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1.  Effects of BMI, Fat Mass, and Lean Mass on Asthma in Childhood: A Mendelian Randomization Study 
PLoS Medicine  2014;11(7):e1001669.
In this study, Granell and colleagues used Mendelian randomization to investigate causal effects of BMI, fat mass, and lean mass on current asthma at age 7½ years in the Avon Longitudinal Study of Parents and Children (ALSPAC) and found that higher BMI increases the risk of asthma in mid-childhood.
Please see later in the article for the Editors' Summary
Observational studies have reported associations between body mass index (BMI) and asthma, but confounding and reverse causality remain plausible explanations. We aim to investigate evidence for a causal effect of BMI on asthma using a Mendelian randomization approach.
Methods and Findings
We used Mendelian randomization to investigate causal effects of BMI, fat mass, and lean mass on current asthma at age 7½ y in the Avon Longitudinal Study of Parents and Children (ALSPAC). A weighted allele score based on 32 independent BMI-related single nucleotide polymorphisms (SNPs) was derived from external data, and associations with BMI, fat mass, lean mass, and asthma were estimated. We derived instrumental variable (IV) estimates of causal risk ratios (RRs). 4,835 children had available data on BMI-associated SNPs, asthma, and BMI. The weighted allele score was strongly associated with BMI, fat mass, and lean mass (all p-values<0.001) and with childhood asthma (RR 2.56, 95% CI 1.38–4.76 per unit score, p = 0.003). The estimated causal RR for the effect of BMI on asthma was 1.55 (95% CI 1.16–2.07) per kg/m2, p = 0.003. This effect appeared stronger for non-atopic (1.90, 95% CI 1.19–3.03) than for atopic asthma (1.37, 95% CI 0.89–2.11) though there was little evidence of heterogeneity (p = 0.31). The estimated causal RRs for the effects of fat mass and lean mass on asthma were 1.41 (95% CI 1.11–1.79) per 0.5 kg and 2.25 (95% CI 1.23–4.11) per kg, respectively. The possibility of genetic pleiotropy could not be discounted completely; however, additional IV analyses using FTO variant rs1558902 and the other BMI-related SNPs separately provided similar causal effects with wider confidence intervals. Loss of follow-up was unlikely to bias the estimated effects.
Higher BMI increases the risk of asthma in mid-childhood. Higher BMI may have contributed to the increase in asthma risk toward the end of the 20th century.
Please see later in the article for the Editors' Summary
Editors' Summary
The global burden of asthma, a chronic (long-term) condition caused by inflammation of the airways (the tubes that carry air in and out of the lungs), has been rising steadily over the past few decades. It is estimated that, nowadays, 200–300 million adults and children worldwide are affected by asthma. Although asthma can develop at any age, it is often diagnosed in childhood—asthma is the most common chronic disease in children. In people with asthma, the airways can react very strongly to allergens such as animal fur or to irritants such as cigarette smoke, becoming narrower so that less air can enter the lungs. Exercise, cold air, and infections can also trigger asthma attacks, which can be fatal. The symptoms of asthma include wheezing, coughing, chest tightness, and shortness of breath. Asthma cannot be cured, but drugs can relieve its symptoms and prevent acute asthma attacks.
Why Was This Study Done?
We cannot halt the ongoing rise in global asthma rates without understanding the causes of asthma. Some experts think obesity may be one cause of asthma. Obesity, like asthma, is increasingly common, and observational studies (investigations that ask whether individuals exposed to a suspected risk factor for a condition develop that condition more often than unexposed individuals) in children have reported that body mass index (BMI, an indicator of body fat calculated by dividing a person's weight in kilograms by their height in meters squared) is positively associated with asthma. Observational studies cannot prove that obesity causes asthma because of “confounding.” Overweight children with asthma may share another unknown characteristic (confounder) that actually causes both obesity and asthma. Moreover, children with asthma may be less active than unaffected children, so they become overweight (reverse causality). Here, the researchers use “Mendelian randomization” to assess whether BMI has a causal effect on asthma. In Mendelian randomization, causality is inferred from associations between genetic variants that mimic the effect of a modifiable risk factor and the outcome of interest. Because gene variants are inherited randomly, they are not prone to confounding and are free from reverse causation. So, if a higher BMI leads to asthma, genetic variants associated with increased BMI should be associated with an increased risk of asthma.
What Did the Researchers Do and Find?
The researchers investigated causal effects of BMI, fat mass, and lean mass on current asthma at age 7½ years in 4,835 children enrolled in the Avon Longitudinal Study of Parents and Children (ALSPAC, a long-term health project that started in 1991). They calculated an allele score for each child based on 32 BMI-related genetic variants, and estimated associations between this score and BMI, fat mass and lean mass (both measured using a special type of X-ray scanner; in children BMI is not a good indicator of “fatness”), and asthma. They report that the allele score was strongly associated with BMI, fat mass, and lean mass, and with childhood asthma. The estimated causal relative risk (risk ratio) for the effect of BMI on asthma was 1.55 per kg/m2. That is, the relative risk of asthma increased by 55% for every extra unit of BMI. The estimated causal relative risks for the effects of fat mass and lean mass on asthma were 1.41 per 0.5 kg and 2.25 per kg, respectively.
What Do These Findings Mean?
These findings suggest that a higher BMI increases the risk of asthma in mid-childhood and that global increases in BMI toward the end of the 20th century may have contributed to the global increase in asthma that occurred at the same time. It is possible that the observed association between BMI and asthma reported in this study is underpinned by “genetic pleiotropy” (a potential limitation of all Mendelian randomization analyses). That is, some of the genetic variants included in the BMI allele score could conceivably also increase the risk of asthma. Nevertheless, these findings suggest that public health interventions designed to reduce obesity may also help to limit the global rise in asthma.
Additional Information
Please access these websites via the online version of this summary at
The US Centers for Disease Control and Prevention provides information on asthma and on all aspects of overweight and obesity (in English and Spanish)
The World Health Organization provides information on asthma and on obesity (in several languages)
The UK National Health Service Choices website provides information about asthma, about asthma in children, and about obesity (including real stories)
The Global Asthma Report 2011 is available
The Global Initiative for Asthma released its updated Global Strategy for Asthma Management and Prevention on World Asthma Day 2014
Information about the Avon Longitudinal Study of Parents and Children is available
MedlinePlus provides links to further information on obesity in children, on asthma, and on asthma in children (in English and Spanish
Wikipedia has a page on Mendelian randomization (note: Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
PMCID: PMC4077660  PMID: 24983943
2.  Genetics of Asthma Susceptibility and Severity 
Clinics in chest medicine  2012;33(3):431-443.
The interaction of genes and environmental exposures influences the development of asthma and determines asthma severity. This review focuses on recent developments in genetic studies of asthma onset and progression. Genome-wide association studies (GWAS) are currently the most effective approach to study genetics of complex diseases. There have been two large meta-analyses of asthma susceptibility, GABRIEL and EVE, which identified the same four chromosomal regions, many of which had also been identified in previous GWAS: loci in the ORMDL3 region of 17q21, IL1RL/IL18R genes on chromosome 2q, the TSLP gene region on 5q22, and IL33 on chromosome 9p24. These regions were associated with asthma in individuals of different ethnic backgrounds. EVE also identified a novel asthma susceptibility locus, PYHIN1, in individuals of African descent. Genome-wide screens for asthma susceptibility in Asian adults and children both identified genetic variants in the major histocompatiblity complex gene region (HLA region) on chromosome 6p21 as highly associated with asthma risk. This locus was one of the first candidate genes identified for asthma and has been a significant predictor of asthma risk in several GWAS.
There is also a need to understand asthma disease heterogeneity as different phenotypes may reflect several pathogenic pathways. Genes that are associated with phenotypes including lung function, biomarker levels and asthma therapeutic responses provide insight into mechanisms of asthma severity progression. For example, the HHIP gene is a significant predictor of pulmonary function changes in asthma and in the normal population. A joint model of risk variants in lung function genes were highly associated with lower FEV1 and increased asthma severity criteria. In addition, a genome-wide screen to discover pharmacogenetic associations related to response to inhaled glucocorticoids identified two correlated SNPs in the GLCCI1 gene that confer a significant lung function response to this asthma therapy.
Future genetic studies for asthma susceptibility and severity will incorporate exome or whole-genome sequencing to identify common and rare genetic variants. Using these variants identified in comprehensively phenotyped asthmatics will lead to the development of personalized therapy in individuals with asthma.
PMCID: PMC3431509  PMID: 22929093
Asthma; genetics; susceptibility; severity; personalized medicine; therapy; lung function
3.  Association of Adenotonsillectomy with Asthma Outcomes in Children: A Longitudinal Database Analysis 
PLoS Medicine  2014;11(11):e1001753.
Rakesh Bhattacharjee and colleagues use data from a US private health insurance database to compare asthma severity measures in children one year before and one year after they underwent adenotonsillectomy with asthma measures in those who did not undergo adenotonsillectomy.
Please see later in the article for the Editors' Summary
Childhood asthma and obstructive sleep apnea (OSA), both disorders of airway inflammation, were associated in recent observational studies. Although childhood OSA is effectively treated by adenotonsillectomy (AT), it remains unclear whether AT also improves childhood asthma. We hypothesized that AT, the first line of therapy for childhood OSA, would be associated with improved asthma outcomes and would reduce the usage of asthma therapies in children.
Methods and Findings
Using the 2003–2010 MarketScan database, we identified 13,506 children with asthma in the United States who underwent AT. Asthma outcomes during 1 y preceding AT were compared to those during 1 y following AT. In addition, 27,012 age-, sex-, and geographically matched children with asthma without AT were included to examine asthma outcomes among children without known adenotonsillar tissue morbidity. Primary outcomes included the occurrence of a diagnostic code for acute asthma exacerbation (AAE) or acute status asthmaticus (ASA). Secondary outcomes included temporal changes in asthma medication prescriptions, the frequency of asthma-related emergency room visits (ARERs), and asthma-related hospitalizations (ARHs). Comparing the year following AT to the year prior, AT was associated with significant reductions in AAE (30.2%; 95% CI: 25.6%–34.3%; p<0.0001), ASA (37.9%; 95% CI: 29.2%–45.6%; p<0.0001), ARERs (25.6%; 95% CI: 16.9%–33.3%; p<0.0001), and ARHs (35.8%; 95% CI: 19.6%–48.7%; p = 0.02). Moreover, AT was associated with significant reductions in most asthma prescription refills, including bronchodilators (16.7%; 95% CI: 16.1%–17.3%; p<0.001), inhaled corticosteroids (21.5%; 95% CI: 20.7%–22.3%; p<0.001), leukotriene receptor antagonists (13.4%; 95% CI: 12.9%–14.0%; p<0.001), and systemic corticosteroids (23.7%; 95% CI: 20.9%–26.5%; p<0.001). In contrast, there were no significant reductions in these outcomes in children with asthma who did not undergo AT over an overlapping follow-up period. Limitations of the MarketScan database include lack of information on race and obesity status. Also, the MarketScan database does not include information on children with public health insurance (i.e., Medicaid) or uninsured children.
In a very large sample of privately insured children, AT was associated with significant improvements in several asthma outcomes. Contingent on validation through prospectively designed clinical trials, this study supports the premise that detection and treatment of adenotonsillar tissue morbidity may serve as an important strategy for improving asthma control.
Please see later in the article for the Editors' Summary
Editors' Summary
The global burden of asthma has been rising steadily over the past few decades. Nowadays, about 200–300 million adults and children worldwide are affected by asthma, a chronic condition caused by inflammation of the airways (the tubes that carry air in and out of the lungs). Although asthma can develop at any age, it is often diagnosed in childhood—asthma is one of the commonest chronic diseases in children. In the US, for example, asthma affects around 7.1 million children under the age of 18 years and is the third leading cause of hospitalization of children under the age of 15 years. In people with asthma, the airways can react very strongly to allergens such as animal fur or to irritants such as cigarette smoke. Exercise, cold air, and infections can trigger asthma attacks, which can be fatal. The symptoms of asthma include wheezing, coughing, chest tightness, and shortness of breath. Asthma cannot be cured, but drugs can relieve its symptoms and prevent acute asthma attacks.
Why Was This Study Done?
Recent studies have found an association between severe childhood asthma and obstructive sleep apnea (OSA). In OSA, airway inflammation promotes hypertrophy (excess growth) of the adenoids and the tonsils, immune system tissues in the upper airway. During sleep, the presence of hypertrophic adenotonsillar tissues predisposes the walls of the throat to collapse, which results in apnea—a brief interruption in breathing. People with OSA often snore loudly and frequently wake from deep sleep as they struggle to breathe. Childhood OSA, which affects 2%–3% of children, can be effectively treated by removal of the adenoids and tonsils (adenotonsillectomy). Given the association between childhood OSA and severe asthma and given the involvement of airway inflammation in both conditions, might adenotonsillectomy also improve childhood asthma? Here, the researchers analyze data from the MarketScan database, a large database of US patients with private health insurance, to investigate whether adenotonsillectomy is associated with improvements in asthma outcomes and with reductions in the use of asthma therapies in children.
What Did the Researchers Do and Find?
The researchers used the database to identify 13,506 children with asthma who had undergone adenotonsillectomy and to obtain information about asthma outcomes among these children for the year before and the year after the operation. Because asthma severity tends to decrease with age, the researchers also used the database to identify 27,012 age-, sex-, and geographically matched children with asthma who did not have the operation so that they could examine asthma outcomes over an equivalent two-year period in the absence of complications related to adenotonsillar hypertrophy. Comparing the year after adenotonsillectomy with the year before the operation, adenotonsillectomy was associated with a 30% reduction in acute asthma exacerbations, a 37.9% reduction in acute status asthmaticus (an asthma attack that is unresponsive to the drugs usually used to treat attacks), a 25.6% reduction in asthma-related emergency room visits, and a 35.8% reduction in asthma-related hospitalizations. By contrast, among the control children, there was only a 2% reduction in acute asthma exacerbations and only a 7% reduction in acute status asthmaticus over an equivalent two-year period. Adenotonsillectomy was also associated with significant reductions (changes unlikely to have occurred by chance) in prescription refills for most types of drugs used to treat asthma, whereas there were no significant reductions in prescription refills among children with asthma who had not undergone adenotonsillectomy. The study was limited by the lack of measures of race and obesity, which are both associated with severity of asthma.
What Do These Findings Mean?
These findings show that in a large sample of privately insured children in the US, adenotonsillectomy was associated with significant improvements in several asthma outcomes. These results do not show, however, that adenotonsillectomy caused a reduction in the severity of childhood asthma. It could be that the children who underwent adenotonsillectomy (but not those who did not have the operation) shared another unknown factor that led to improvements in their asthma over time. To prove a causal link, it will be necessary to undertake a randomized controlled trial in which the outcomes of groups of children with asthma who are chosen at random to undergo or not undergo adenotonsillectomy are compared. However, with the proviso that there are some risks associated with adenotonsillectomy, these findings suggest that the detection and treatment of adenotonsillar hypertrophy may help to improve asthma control in children.
Additional Information
Please access these websites via the online version of this summary at
The US Centers for Disease Control and Prevention provides information on asthma, including videos, games, and links to other resources for children with asthma
The American Lung Association provides detailed information about asthma and a fact sheet on asthma in children; it also has information about obstructive sleep apnea
The National Sleep Foundation provides information on snoring and obstructive sleep apnea in children
The UK National Health Service Choices website provides information (including some personal stories) about asthma, about asthma in children, and about obstructive sleep apnea
The “Global Asthma Report 2014” will be available in October 2014
MedlinePlus provides links to further information on asthma, on asthma in children, on sleep apnea, and on tonsils and adenoids (in English and Spanish)
PMCID: PMC4219664  PMID: 25369282
4.  Preterm Birth and Childhood Wheezing Disorders: A Systematic Review and Meta-Analysis 
PLoS Medicine  2014;11(1):e1001596.
In a systematic review and meta-analysis, Jasper Been and colleagues investigate the association between preterm birth and the development of wheezing disorders in childhood.
Please see later in the article for the Editors' Summary
Accumulating evidence implicates early life factors in the aetiology of non-communicable diseases, including asthma/wheezing disorders. We undertook a systematic review investigating risks of asthma/wheezing disorders in children born preterm, including the increasing numbers who, as a result of advances in neonatal care, now survive very preterm birth.
Methods and Findings
Two reviewers independently searched seven online databases for contemporaneous (1 January 1995–23 September 2013) epidemiological studies investigating the association between preterm birth and asthma/wheezing disorders. Additional studies were identified through reference and citation searches, and contacting international experts. Quality appraisal was undertaken using the Effective Public Health Practice Project instrument. We pooled unadjusted and adjusted effect estimates using random-effects meta-analysis, investigated “dose–response” associations, and undertook subgroup, sensitivity, and meta-regression analyses to assess the robustness of associations.
We identified 42 eligible studies from six continents. Twelve were excluded for population overlap, leaving 30 unique studies involving 1,543,639 children. Preterm birth was associated with an increased risk of wheezing disorders in unadjusted (13.7% versus 8.3%; odds ratio [OR] 1.71, 95% CI 1.57–1.87; 26 studies including 1,500,916 children) and adjusted analyses (OR 1.46, 95% CI 1.29–1.65; 17 studies including 874,710 children). The risk was particularly high among children born very preterm (<32 wk gestation; unadjusted: OR 3.00, 95% CI 2.61–3.44; adjusted: OR 2.81, 95% CI 2.55–3.12). Findings were most pronounced for studies with low risk of bias and were consistent across sensitivity analyses. The estimated population-attributable risk of preterm birth for childhood wheezing disorders was ≥3.1%.
Key limitations related to the paucity of data from low- and middle-income countries, and risk of residual confounding.
There is compelling evidence that preterm birth—particularly very preterm birth—increases the risk of asthma. Given the projected global increases in children surviving preterm births, research now needs to focus on understanding underlying mechanisms, and then to translate these insights into the development of preventive interventions.
Review Registration
PROSPERO CRD42013004965
Please see later in the article for the Editors' Summary
Editors' Summary
Most pregnancies last around 40 weeks, but worldwide, more than 11% of babies are born before 37 weeks of gestation (the period during which a baby develops in its mother's womb). Preterm birth is a major cause of infant death—more than 1 million babies die annually from preterm birth complications—and the number of preterm births is increasing globally. Multiple pregnancies, infections, and chronic (long-term) maternal conditions such as diabetes can all cause premature birth, but the cause of many preterm births is unknown. The most obvious immediate complication that is associated with preterm birth is respiratory distress syndrome. This breathing problem, which is more common in early preterm babies than in near-term babies, occurs because the lungs of premature babies are structurally immature and lack pulmonary surfactant, a unique mixture of lipids and proteins that coats the inner lining of the lungs and helps to prevent the collapse of the small air sacs in the lungs that absorb oxygen from the air. Consequently, preterm babies often need help with their breathing and oxygen supplementation.
Why Was This Study Done?
Improvements in the management of prematurity mean that more preterm babies survive today than in the past. However, accumulating evidence suggests that early life events are involved in the subsequent development of non-communicable diseases (non-infectious chronic diseases). Given the increasing burden of preterm birth, a better understanding of the long-term effects of preterm birth is essential. Here, the researchers investigate the risks of asthma and wheezing disorders in children who are born preterm by undertaking a systematic review (a study that uses predefined criteria to identify all the research on a given topic) and a meta-analysis (a statistical method for combining the results of several studies). Asthma is a chronic condition that is caused by inflammation of the airways. In people with asthma, the airways can react very strongly to allergens such as animal fur and to irritants such as cigarette smoke. Exercise, cold air, and infections can also trigger asthma attacks, which can sometimes be fatal. The symptoms of asthma include wheezing (a high-pitched whistling sound during breathing), coughing, chest tightness, and shortness of breath. Asthma cannot be cured, but drugs can relieve its symptoms and prevent acute asthma attacks.
What Did the Researchers Do and Find?
The researchers identified 30 studies undertaken between 1995 and the present (a time span chosen to allow for recent changes in the management of prematurity) that investigated the association between preterm birth and asthma/wheezing disorders in more than 1.5 million children. Across the studies, 13.7% of preterm babies developed asthma/wheezing disorders during childhood, compared to only 8.3% of babies born at term. Thus, the risk of preterm babies developing asthma or a wheezing disorder during childhood was 1.71 times higher than the risk of term babies developing these conditions (an unadjusted odds ratio [OR] of 1.71). In analyses that allowed for confounding factors—other factors that affect the risk of developing asthma/wheezing disorders such as maternal smoking—the risk of preterm babies developing asthma or a wheezing disorder during childhood was 1.46 times higher than that of babies born at term (an adjusted OR of 1.46). Notably, compared to children born at term, children born very early (before 32 weeks of gestation) had about three times the risk of developing asthma/wheezing disorders in unadjusted and adjusted analyses. Finally, the population-attributable risk of preterm birth for childhood wheezing disorders was more than 3.1%. That is, if no preterm births had occurred, there would have been more than a 3.1% reduction in childhood wheezing disorders.
What Do These Findings Mean?
These findings strongly suggest that preterm birth increases the risk of asthma and wheezing disorders during childhood and that the risk of asthma/wheezing disorders increases as the degree of prematurity increases. The accuracy of these findings may be affected, however, by residual confounding. That is, preterm children may share other, unknown characteristics that increase their risk of developing asthma/wheezing disorders. Moreover, the generalizability of these findings is limited by the lack of data from low- and middle-income countries. However, given the projected global increases in children surviving preterm births, these findings highlight the need to undertake research into the mechanisms underlying the association between preterm birth and asthma/wheezing disorders and the need to develop appropriate preventative and therapeutic measures.
Additional Information
Please access these websites via the online version of this summary at
The March of Dimes, a nonprofit organization for pregnancy and baby health, provides information on preterm birth (in English and Spanish)
Nemours, another nonprofit organization for child health, also provides information (in English and Spanish) on premature babies and on asthma (including personal stories)
The UK National Health Service Choices website provides information about premature labor and birth and a real story about having a preterm baby; it provides information about asthma in children (including real stories)
The MedlinePlus Encyclopedia has pages on preterm birth, asthma, asthma in children, and wheezing (in English and Spanish); MedlinePlus provides links to further information on premature birth, asthma, and asthma in children (in English and Spanish)
PMCID: PMC3904844  PMID: 24492409
5.  Genome-Wide Association Study Implicates Chromosome 9q21.31 as a Susceptibility Locus for Asthma in Mexican Children 
PLoS Genetics  2009;5(8):e1000623.
Many candidate genes have been studied for asthma, but replication has varied. Novel candidate genes have been identified for various complex diseases using genome-wide association studies (GWASs). We conducted a GWAS in 492 Mexican children with asthma, predominantly atopic by skin prick test, and their parents using the Illumina HumanHap 550 K BeadChip to identify novel genetic variation for childhood asthma. The 520,767 autosomal single nucleotide polymorphisms (SNPs) passing quality control were tested for association with childhood asthma using log-linear regression with a log-additive risk model. Eleven of the most significantly associated GWAS SNPs were tested for replication in an independent study of 177 Mexican case–parent trios with childhood-onset asthma and atopy using log-linear analysis. The chromosome 9q21.31 SNP rs2378383 (p = 7.10×10−6 in the GWAS), located upstream of transducin-like enhancer of split 4 (TLE4), gave a p-value of 0.03 and the same direction and magnitude of association in the replication study (combined p = 6.79×10−7). Ancestry analysis on chromosome 9q supported an inverse association between the rs2378383 minor allele (G) and childhood asthma. This work identifies chromosome 9q21.31 as a novel susceptibility locus for childhood asthma in Mexicans. Further, analysis of genome-wide expression data in 51 human tissues from the Novartis Research Foundation showed that median GWAS significance levels for SNPs in genes expressed in the lung differed most significantly from genes not expressed in the lung when compared to 50 other tissues, supporting the biological plausibility of our overall GWAS findings and the multigenic etiology of childhood asthma.
Author Summary
Asthma is a leading chronic childhood disease with a presumed strong genetic component, but no genes have been definitely shown to influence asthma development. Few genetic studies of asthma have included Hispanic populations. Here, we conducted a genome-wide association study of asthma in 492 Mexican children with asthma, predominantly atopic by skin prick test, and their parents to identify novel genetic variation for childhood asthma. We implicated several polymorphisms in or near TLE4 on chromosome 9q21.31 (a novel candidate region for childhood asthma) and replicated one polymorphism in an independent study of childhood-onset asthmatics with atopy and their parents of Mexican ethnicity. Hispanics have differing proportions of Native American, European, and African ancestries, and we found less Native American ancestry than expected at chromosome 9q21.31. This suggests that chromosome 9q21.31 may underlie ethnic differences in childhood asthma and that future replication would be most effective in populations with Native American ancestry. Analysis of publicly available genome-wide expression data revealed that association signals in genes expressed in the lung differed most significantly from genes not expressed in the lung when compared to 50 other tissues, supporting the biological plausibility of the overall GWAS findings and the multigenic etiology of asthma.
PMCID: PMC2722731  PMID: 19714205
6.  Comparison of Temporal Transcriptomic Profiles from Immature Lungs of Two Rat Strains Reveals a Viral Response Signature Associated with Chronic Lung Dysfunction 
PLoS ONE  2014;9(12):e112997.
Early life respiratory viral infections and atopic characteristics are significant risk factors for the development of childhood asthma. It is hypothesized that repeated respiratory viral infections might induce structural remodeling by interfering with the normal process of lung maturation; however, the specific molecular processes that underlie these pathological changes are not understood. To investigate the molecular basis for these changes, we used an established Sendai virus infection model in weanling rats to compare the post-infection transcriptomes of an atopic asthma susceptible strain, Brown Norway, and a non-atopic asthma resistant strain, Fischer 344. Specific to this weanling infection model and not described in adult infection models, Sendai virus in the susceptible, but not the resistant strain, results in morphological abnormalities in distal airways that persist into adulthood. Gene expression data from infected and control lungs across five time points indicated that specific features of the immune response following viral infection were heightened and prolonged in lungs from Brown Norway rats compared with Fischer 344 rats. These features included an increase in macrophage cell number and related gene expression, which then transitioned to an increase in mast cell number and related gene expression. In contrast, infected Fischer F344 lungs exhibited more efficient restoration of the airway epithelial morphology, with transient appearance of basal cell pods near distal airways. Together, these findings indicate that the pronounced macrophage and mast cell responses and abnormal re-epithelialization precede the structural defects that developed and persisted in Brown Norway, but not Fischer 344 lungs.
PMCID: PMC4249857  PMID: 25437859
7.  Perinatal nicotine exposure induces asthma in second generation offspring 
BMC Medicine  2012;10:129.
By altering specific developmental signaling pathways that are necessary for fetal lung development, perinatal nicotine exposure affects lung growth and differentiation, resulting in the offsprings' predisposition to childhood asthma; peroxisome proliferator-activated receptor gamma (PPARγ) agonists can inhibit this effect. However, whether the perinatal nicotine-induced asthma risk is restricted to nicotine-exposed offspring only; whether it can be transmitted to the next generation; and whether PPARγ agonists would have any effect on this process are not known.
Time-mated Sprague Dawley rat dams received either placebo or nicotine (1 mg/kg, s.c.), once daily from day 6 of gestation to postnatal day (PND) 21. Following delivery, at PND21, generation 1 (F1) pups were either subjected to pulmonary function tests, or killed to obtain their lungs, tracheas, and gonads to determine the relevant protein markers (mesenchymal contractile proteins), global DNA methylation, histone 3 and 4 acetylation, and for tracheal tension studies. Some F1 animals were used as breeders to generate F2 pups, but without any exposure to nicotine in the F1 pregnancy. At PND21, F2 pups underwent studies similar to those performed on F1 pups.
Consistent with the asthma phenotype, nicotine affected lung function in both male and female F1 and F2 offspring (maximal 250% increase in total respiratory system resistance, and 84% maximal decrease in dynamic compliance following methacholine challenge; P < 0.01, nicotine versus control; P < 0.05, males versus females; and P > 0.05, F1 versus F2), but only affected tracheal constriction in males (51% maximal increase in tracheal constriction following acetylcholine challenge, P < 0.01, nicotine versus control; P < 0.0001, males versus females; P > 0.05, F1 versus F2); nicotine also increased the contractile protein content of whole lung (180% increase in fibronectin protein levels, P < 0.01, nicotine versus control, and P < 0.05, males versus females) and isolated lung fibroblasts (for example, 45% increase in fibronectin protein levels, P < 0.05, nicotine versus control), along with decreased PPARγ expression (30% decrease, P < 0.05, nicotine versus control), but only affected contractile proteins in the male trachea (P < 0.05, nicotine versus control, and P < 0.0001, males versus females). All of the nicotine-induced changes in the lung and gonad DNA methylation and histone 3 and 4 acetylation were normalized by the PPARγ agonist rosiglitazone except for the histone 4 acetylation in the lung.
Germline epigenetic marks imposed by exposure to nicotine during pregnancy can become permanently programmed and transferred through the germline to subsequent generations, a ground-breaking finding that shifts the current asthma paradigm, opening up many new avenues to explore.
PMCID: PMC3568737  PMID: 23106849
nicotine; lung; epigenetic; asthma; multigenerational; gender difference
8.  Prenatal Secondhand Cigarette Smoke Promotes Th2 polarization and impairs goblet cell differentiation and airway mucus formation 
Parental, particularly maternal, smoking increases the risk of childhood allergic asthma and infection. Similarly, in a murine allergic asthma model, prenatal plus early postnatal exposure to secondhand cigarette smoke (SS) exacerbates airway hyperreactivity and Th2 responses in the lung. However, the mechanism and contribution of prenatal versus early postnatal SS exposure on allergic asthma remains unresolved. To identify the effects of prenatal and/or early postnatal SS on allergic asthma, BALB/c dams and their offspring were exposed gestationally and/or 8–10 weeks post-birth to filtered air or SS. Prenatal, but not postnatal SS strongly increased methacholine and allergen (Aspergillus)-induced airway resistance, Th2-cytokines levels and atopy, and activated the Th2 polarizing pathway GATA3/Lck/ERK1/2/STAT6. Either prenatal and/or early postnatal SS downregulated the Th1-specific transcription factor T-bet and, surprisingly, in spite of high levels of IL-4/IL-13, dramatically blocked the allergen-induced mucous cell metaplasia, airway mucus formation, and the expression of mucus-related genes/proteins: Muc5ac, GABAA-receptors, and SPDEF. Given that SS/nicotine exposure of normal adult mice promotes mucus formation, the results suggest that fetal and neonatal lung are highly sensitive to cigarette smoke. Thus, while the gestational SS promotes Th2 polarization/allergic asthma, it may also impair and/or delay the development of fetal and neonatal lung, affecting mucociliary clearance and Th1 responses. Together, this may explain the increased susceptibility of children from smoking parents to allergic asthma and childhood respiratory infections.
PMCID: PMC3197944  PMID: 21930963
Environmental (secondhand) tobacco smoke; airways hyperreactivity; allergic asthma; Th2 polarization; airway mucus
9.  Inflammatory cytokines, goblet cell hyperplasia and altered lung mechanics in Lgl1+/- mice 
Respiratory Research  2009;10(1):83.
Neonatal lung injury, a leading cause of morbidity in prematurely born infants, has been associated with arrested alveolar development and is often accompanied by goblet cell hyperplasia. Genes that regulate alveolarization and inflammation are likely to contribute to susceptibility to neonatal lung injury. We previously cloned Lgl1, a developmentally regulated secreted glycoprotein in the lung. In rat, O2 toxicity caused reduced levels of Lgl1, which normalized during recovery. We report here on the generation of an Lgl1 knockout mouse in order to determine whether deficiency of Lgl1 is associated with arrested alveolarization and contributes to neonatal lung injury.
An Lgl1 knockout mouse was generated by introduction of a neomycin cassette in exon 2 of the Lgl1 gene. To evaluate the pulmonary phenotype of Lgl1+/- mice, we assessed lung morphology, Lgl1 RNA and protein, elastin fibers and lung function. We also analyzed tracheal goblet cells, and expression of mucin, interleukin (IL)-4 and IL-13 as markers of inflammation.
Absence of Lgl1 was lethal prior to lung formation. Postnatal Lgl1+/- lungs displayed delayed histological maturation, goblet cell hyperplasia, fragmented elastin fibers, and elevated expression of TH2 cytokines (IL-4 and IL-13). At one month of age, reduced expression of Lgl1 was associated with elevated tropoelastin expression and altered pulmonary mechanics.
Our findings confirm that Lgl1 is essential for viability and is required for developmental processes that precede lung formation. Lgl1+/- mice display a complex phenotype characterized by delayed histological maturation, features of inflammation in the post-natal period and altered lung mechanics at maturity. Lgl1 haploinsufficiency may contribute to lung disease in prematurity and to increased risk for late-onset respiratory disease.
PMCID: PMC2760518  PMID: 19772569
10.  Early diagnosis of asthma in young children by using non-invasive biomarkers of airway inflammation and early lung function measurements: study protocol of a case-control study 
BMC Public Health  2009;9:210.
Asthma is the most common chronic disease in childhood, characterized by chronic airway inflammation. There are problems with the diagnosis of asthma in young children since the majority of the children with recurrent asthma-like symptoms is symptom free at 6 years, and does not have asthma. With the conventional diagnostic tools it is not possible to differentiate between preschool children with transient symptoms and children with asthma. The analysis of biomarkers of airway inflammation in exhaled breath is a non-invasive and promising technique to diagnose asthma and monitor inflammation in young children. Moreover, relatively new lung function tests (airway resistance using the interrupter technique) have become available for young children. The primary objective of the ADEM study (Asthma DEtection and Monitoring study), is to develop a non-invasive instrument for an early asthma diagnosis in young children, using exhaled inflammatory markers and early lung function measurements. In addition, aetiological factors, including gene polymorphisms and gene expression profiles, in relation to the development of asthma are studied.
A prospective case-control study is started in 200 children with recurrent respiratory symptoms and 50 control subjects without respiratory symptoms. At 6 years, a definite diagnosis of asthma is made (primary outcome measure) on basis of lung function assessments and current respiratory symptoms ('golden standard'). From inclusion until the definite asthma diagnosis, repeated measurements of lung function tests and inflammatory markers in exhaled breath (condensate), blood and faeces are performed. The study is registered and ethically approved.
This article describes the study protocol of the ADEM study. The new diagnostic techniques applied in this study could make an early diagnosis of asthma possible. An early and reliable asthma diagnosis at 2–3 years will have consequences for the management of the large group of young children with asthma-like symptoms. It will avoid both over-treatment of children with transient wheeze and under-treatment of children with asthma. This might have a beneficial influence on the prognosis of asthma in these young children. Besides, insight into the pathophysiology and aetiology of asthma will be obtained.
This study is registered by (NCT00422747).
PMCID: PMC2711088  PMID: 19563637
11.  Multiple Roles and Interactions of Tbx4 and Tbx5 in Development of the Respiratory System 
PLoS Genetics  2012;8(8):e1002866.
Normal development of the respiratory system is essential for survival and is regulated by multiple genes and signaling pathways. Both Tbx4 and Tbx5 are expressed throughout the mesenchyme of the developing lung and trachea; and, although multiple genes are known to be required in the epithelium, only Fgfs have been well studied in the mesenchyme. In this study, we investigated the roles of Tbx4 and Tbx5 in lung and trachea development using conditional mutant alleles and two different Cre recombinase transgenic lines. Loss of Tbx5 leads to a unilateral loss of lung bud specification and absence of tracheal specification in organ culture. Mutants deficient in Tbx4 and Tbx5 show severely reduced lung branching at mid-gestation. Concordant with this defect, the expression of mesenchymal markers Wnt2 and Fgf10, as well as Fgf10 target genes Bmp4 and Spry2, in the epithelium is downregulated. Lung branching undergoes arrest ex vivo when Tbx4 and Tbx5 are both completely lacking. Lung-specific Tbx4 heterozygous;Tbx5 conditional null mice die soon after birth due to respiratory distress. These pups have small lungs and show severe disruptions in tracheal/bronchial cartilage rings. Sox9, a master regulator of cartilage formation, is expressed in the trachea; but mesenchymal cells fail to condense and consequently do not develop cartilage normally at birth. Tbx4;Tbx5 double heterozygous mutants show decreased lung branching and fewer tracheal cartilage rings, suggesting a genetic interaction. Finally, we show that Tbx4 and Tbx5 interact with Fgf10 during the process of lung growth and branching but not during tracheal/bronchial cartilage development.
Author Summary
Defective development of the mammalian respiratory system can lead to tracheal, bronchial, or pulmonary malformations causing severe consequences at birth or during postnatal life. Studies using mouse genetics have begun to reveal complex regulatory mechanisms that guide the development of the respiratory system, but understanding how disruption of these mechanisms leads to malformations is far from complete. In this study, we analyze the role of two T-box transcription factors, Tbx4 and Tbx5, in three processes essential to the development of the respiratory system: the specification of the lung and trachea primordia, the growth and branching of the airways to form the lung, and formation of cartilage rings of the trachea and bronchi. In the absence of Tbx5, only one lung is specified, and no trachea. Both Tbx4 and Tbx5 regulate the process of lung branching by controlling the expression of the secreted growth factor Fgf10 and activation of Fgf10 signaling. In the trachea, both Tbx4 and Tbx5 are important for condensation of cells to form cartilage rings, although this is regulated by a distinct pathway that does not involve Fgf10.
PMCID: PMC3410851  PMID: 22876201
12.  Redox Control of Asthma: Molecular Mechanisms and Therapeutic Opportunities 
Antioxidants & Redox Signaling  2010;12(1):93-124.
An imbalance in reducing and oxidizing (redox) systems favoring a more oxidative environment is present in asthma and linked to the pathophysiology of the defining symptoms and signs including airflow limitation, hyper-reactivity, and airway remodeling. High levels of hydrogen peroxide, nitric oxide (•NO), and 15-F2t-isoprostane in exhaled breath, and excessive oxidative protein products in lung epithelial lining fluid, peripheral blood, and urine provide abundant evidence for pathologic oxidizing processes in asthma. Parallel studies document loss of reducing potential by nonenzymatic and enzymatic antioxidants. The essential first line antioxidant enzymes superoxide dismutases (SOD) and catalase are reduced in asthma as compared to healthy individuals, with lowest levels in those patients with the most severe asthma. Loss of SOD and catalase activity is related to oxidative modifications of the enzymes, while other antioxidant gene polymorphisms are linked to susceptibility to develop asthma. Monitoring of exhaled •NO has entered clinical practice because it is useful to optimize asthma care, and a wide array of other biochemical oxidative and nitrative biomarkers are currently being evaluated for asthma monitoring and phenotyping. Novel therapeutic strategies that target correction of redox abnormalities show promise for the treatment of asthma. Antioxid. Redox Signal. 12, 93–124.
Redox Reactions Form the Basis for Aerobic Life
Redox Systems in the Lung
ROS and RNS production in the lung
Endogenous reactive oxygen species
Hydrogen peroxide (H2O2)
Hydroxyl radical (OH)
Protein modifications by MPO and EPO
Reactive nitrogen species
Environmental exposures
Atmospheric ozone (O3) and particulate matter pollution
Cigarette smoke and environmental tobacco smoke
Oxidative processes in biology
Antioxidants in the lung
Nonenzymatic lung antioxidants
Vitamin E (alpha-tocopherol)
Vitamin C (ascorbic acid)
Enzymatic lung antioxidants
Superoxide dismutases (SOD)
Glutathione system
Thioredoxin system
Glutaredoxin system
The role of protein thiolation (Pr-SH); S-glutathionylation in redox signaling
Heme oxygenase
The Role of Redox in Asthma
Pathophysiology of asthma
Production of ROS in asthma
Inhalation of exogenous ROS or RNS: Contribution to asthma severity
Nitric oxide in the lungs: Relation to oxidative modifications
Redox imbalance in asthma
Oxidative stress
Antioxidant deficiency in asthma
SOD deficiency
Catalase inactivation
Glutathione systems in asthma
Redox-dependent transcriptional regulation
Transcription factors NF-κB and AP1
Redox-dependent activation of JAK/STAT pathway
Genetics of redox in asthma
Clinical Implications
Clinical monitoring of redox in asthma
Antioxidant therapeutic strategies
Redox-sensitive transcription factors
SOD therapies
Glutathione system
Dietary antioxidants
Conclusions and Future Directions
PMCID: PMC2824520  PMID: 19634987
13.  Expression analysis of asthma candidate genes during human and murine lung development 
Respiratory Research  2011;12(1):86.
Little is known about the role of most asthma susceptibility genes during human lung development. Genetic determinants for normal lung development are not only important early in life, but also for later lung function.
To investigate the role of expression patterns of well-defined asthma susceptibility genes during human and murine lung development. We hypothesized that genes influencing normal airways development would be over-represented by genes associated with asthma.
Asthma genes were first identified via comprehensive search of the current literature. Next, we analyzed their expression patterns in the developing human lung during the pseudoglandular (gestational age, 7-16 weeks) and canalicular (17-26 weeks) stages of development, and in the complete developing lung time series of 3 mouse strains: A/J, SW, C57BL6.
In total, 96 genes with association to asthma in at least two human populations were identified in the literature. Overall, there was no significant over-representation of the asthma genes among genes differentially expressed during lung development, although trends were seen in the human (Odds ratio, OR 1.22, confidence interval, CI 0.90-1.62) and C57BL6 mouse (OR 1.41, CI 0.92-2.11) data. However, differential expression of some asthma genes was consistent in both developing human and murine lung, e.g. NOD1, EDN1, CCL5, RORA and HLA-G. Among the asthma genes identified in genome wide association studies, ROBO1, RORA, HLA-DQB1, IL2RB and PDE10A were differentially expressed during human lung development.
Our data provide insight about the role of asthma susceptibility genes during lung development and suggest common mechanisms underlying lung morphogenesis and pathogenesis of respiratory diseases.
PMCID: PMC3141421  PMID: 21699702
Asthma; Development; Expression; Genetics; Lung
14.  T-Bet Polymorphisms Are Associated with Asthma and Airway Hyperresponsiveness 
Rationale: T-bet (TBX21 or T-box 21) is a critical regulator of T-helper 1 lineage commitment and IFN-γ production. Knockout mice lacking T-bet develop airway hyperresponsiveness (AHR) to methacholine, peribronchial eosinophilic and lymphocytic inflammation, and increased type III collagen deposition below the bronchial epithelium basement membrane, reminiscent of both acute and chronic asthma histopathology. Little is known regarding the role of genetic variation surrounding T-bet in the development of human AHR.
Objectives: To assess the relationship between T-bet polymorphisms and asthma-related phenotypes using family-based association.
Methods: Single nucleotide polymorphism discovery was performed by resequencing the T-bet genomic locus in 30 individuals (including 22 patients with asthma). Sixteen variants were genotyped in 580 nuclear families ascertained through offspring with asthma from the Childhood Asthma Management Program clinical trial. Haplotype patterns were determined from this genotype data. Family-based tests of association were performed with asthma, AHR, lung function, total serum immunoglobulin E, and blood eosinophil levels.
Main Results: We identified 24 variants. Evidence of association was observed between c.−7947 and asthma in white families using both additive (p = 0.02) or dominant models (p = 0.006). c.−7947 and three other variants were also associated with AHR (log-methacholine PC20, p = 0.02–0.04). Haplotype analysis suggested that an AHR locus is in linkage disequilibrium with variants in the 3′UTR. Evidence of association of AHR with c.−7947, but not with other 3′UTR SNPs, was replicated in an independent cohort of adult males with AHR.
Conclusions: These data suggest that T-bet variation contributes to airway responsiveness in asthma.
PMCID: PMC2662983  PMID: 16179640
immunoglobulin E; single nucleotide polymorphism; T-box; TBX21
15.  Ficolin-2 Defends against Virulent Mycobacteria Tuberculosis Infection In Vivo, and Its Insufficiency Is Associated with Infection in Humans 
PLoS ONE  2013;8(9):e73859.
Human ficolin-2 (ficolin-2/P35) is a lectin complement pathway activator that is present in normal human plasma and is associated with infectious diseases; however, little is known regarding the roles and mechanisms of ficolin-2 during Mycobacterium tuberculosis (Mtb) infection. Here, we describe our novel findings that the ficolin-2 serum levels of 107 pulmonary tuberculosis (TB) patients were much lower compared with 107 healthy controls. In vitro analysis showed that ficolin-2 bound to the virulent Mtb H37Rv strain much more strongly than to the non-virulent M. bovis BCG and M. smegmatis. Ficolin-2 bound to the surface glycolipid portion of H37Rv and blocked H37Rv infection in human lung A549 cells. Opsonophagocytosis was also promoted by ficolin-2. Importantly, we found that administration of exogenous ficolin-2 had a remarkable protective effect against virulent Mtb H37Rv infection in both C57BL/6J and BALB/c mice. Ficolin-A (a ficolin-2-like molecule in mouse) knockout mice exhibited increased susceptibility to H37Rv infection. We further demonstrated that ficolin-2 could defend against virulent Mtb H37Rv infection at least partially by activating JNK phosphorylation and stimulating the secretion of interferon (IFN)-γ, interleukin (IL)-17, IL-6, tumor necrosis factor (TNF)-α, and nitric oxide (NO) production by macrophages. Our data provide a new immunotherapeutic strategy against TB based on the innate immune molecule ficolin-2 and indicate that ficolin-2 insufficiency is associated with higher susceptibility to infection in humans.
PMCID: PMC3767610  PMID: 24040095
16.  Multifactorial intervention for children with asthma and overweight (Mikado): study design of a randomised controlled trial 
BMC Public Health  2013;13:494.
In children, the prevalence’s of both obesity and asthma are disconcertingly high. Asthmatic children with obesity are characterised by less asthma control and a high need for asthma medication. As the obese asthmatic child is becoming more common in the clinical setting and the disease burden of the asthma-obesity phenotype is high, there is an increasing need for effective treatment in these children. In adults, weight reduction resulted in improved lung function, better asthma control and less need for asthma medication. In children this is hardly studied. The Mikado study aims to evaluate the effectiveness of a long term multifactorial weight reduction intervention, on asthma characteristics in children with asthma and a high body weight.
The Mikado study is a two-armed, randomised controlled trial. In total, 104 participants will be recruited via online questionnaires, pulmonary paediatricians, the youth department of the Municipal Health Services and cohorts of existing studies. All participants will be aged 6–16 years, will have current asthma, a Body Mass Index in the overweight or obesity range, and no serious comorbidities (such as diabetes, heart diseases). Participants in the intervention arm will receive a multifactorial intervention of 18 months consisting of sessions concerning sports, parental involvement, individual counselling and lifestyle advices including dietary advices and cognitive behavioural therapy. The control group will receive usual care. The primary outcome variables will include Forced Expiratory Volume in one second and Body Mass Index - Standard Deviation Score. Secondary outcomes will include other lung function parameters (including dynamic and static lung function parameters), asthma control, asthma-specific quality of life, use of asthma medication and markers of systemic inflammation and airway inflammation.
In this randomised controlled trial we will study the potential of a multifactorial weight reduction intervention to improve asthma-related outcome measures in asthmatic children with overweight. Moreover, it will provide information about the underlying mechanisms in the relationship between asthma and a high body weight in children. These findings can contribute to optimal management programs and better clinical guidelines for children with asthma and overweight.
Trial registration NCT00998413
PMCID: PMC3682864  PMID: 23692648
Asthmatic; BMI; Child; Obesity; Paediatric; Weight loss
17.  Copy number variation prevalence in known asthma genes and their impact on asthma susceptibility 
Genetic studies have identified numerous genes reproducibly associated with asthma, yet these studies have focused almost entirely on single nucleotide polymorphisms (SNPs), and virtually ignored another highly prevalent form of genetic variation: Copy Number Variants (CNVs).
To survey the prevalence of CNVs in genes previously associated with asthma, and to assess whether CNVs represent the functional asthma-susceptibility variants at these loci.
We genotyped 383 asthmatic trios participating in the Childhood Asthma Management Program (CAMP) using a competitive genomic hybridization (CGH) array designed to interrogate 20,092 CNVs. To ensure comprehensive assessment of all potential asthma candidate genes, we purposely used liberal asthma gene inclusion criteria, resulting in consideration of 270 candidate genes previously implicated in asthma. We performed statistical testing using FBAT-CNV.
Copy number variation in asthma candidate genes was prevalent, with 21% of tested genes residing near or within one of 69 CNVs. In 6 instances, the complete candidate gene sequence resides within the CNV boundaries. On average, asthmatic probands carried 6 asthma-candidate CNVs (range 1–29). However, the vast majority of identified CNVs were of rare frequency (< 5%), and were not statistically associated with asthma. Modest evidence for association with asthma was observed for 2 CNVs near NOS1 and SERPINA3. Linkage disequilibrium analysis suggests that CNV effects are unlikely to explain previously detected SNP associations with asthma.
Although a substantial proportion of asthma-susceptibility genes harbor polymorphic CNVs, the majority of these variants do not confer increased asthma risk. The lack of linkage disequilibrium (LD) between CNVs and asthma-associated SNPs suggests that these CNVs are unlikely to represent the functional variant responsible for most known asthma associations.
PMCID: PMC3609036  PMID: 23517041
18.  Mast cells can promote the development of multiple features of chronic asthma in mice 
Journal of Clinical Investigation  2006;116(6):1633-1641.
Bronchial asthma, the most prevalent cause of significant respiratory morbidity in the developed world, typically is a chronic disorder associated with long-term changes in the airways. We developed a mouse model of chronic asthma that results in markedly increased numbers of airway mast cells, enhanced airway responses to methacholine or antigen, chronic inflammation including infiltration with eosinophils and lymphocytes, airway epithelial goblet cell hyperplasia, enhanced expression of the mucin genes Muc5ac and Muc5b, and increased levels of lung collagen. Using mast cell–deficient (KitW-sh/W-sh and/or KitW/W-v) mice engrafted with FcRγ+/+ or FcRγ–/– mast cells, we found that mast cells were required for the full development of each of these features of the model. However, some features also were expressed, although usually at less than wild-type levels, in mice whose mast cells lacked FcRγ and therefore could not be activated by either antigen- and IgE-dependent aggregation of FcεRI or the binding of antigen-IgG1 immune complexes to FcγRIII. These findings demonstrate that mast cells can contribute to the development of multiple features of chronic asthma in mice and identify both FcRγ-dependent and FcRγ-independent pathways of mast cell activation as important for the expression of key features of this asthma model.
PMCID: PMC1462940  PMID: 16710480
19.  Fetal exposure to bisphenol A as a risk factor for the development of childhood asthma: an animal model study 
Environmental Health  2012;11:8.
The prevalence of asthma in industrialized countries has been increasing dramatically and asthma is now the most common chronic disease of children in the United States. The rapidity of the increase strongly suggests that changes in environmental exposures are the likely cause of this epidemic. Further, the early onset of allergic manifestations suggests that these exposures may act on the prenatal development of the immune system. We have focused on the potential effects of bisphenol A (BPA), a chemical pollutant with one of the largest productions, on the development of childhood asthma. We have reported that perinatal BPA exposure promotes the development of allergic asthma in a mouse model. The current study was designed to identify a critical period of BPA exposure and to begin elucidating the mechanisms for this susceptibility.
Female BALB/c mice received 10 micro g/ml BPA in their drinking water from one week before pregnancy until the end of the study. Some of the pups were transferred in the first 48 h of life from their BPA-loaded mother to an unexposed mother, or vice versa. Half of the pups were sensitized with a low dose of the experimental allergen ovalbumin (OVA), the rest received PBS as an unsensitized controls. On day 22, the pups were challenged by inhalations of ovalbumin or PBS followed by quantification of eosinophils in and hyperreactivity of their airways, major indicators of experimental asthma in this classical mouse model. Hepatic expression of two isoforms of UDP-glucuronosyltransferase (Ugt) was quantified by quantitative RT-PCR at various ages.
Pups exposed to BPA in utero and through breast milk, or in utero only, displayed an asthma phenotype in response to their "suboptimal" allergic sensitization, whereas, pups only exposed to BPA postnatally from breast milk, did not. The expression of Ugt2b1, an isoform related to BPA clearance in rats, was not detectable in mouse fetuses and newborn pups, but increased by day 5 and approached adult levels by day 25.
Prenatal exposures that produce environmentally relevant burdens of BPA, followed by postnatal allergic sensitization and challenges, promote the development of experimental allergic asthma. Delayed expression of BPA-metabolizing enzymes may explain, at least in part, the enhanced fetal susceptibility to this common environmental contaminant.
PMCID: PMC3306825  PMID: 22353195
Animal model; Asthma; Bisphenol A; BPA; Environmental estrogen; Enzyme; Fetal exposure; Metabolism; Ovalbumin; Ugt2b1
20.  Skin-Derived TSLP Triggers Progression from Epidermal-Barrier Defects to Asthma 
PLoS Biology  2009;7(5):e1000067.
A skin-derived cytokine with high systemic availability provides a mechanistic explanation for atopic march and highlights a potential therapeutic target for preventing the development of asthma among people with atopic dermatitis.
Asthma is a common allergic lung disease frequently affecting individuals with a prior history of eczema/atopic dermatitis (AD); however, the mechanism underlying the progression from AD to asthma (the so-called “atopic march”) is unclear. Here we show that, like humans with AD, mice with skin-barrier defects develop AD-like skin inflammation and are susceptible to allergic asthma. Furthermore, we show that thymic stromal lymphopoietin (TSLP), overexpressed by skin keratinocytes, is the systemic driver of this bronchial hyper-responsiveness. As an AD-like model, we used mice with keratinocyte-specific deletion of RBP-j that sustained high systemic levels of TSLP. Antigen-induced allergic challenge to the lung airways of RBP-j–deficient animals resulted in a severe asthmatic phenotype not seen in similarly treated wild-type littermates. Elimination of TSLP signaling in these animals blocked the atopic march, demonstrating that high serum TSLP levels were required to sensitize the lung to allergic inflammation. Furthermore, we analyzed outbred K14-TSLPtg mice that maintained high systemic levels of TSLP without developing any skin pathology. Importantly, epidermal-derived TSLP was sufficient to trigger the atopic march, sensitizing the lung airways to inhaled allergens in the absence of epicutaneous sensitization. Based on these findings, we propose that in addition to early treatment of the primary skin-barrier defects, selective inhibition of systemic TSLP may be the key to blocking the development of asthma in AD patients.
Author Summary
Eczema (atopic dermatitis) is a common allergic skin inflammation that has a particularly high prevalence among children. Importantly, a large proportion of people suffering from eczema go on to develop asthma later in life. Although the susceptibility of eczema patients to asthma is well documented, the mechanism that mediates “atopic march”—the progression from eczema to asthma—is unclear. We used genetic engineering to generate mice with chronic skin-barrier defects and a subsequent eczema-like disorder. With these mice, we were able to investigate how skin-specific defects predisposed the lungs to allergic asthma. We identified thymic stromal lymphopoietin (TSLP), a cytokine that is secreted by barrier-defective skin into the systemic circulation, as the agent sensitizing the lung to allergens. We demonstrated that high systemic levels of skin-derived TSLP were both required and sufficient to render lung airways hypersensitive to allergens. Thus, these data suggest that early treatment of skin-barrier defects to prevent TSLP overexpression, and systemic inhibition of TSLP, may be crucial in preventing the progression from eczema to asthma.
PMCID: PMC2700555  PMID: 19557146
21.  Significance of the microbiome in obstructive lung disease 
Thorax  2012;67(5):456-463.
The composition of the lung microbiome contributes to both health and disease, including obstructive lung disease. Because it has been estimated that over 70% of the bacterial species on body surfaces cannot be cultured by currently available techniques, traditional culture techniques are no longer the gold standard for microbial investigation. Advanced techniques that identify bacterial sequences, including the 16S ribosomal RNA gene, have provided new insights into the depth and breadth of microbiota present both in the diseased and normal lung. In asthma, the composition of the microbiome of the lung and gut during early childhood development may play a key role in the development of asthma, while specific airway microbiota are associated with chronic asthma in adults. Early bacterial stimulation appears to reduce asthma susceptibility by helping the immune system develop lifelong tolerance to innocuous antigens. By contrast, perturbations in the microbiome from antibiotic use may increase the risk for asthma development. In chronic obstructive pulmonary disease, bacterial colonisation has been associated with a chronic bronchitic phenotype, increased risk of exacerbations, and accelerated loss of lung function. In cystic fibrosis, studies utilising culture-independent methods have identified associations between decreased bacterial community diversity and reduced lung function; colonisation with Pseudomonas aeruginosa has been associated with the presence of certain CFTR mutations. Genomic analysis of the lung microbiome is a young field, but has the potential to define the relationship between lung microbiome composition and disease course. Whether we can manipulate bacterial communities to improve clinical outcomes remains to be seen.
PMCID: PMC3578398  PMID: 22318161
22.  Peptidoglycan Recognition Protein Pglyrp1 Enhances Experimental Asthma by Promoting Th2 and Th17 and Limiting Regulatory T Cell and Plasmacytoid Dendritic Cell Responses 
Asthma is a common inflammatory disease involving crosstalk between innate and adaptive immunity. We reveal that antibacterial innate immunity protein, peptidoglycan recognition protein 1 (Pglyrp1), is involved in the development of allergic asthma. Pglyrp1−/− mice developed less severe asthma than wild type (WT) mice following sensitization with house dust mite (HDM) allergen. HDM-sensitized Pglyrp1−/− mice, compared with WT mice, had diminished: bronchial hyper-responsiveness (lung airway resistance); numbers of eosinophils, neutrophils, lymphocytes, and macrophages in bronchoalveolar lavage fluid and lungs; inflammatory cell infiltrates in the lungs around bronchi, bronchioles, and pulmonary arteries and veins; lung remodeling (mucin-producing goblet cell hyperplasia and metaplasia and smooth muscle hypertrophy and fibrosis); levels of IgE, eotaxins, IL-4, IL-5, and IL-17 in the lungs; and numbers of Th2 and Th17 cells and expression of their marker genes in the lungs. The mechanism underlying this decreased sensitivity of Pglyrp1−/− mice to asthma was increased generation and activation of CD8α+β+ and CD8α+β− plasmacytoid dendritic cells (pDC) and increased recruitment and activity of regulatory T (Treg) cells in the lungs. In vivo depletion of pDC in HDM-sensitized Pglyrp1−/− mice reversed the low responsive asthma phenotype of Pglyrp1−/− mice to resemble the more severe WT phenotype. Thus, Pglyrp1−/− mice efficiently control allergic asthma by up-regulating pDC and Treg cells in the lungs, whereas in WT mice Pglyrp1 is proinflammatory and decreases pDC and Treg, and increases pro-asthmatic Th2 and Th17 responses. Blocking Pglyrp1 or enhancing pDC in the lungs may be beneficial for prevention and treatment of asthma.
PMCID: PMC3608703  PMID: 23420883
23.  Absence of α4 but not β2 integrins restrains development of chronic allergic asthma using mouse genetic models 
Experimental hematology  2009;37(6):715-727.e3.
Chronic asthma is characterized by ongoing recruitment of inflammatory cells and airway hyperresponsiveness leading to structural airway remodeling. Although α4β1 and β2 integrins regulate leukocyte migration in inflammatory diseases and play decisive roles in acute asthma, their role has not been explored under the chronic asthma setting. To extend our earlier studies with α4Δ/Δ and β2−/− mice, which showed that both a4 and b2 integrins have nonredundant regulatory roles in acute ovalbumin (OVA)-induced asthma, we explored to what extent these molecular pathways control development of structural airway remodeling in chronic asthma.
Materials and Methods
Control, α4Δ/Δ, and β2−/−mouse groups, sensitized by intraperitoneal OVA as allergen, received intratracheal OVA periodically over days 8 to 55 to induce a chronic asthma phenotype. Post-OVA assessment of inflammation and pulmonary function (airway hyperresponsiveness), together with airway modeling measured by goblet cell metaplasia, collagen content of lung, and transforming growth factor β1 expression in lung homogenates, were evaluated.
In contrast to control and β2−/− mice, α4Δ/Δ mice failed to develop and maintain the composite chronic asthma phenotype evaluated as mentioned and subepithelial collagen content was comparable to baseline. These data indicate that β2 integrins, although required for inflammatory migration in acute asthma, are dispensable for structural remodeling in chronic asthma.
α4 integrins appear to have a regulatory role in directing transforming growth factor β-induced collagen deposition and structural alterations in lung architecture likely through interactions of Th2 cells, eosinophils, or mast cells with endothelium, resident airway cells, and/or extracellular matrix.
PMCID: PMC3696022  PMID: 19463772
24.  Respiratory symptoms in adults are related to impaired quality of life, regardless of asthma and COPD: results from the European community respiratory health survey 
Respiratory symptoms are common in the general population, and their presence is related to Health-related quality of life (HRQoL). The objective was to describe the association of respiratory symptoms with HRQoL in subjects with and without asthma or COPD and to investigate the role of atopy, bronchial hyperresponsiveness (BHR), and lung function in HRQoL.
The European Community Respiratory Health Survey (ECRHS) I and II provided data on HRQoL, lung function, respiratory symptoms, asthma, atopy, and BHR from 6009 subjects. Generic HRQoL was assessed through the physical component summary (PCS) score and the mental component summary (MCS) score of the SF-36.
Factor analyses and linear regressions adjusted for age, gender, smoking, occupation, BMI, comorbidity, and study centre were conducted.
Having breathlessness at rest in ECRHS II was associated with mean score (95% CI) impairment in PCS of -8.05 (-11.18, -4.93). Impairment in MCS score in subjects waking up with chest tightness was -4.02 (-5.51, -2.52). The magnitude of HRQoL impairment associated with respiratory symptoms was similar for subjects with and without asthma/COPD. Adjustments for atopy, BHR, and lung function did not explain the association of respiratory symptoms and HRQoL in subjects without asthma and/or COPD.
Subjects with respiratory symptoms had poorer HRQoL; including subjects without a diagnosis of asthma or COPD. These findings suggest that respiratory symptoms in the absence of a medical diagnosis of asthma or COPD are by no means trivial, and that clarifying the nature and natural history of respiratory symptoms is a relevant challenge.
Several community studies have estimated the prevalence of common respiratory symptoms like cough, dyspnoea, and wheeze in adults [1-3]. Although the prevalence varies to a large degree between studies and geographical areas, respiratory symptoms are quite common. The prevalences of respiratory symptoms in the European Community Respiratory Health Study (ECRHS) varied from one percent to 35% [1]. In fact, two studies have reported that more than half of the adult population suffers from one or more respiratory symptoms [4,5].
Respiratory symptoms are important markers of the risk of having or developing disease. Respiratory symptoms have been shown to be predictors for lung function decline [6-8], asthma [9,10], and even all-cause mortality in a general population study [11]. In patients with a known diagnosis of asthma or chronic obstructive pulmonary disease (COPD), respiratory symptoms are important determinants of reduced health related quality of life (HRQoL) [12-15]. The prevalence of respiratory symptoms exceeds the combined prevalences of asthma and COPD, and both asthma and COPD are frequently undiagnosed diseases [16-18]. Thus, the high prevalence of respipratory symptoms may mirror undiagnosed and untreated disease.
The common occurrence of respiratory symptoms calls for attention to how these symptoms affect health also in subjects with no diagnosis of obstructive airways disease. Impaired HRQoL in the presence of respiratory symptoms have been found in two population-based studies [6,19], but no study of respiratory sypmtoms and HRQoL have separate analyses for subjects with and without asthma and COPD, and no study provide information about extensive objective measurements of respiratory health.
The ECRHS is a randomly sampled, multi-cultural, population based cohort study. The ECRHS included measurements of atopy, bronchial hyperresponsiveness (BHR), and lung function, and offers a unique opportunity to investigate how respiratory symptoms affect HRQoL among subjects both with and without obstructive lung disease.
In the present paper we aimed to: 1) Describe the relationship between respiratory symptoms and HRQoL in an international adult general population and: 2) To assess whether this relationship varied with presence of asthma and/or COPD, or presence of objective functional markers like atopy and BHR.
PMCID: PMC2954977  PMID: 20875099
25.  Mesenchymal Stem Cell Transfer Suppresses Airway Remodeling in a Toluene Diisocyanate-Induced Murine Asthma Model 
Severe asthma is characterized by high medication requirements to maintain good disease control or by persistent symptoms despite high medication use. The transfer of bone marrow-derived mesenchymal stem cells (BMDMSCs) to the injured lungs is a possible treatment for severe asthma. This study investigated the therapeutic effects of BMDMSCs in airway remodeling and inflammation in an experimental toluene diisocyanate (TDI)-induced asthma animal model of severe asthma.
BMDMSCs were transferred into rats after TDI inhalation. Bronchoalveolar lavage (BAL) cell profiles, histological changes including an inflammatory index and goblet cell hyperplasia, and the airway response to methacholine using plethysmography were analyzed. Smooth muscle actin (SMA) and proliferating cell nuclear antigen (PCNA) protein expression were observed in lung tissue using immunohistochemical staining. The collagen content was measured in lung tissue sections and lung extracts using Masson's trichrome staining and an immunoassay kit.
The numbers of inflammatory cells in BAL fluid, histological inflammatory index, airway response to methacholine, number of goblet cells, and amount of collagen were increased in TDI-treated rats compared with sham rats (P=0.05-0.002). BMDMSC transfer significantly reduced the TDI-induced increase in the inflammatory index and numbers of eosinophils and neutrophils in BAL fluid to levels seen in sham-treated rats (P<0.05). BMDMSC transfer significantly reduced the number of goblet cells, collagen deposition, and immune staining for SMA and PCNA with concomitant normalization of the airway response to methacholine.
The systemic transfer of BMDMSCs effectively reduced experimental TDI-induced airway inflammation and remodeling and airway hyperreactivity.
PMCID: PMC3121063  PMID: 21738887
Airway remodeling; asthma; collagen; stem cells; TDI

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