; however, infection increased airway neutrophil influx in response to OVA challenge. Augmented neutrophilic inflammation correlated with increased IL-17 responses and IL-17 expressing macrophages and neutrophils (early, innate) and T lymphocytes (late, adaptive) in the lung. Significantly, depletion of IL-17 completely abrogated infection-induced neutrophilic inflammation during AAD. In conclusion, H. influenzae infection synergizes with AAD to induce Th17 immune responses that drive the development of neutrophilic and suppress eosinophilic inflammation during AAD. This results in a phenotype that is similar to neutrophilic asthma. Infection-induced neutrophilic inflammation in AAD is mediated by IL-17 responses.
Author Summary
Approximately 50% of asthmatics have non-eosinophilic inflammation, and 20% of these patients have severe neutrophilic inflammation and increased IL-8 levels. These so-called neutrophilic asthmatics have persistent airway colonization with bacteria, and Haemophilus influenzae is one of the bacteria most commonly isolated. However, how H. influenzae is associated with the pathogenesis of neutrophilic asthma is unknown. In this study we used mouse models to investigate the relationship between H. influenzae infection and allergic airways disease (AAD). We showed that infection promoted the development of hallmark features of neutrophilic asthma. Infection suppressed Th2 cytokines, eosinophilic inflammation, and AHR in AAD, while increasing neutrophilic inflammation and IL-17 responses. Importantly, inhibition of IL-17 during AAD reduced airway neutrophils and neutrophil chemokines, suggesting that infection drives the development of neutrophilic inflammation through an IL-17-mediated mechanism. This provides novel insights into the mechanisms that may underpin infection-induced neutrophilic asthma. These data also suggest that treatments targeting infection may lead to improved management of neutrophilic asthma.
doi:10.1371/journal.ppat.1002244
PMCID: PMC3188527  PMID: 21998577
23.  Rhinovirus Exacerbates House-Dust-Mite Induced Lung Disease in Adult Mice 
PLoS ONE  2014;9(3):e92163.
Human rhinovirus is a key viral trigger for asthma exacerbations. To date, murine studies investigating rhinovirus-induced exacerbation of allergic airways disease have employed systemic sensitisation/intranasal challenge with ovalbumin. In this study, we combined human-rhinovirus infection with a clinically relevant mouse model of aero-allergen exposure using house-dust-mite in an attempt to more accurately understand the links between human-rhinovirus infection and exacerbations of asthma. Adult BALB/c mice were intranasally exposed to low-dose house-dust-mite (or vehicle) daily for 10 days. On day 9, mice were inoculated with human-rhinovirus-1B (or UV-inactivated human-rhinovirus-1B). Forty-eight hours after inoculation, we assessed bronchoalveolar cellular inflammation, levels of relevant cytokines/serum antibodies, lung function and responsiveness/sensitivity to methacholine. House-dust-mite exposure did not result in a classical TH2-driven response, but was more representative of noneosinophilic asthma. However, there were significant effects of house-dust-mite exposure on most of the parameters measured including increased cellular inflammation (primarily macrophages and neutrophils), increased total IgE and house-dust-mite-specific IgG1 and increased responsiveness/sensitivity to methacholine. There were limited effects of human-rhinovirus-1B infection alone, and the combination of the two insults resulted in additive increases in neutrophil levels and lung parenchymal responses to methacholine (tissue elastance). We conclude that acute rhinovirus infection exacerbates house-dust-mite-induced lung disease in adult mice. The similarity of our results using the naturally occurring allergen house-dust-mite, to previous studies using ovalbumin, suggests that the exacerbation of allergic airways disease by rhinovirus infection could act via multiple or conserved mechanisms.
doi:10.1371/journal.pone.0092163
PMCID: PMC3954893  PMID: 24632596
24.  Exposure of neonates to Respiratory Syncytial Virus is critical in determining subsequent airway response in adults 
Respiratory Research  2006;7(1):107.
Background
Respiratory syncytial virus (RSV) is the most common cause of acute bronchiolitis in infants and the elderly. Furthermore, epidemiological data suggest that RSV infection during infancy is a potent trigger of subsequent wheeze and asthma development. However, the mechanism by which RSV contributes to asthma is complex and remains largely unknown. A recent study indicates that the age of initial RSV infection is a key factor in determining airway response to RSV rechallenge. We hypothesized that severe RSV infection during neonatal development significantly alters lung structure and the pulmonary immune micro-environment; and thus, neonatal RSV infection is crucial in the development of or predisposition to allergic inflammatory diseases such as asthma.
Methods
To investigate this hypothesis the present study was conducted in a neonatal mouse model of RSV-induced pulmonary inflammation and airway dysfunction. Seven-day-old mice were infected with RSV (2 × 105 TCID50/g body weight) and allowed to mature to adulthood. To determine if neonatal RSV infection predisposed adult animals to enhanced pathophysiological responses to allergens, these mice were then sensitized and challenged with ovalbumin. Various endpoints including lung function, histopathology, cytokine production, and cellularity in bronchoalveolar lavage were examined.
Results
RSV infection in neonates alone led to inflammatory airway disease characterized by airway hyperreactivity, peribronchial and perivascular inflammation, and subepithelial fibrosis in adults. If early RSV infection was followed by allergen exposure, this pulmonary phenotype was exacerbated. The initial response to neonatal RSV infection resulted in increased TNF-α levels in bronchoalveolar lavage. Interestingly, increased levels of IL-13 and mucus hyperproduction were observed almost three months after the initial infection with RSV.
Conclusion
Neonatal RSV exposure results in long term pulmonary inflammation and exacerbates allergic airways disease. The early increase in TNF-α in the bronchoalveolar lavage implicates this inflammatory cytokine in orchestrating these events. Finally, the data presented emphasize IL-13 and TNF-α as potential therapeutic targets for treating RSV induced-asthma.
doi:10.1186/1465-9921-7-107
PMCID: PMC1563465  PMID: 16893457
25.  Associations of airway inflammation and responsiveness markers in non asthmatic subjects at start of apprenticeship 
Background
Bronchial Hyperresponsiveness (BHR) is considered a hallmark of asthma. Other methods are helpful in epidemiological respiratory health studies including Fractional Exhaled Nitric Oxide (FENO) and Eosinophils Percentage (EP) in nasal lavage fluid measuring markers for airway inflammation along with the Forced Oscillatory Technique measuring Airway resistance (AR). Can their outcomes discriminate profiles of respiratory health in healthy subjects starting apprenticeship in occupations with a risk of asthma?
Methods
Rhinoconjunctivitis, asthma-like symptoms, FEV1 and AR post-Methacholine Bronchial Challenge (MBC) test results, FENO measurements and EP were all investigated in apprentice bakers, pastry-makers and hairdressers not suffering from asthma. Multiple Correspondence Analysis (MCA) was simultaneously conducted in relation to these groups and this generated a synthetic partition (EI). Associations between groups of subjects based on BHR and EI respectively, as well as risk factors, symptoms and investigations were also assessed.
Results
Among the 441 apprentice subjects, 45 (10%) declared rhinoconjunctivitis-like symptoms, 18 (4%) declared asthma-like symptoms and 26 (6%) suffered from BHR. The mean increase in AR post-MBC test was 21% (sd = 20.8%). The median of FENO values was 12.6 ppb (2.6-132 range). Twenty-six subjects (6.7%) had EP exceeding 14%. BHR was associated with atopy (p < 0.01) and highest FENO values (p = 0.09). EI identified 39 subjects with eosinophilic inflammation (highest values of FENO and eosinophils), which was associated with BHR and atopy.
Conclusions
Are any of the identified markers predictive of increased inflammatory responsiveness or of development of symptoms caused by occupational exposures? Analysis of population follow-up will attempt to answer this question.
doi:10.1186/1471-2466-10-37
PMCID: PMC2913998  PMID: 20604945

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