Leonardo Araújo Pinto

TLR4 genotype and environmental LPS mediate RSV bronchiolitis through Th2 polarization

While 30%-70% of RSV-infected infants develop bronchiolitis, 2% require hospitalization. It is not clear why disease severity differs among healthy, full-term infants; however, virus titers, inflammation, and Th2 bias are proposed explanations. While TLR4 is associated with these disease phenotypes, the role of this receptor in respiratory syncytial virus (RSV) pathogenesis is controversial. Here, we evaluated the interaction between TLR4 and environmental factors in RSV disease and defined the immune mediators associated with severe illness. Two independent populations of infants with RSV bronchiolitis revealed that the severity of RSV infection is determined by the TLR4 genotype of the individual and by environmental exposure to LPS. RSV-infected infants with severe disease exhibited a high GATA3/T-bet ratio, which manifested as a high IL-4/IFN-γ ratio in respiratory secretions. The IL-4/IFN-γ ratio present in infants with severe RSV is indicative of Th2 polarization. Murine models of RSV infection confirmed that LPS exposure, Tlr4 genotype, and Th2 polarization influence disease phenotypes. Together, the results of this study identify environmental and genetic factors that influence RSV pathogenesis and reveal that a high IL-4/IFN-γ ratio is associated with severe disease. Moreover, these molecules should be explored as potential targets for therapeutic intervention.

Impact of 10-valent pneumococcal non-typeable Haemophilus influenzae protein D conjugate vaccine (PHiD-CV) on childhood pneumonia hospitalizations in Brazil two years after introduction.

INTRODUCTION Pneumococcal disease is a major public health problem worldwide. From March to September of 2010, 10-valent pneumococcal non-typeable Haemophilus influenzae protein conjugate vaccine (PHiD-CV) was introduced in the Brazilian childhood National Immunization Program (NIP) in all 27 Brazilian states. The aim of the present study is to report national time-trends in incidence of hospital admissions for childhood pneumonia in Brazil before and after two years of introduction of this new pneumococcal conjugate vaccine. METHODS Analysis of hospitalization data of children aged 0-4 years in Brazilian public health system with an admission diagnosis of pneumonia from 2002 to 2012 was performed comparing pre (2002-2009) and post-vaccination periods (2011-2012). Hospital number of admission due to pneumonia and all non-respiratory diseases were obtained from DATASUS, the Brazilian government open-access public health database system. Incidence of pneumonia hospitalization was compared to incidence of all non-respiratory admissions. RESULTS Admission rates for pneumonia decreased steadily from 2010 to 2012. In children aged less than four years, incidence of pneumonia hospitalizations decreased 12.65% when pre (2002-2009) and post-vaccination introduction periods (2011-2012) were compared and adjusted for seasonality and secular-trend (p<0.001). On the other hand, non-respiratory admission rates remained stable comparing both periods (p=0.39). CONCLUSION Childhood pneumonia hospitalization rates were fluctuating prior to 2010 and decreased significantly in the two years after PHiD-CV introduction. Conversely, rate of non-respiratory admissions has shown no decrease. These data are an evidence of the effectiveness and public health impact of this new pneumococcal vaccine.

MMP-9 gene variants increase the risk for non-atopic asthma in children

BackgroundAtopic and non-atopic wheezing may be caused by different etiologies: while eosinophils are more important in atopic asthmatic wheezers, neutrophils are predominantly found in BAL samples of young children with wheezing. Both neutrophils as well as eosinophils may secrete matrix metalloproteinase 9 (MMP-9). Considering that MMP-9 plays an important role in airway wall thickening and airway inflammation, it may influence the development of obstructive airway phenotypes in children. In the present study we investigated whether genetic variations in MMP-9 influence the development of different forms of childhood asthma.MethodsGenotyping of four HapMap derived tagging SNPs in the MMP-9 gene was performed using MALDI-TOF MS in three cross sectional study populations of German children (age 9-11; N = 4,264) phenotyped for asthma and atopic diseases according to ISAAC standard procedures. Effects of single SNPs and haplotypes were studied using SAS 9.1.3 and Haploview.ResultsSNP rs2664538 significantly increased the risk for non-atopic wheezing (OR 2.12, 95%CI 1.40-3.21, p < 0.001) and non-atopic asthma (OR 1.66, 95%CI 1.12-2.46, p = 0.011). Furthermore, the minor allele of rs3918241 may be associated with decreased expiratory flow measurements in non-atopic children. No significant effects on the development of atopy or total serum IgE levels were observed.ConclusionsOur results have shown that homozygocity for MMP-9 variants increase the risk to develop non-atopic forms of asthma and wheezing, which may be explained by a functional role of MMP-9 in airway remodeling. These results suggest that different wheezing disorders in childhood are affected differently by genetic alterations.

Azithromycin therapy in hospitalized infants with acute bronchiolitis is not associated with better clinical outcomes: a randomized, double-blinded, and placebo-controlled clinical trial.

OBJECTIVE To test the hypothesis that azithromycin reduces the length of hospitalization and oxygen requirement in infants with acute viral bronchiolitis (AB). STUDY DESIGN We performed a randomized, double-blinded, placebo-controlled trial in southern Brazil, from 2009 to 2011. Infants (<12 months of age) hospitalized with AB were recruited in 2 hospitals. Patients were randomized to receive either azithromycin or placebo, administered orally, for 7 days. At enrollment, clinical data were recorded and nasopharyngeal samples were collected for viral identification through immunofluorescence. Main outcomes were duration of oxygen requirement and length of hospitalization. RESULTS One hundred eighty-four patients were included in the study (azithromycin 88 subjects, placebo 96 subjects). Baseline clinical characteristics and viral identification were not different between the groups studied. A virus was detected in 112 (63%) patients, and of those, 92% were positive for respiratory syncytial virus. The use of azithromycin did not reduce the median number of days of either hospitalization (P = .28) or oxygen requirement (P = .47). CONCLUSIONS Azithromycin did not improve major clinical outcomes in a large sample of hospitalized infants with AB, even when restricting the findings to those with positive respiratory syncytial virus samples. Azithromycin therapy should not be given for AB because it provides no benefit and overuse increases overall antibiotic resistance.

IL10 polymorphisms influence neonatal immune responses, atopic dermatitis, and wheeze at age 3 years.

BACKGROUND IL10 encodes for IL-10, an important anti-inflammatory cytokine with pleiotropic effects. It is crucial for development of immune tolerance, downregulates expression of TH1 cytokines, and is relevant for T-cell regulation. Several IL10 single nucleotide polymorphisms (SNPs) were associated with inflammatory diseases, such as atopic diseases, which might have their onset during early immune maturation. OBJECTIVE We hypothesized that IL10 SNPs are associated with decreased regulatory T (Treg) cell numbers, TH2-skewed immune responses, and decreased IFN-γ levels in cord blood parallel with increased proinflammatory markers, subsequently leading to increased atopic diseases until 3 years. METHODS Eight genetic IL10 variants, represented by 4 linkage disequilibrium blocks (R(2) > 0.80) and 2 distal promoter SNPs, were genotyped in cord blood mononuclear cells of 200 healthy neonates. Cord blood mononuclear cells were cultured unstimulated or after stimulation with lipid A, peptidoglycan, PHA, house dust mite (Der p 1), or Der p 1 plus lipid A. mRNA expression of Treg cell-associated genes (forkhead box protein P3 [FOXP3], glucocorticoid-induced TNF receptor [GITR], lymphocyte activation gene 3 [LAG3]), TH1/TH2 cytokines, TNF-α, and GM-CSF were assessed. Atopic and respiratory outcomes (atopic dermatitis [AD] and wheeze) were assessed by means of questionnaire at age 3 years. RESULTS Carriers of 3 IL10 SNP blocks and both distal promoter SNPs showed reduced expression of Treg cell markers, reduced IL-5 levels, proinflammatory TNF-α and GM-CSF, and partially increased IFN-γ levels. The same SNPs presented as determinant for AD, wheeze, or symptoms of AD, wheeze, or both at age 3 years. CONCLUSIONS Polymorphisms in IL10 influenced Treg cell marker expression and TH1/TH2 and proinflammatory cytokine secretion early in life. This was relevant for further development of immune-mediated diseases, such as AD and wheeze, in early childhood.

Impact of genetics in childhood asthma

OBJECTIVE To present the most important and recent results of studies on asthma genetics. These data may help general physicians understand the impact of genetics on this complex disorder and how genes and polymorphisms influence asthma and atopy. SOURCES Data were collected from MEDLINE. Genetic association studies were selected from the Genetic Association Database, which is an archive of human genetic association studies of complex diseases and disorders organized by the National Institutes of Health. SUMMARY OF THE FINDINGS Considering the data from several important twin studies on asthma genetics, heritability, which measures the contribution of genetic factors to the variance of asthma, may be estimated in 0.48-0.79. A huge number of genetic association studies have been trying to identify asthma susceptibility genes. The most replicated results in the genetic association studies involve the following five regions of the human genome: 5q31-32, 6p21, 11q12-13, 16p11-12, and 20p13. Only recently a new asthma susceptibility gene (ORMDL3) has been identified by a whole genome association study, considered to be a major determinant for childhood asthma. CONCLUSIONS Genetic contribution to asthma may be estimated ranging from 48 to 79%. Several loci seem to influence asthma susceptibility. Genes located on chromosome 5q (ADRB2, IL13 and IL4) and the recently identified ORMDL3, on chromosome 17, seem to be determinants of childhood asthma. Diagnostics and pharmacogenetics may be the first clinical implication of extensive studies on asthma genetics.

The effect of BDNF gene variants on asthma in German children.

Background:  Allergic inflammation can trigger neuronal dysfunction and structural changes in the airways and the skin. Levels of brain‐derived neurotrophic factor (BDNF) are strongly up regulated at the location of allergic inflammation.

HLX1 gene variants influence the development of childhood asthma.

BACKGROUND Major transcription factors controlling T(H)1 and T(H)2 development, such as T-box transcription factor and GATA3, might be centrally involved in asthma and atopic diseases. Only recently, the homeobox transcription factor H.20-like homeobox 1 (HLX1), interacting closely with T-box transcription factor, has been identified as an important regulator of T(H)1 differentiation and suppressor of T(H)2 commitment. OBJECTIVE We investigated whether genetic variations in the HLX1 gene exist and whether these could affect the development of childhood asthma. METHODS The HLX1 gene was resequenced in 80 chromosomes. Associations between identified polymorphisms, asthma, and atopic diseases were investigated in German children (total n = 3099) from the cross-sectional International Study of Asthma and Allergy in Childhood phase II. Functional properties of polymorphisms were studied by using luciferase reporter gene assays and electrophoretic mobility shift assays in T cells. All statistical analyses were performed with SAS/Genetics software (SAS Institute, Inc, Cary, NC). RESULTS Nineteen polymorphisms were identified in the HLX1 gene, and 2 tagging single nucleotide polymorphisms representing 7 polymorphisms were associated with childhood asthma in our study population. Two promoter polymorphisms, C-1407T and C-742G, contained in 1 tagging block were associated with asthma (odds ratio, 1.44; 95% CI, 1.11-1.86; P = .0061), significantly decrease promoter transactivation, and disrupt specificity protein-transcription factor binding in in vitro experiments. CONCLUSIONS Our data suggest that polymorphisms in the HLX1 gene increase the risk for childhood asthma. On the cellular level, altered binding of specificity protein-transcription factors to the HLX1 promoter and subsequent changes in HLX1 gene expression might contribute to these effects.

Viabilidade da realização de espirometria em pré-escolares

OBJECTIVE: To determine the rate at which satisfactory spirometry results are obtained (spirometry success rate) in preschool children. METHODS: We analyzed the spirometry results of children 0.05). An average of 6.6 attempts/test were needed in order to achieve acceptable, reproducible curves. All 61 successful tests produced satisfactory FEV0.5 and FEV1 values. By calculating Z scores, we found that 21.6% of the children presented with an obstructive pattern. CONCLUSIONS: In our sample, the spirometry success rate was high, showing that spirometry is a valid method for assessing pulmonary function in preschool children. The high success rate in our sample might be attributable to the use of an incentive and to the fact that the tests were performed by professionals specializing in pediatrics.

Intestinal helminth infestation is associated with increased bronchial responsiveness in children

Non‐atopic asthma is the predominant phenotype in non‐affluent parts of Latin America. We recently reported that infestation with Ascaris lumbricoides increased the risk of non‐atopic asthma in less affluent areas of Brazil but the mechanism is unclear. The present study was conducted to determine whether helminth infestation is associated with heightened bronchial responsiveness (BHR), a common finding in asthma. A random sample of 50 asthmatic and 50 non‐asthmatic controls (mean age 10.1 years) were selected from a larger cohort (n = 1,011) without knowledge of their helminth infestation status. Three stool samples were collected from each child on different days and each sample was analyzed by the Kato‐Katz method for quantitative determination of helminth eggs. Bronchial provocation tests were performed with inhaled 4.5% hypertonic saline using the ISAAC Phase II standardized protocol. There was no difference between the prevalence of positive BHR in the asthmatics (20.4%) compared with the controls (14.6%) (P = 1.0). Helminth infestation was detected in 24.0% of children, with A. lumbricoides being the most common. Children with high load infestation (≥100 eggs/g) were five times more likely to have BHR than children with low load or no infestation. Despite the small sample size the results of the present study suggest that the link between high load helminth infestation and non‐atopic asthma may be mediated via heightened bronchial responsiveness, possibly due to an inflammatory response to the pulmonary phase of the helminth life cycle. Pediatr Pulmonol. 2008; 43:662–665.