Rodrigo Godinho de Souza

Recombinant human deoxyribonuclease therapy improves airway resistance and reduces DNA extracellular traps in a murine acute asthma model.

ABSTRACT Purpose: Asthma is a highly prevalent chronic inflammatory lung disease characterized by airway hyperresponsiveness to allergens, airway edema, and increased mucus secretion. Such mucus can be liquefied by recombinant human deoxyribonuclease (rhDNase), in which efficacy of rhDNase has been well documented in patients with cystic fibrosis, but little studied in asthma. In the present study, we investigated whether rhDNase intranasal administration improved inflammation and pulmonary function in an experimental model of asthma. Methods: Mice were sensitized by two subcutaneous injections of ovalbumin (OVA), on days 0 and 7, followed by three intranasal challenges with OVA on days 14, 15, and 16. A control group, replacing OVA by DPBS, was included. On days 15 and 16, after 2 hours of OVA challenge, mice received 1 mg/mL of intranasal rhDNase. Results: We showed that rhDNase decreased significantly the airway resistance and reduced EETs formation and globet cells hyperplasia. Conclusions: Our results suggest that extracellular DNA in mucus play a role in lower airways obstruction in OVA asthma protocol and that the treatment with rhDNase improved lung function and DNA extracellular traps, with no direct cellular anti-inflammatory effects.

Extracellular DNA traps in bronchoalveolar fluid from a murine eosinophilic pulmonary response

Asthma is associated with a loss of the structural integrity of airway epithelium and dysfunction of the physical barrier, which protects airways from external harmful factors. Granulocyte activation causes the formation of extracellular traps, releasing web‐like structures of DNA and proteins, being important to kill pathogens extracellularly. We investigated whether eosinophils infiltrating airways in an experimental model of asthma would induce eosinophil extracellular traps (EETs) in bronchoalveolar lavage fluid and lung tissue. We showed that an ovalbumin (OVA) asthma protocol presented a significant increase in eosinophil counts with increased extracellular DNA in bronchoalveolar lavage fluid as well as in lung tissue, confirming the presence of DNA traps colocalized with eosinophil peroxidase. EETs formation was reversed by DNase treatment. With these approaches, we demonstrated for the first time that OVA‐challenged mice release extracellular DNA traps, which could aggravate pulmonary dysfunction.

Proposta de um modelo murino de curta duração de resposta pulmonar alérgica aguda sem utilização de adjuvante

OBJECTIVE To determine whether a short-term protocol using subcutaneous sensitization with ovalbumin, without the use of adjuvants, would induce an eosinophilic response in the lungs of mice similar to that observed in previous, well-established protocols. METHODS Adult female BALB/c mice were randomized and divided into groups according to the number of sensitizations with ovalbumin and the number/dosage of intranasal ovalbumin challenges. The short-term protocol (10 days) consisted of one sensitization with ovalbumin and three ovalbumin challenges (100 µg). Total and differential cell counts in BAL fluid, levels of eosinophil peroxidase in lung tissue, and histopathological examination of the lungs were performed 24 h after the last ovalbumin challenge. RESULTS No significant differences were found among the groups regarding the variables studied. The short-term protocol, as well as the other protocols studied, induced an eosinophilic response similar to that obtained in the positive control. CONCLUSIONS Subcutaneous sensitization with ovalbumin and without the use of adjuvants resulted in a significant allergic response in the lungs of mice, even in the short-term protocol group. Our findings suggest that this short-term protocol can be used as a first-line pre-clinical test for the study of new medications, reducing the costs and observation periods.

Acute and chronic exposure to Tyrophagus putrescentiae induces allergic pulmonary response in a murine model.

Background Tyrophagus putrescentiae (Tp) is a source of aeroallergen that causes allergic diseases. Objective To describe an acute and chronic murine model of allergic asthma with Tp extract with no systemic sensitization and no use of adjuvant. Methods Mites from dust sample were cultured and a raw extract was produced. Female BALB/c mice (6-8 weeks) were challenged intranasally with Tp extract or Dulbeccos phosphate-buffered saline, for 10 consecutive days (acute protocol) or for 6 weeks (chronic protocol). Twenty-four hours after the last intranasal challenge, bronchoalveolar lavage fluid (BALF) was performed for total and differential cells count, cytokine analysis, and eosinophil peroxidase activity. Lung tissue was also removed for histopathologic analysis. Results Tp extract has shown a significant increase in total cells count from BALF as well as an increase in absolute eosinophils count, eosinophil peroxidase activity, interleukin (IL)-5 and IL-13 levels, in both acute and chronic protocols. Peribronchovascular infiltrate, goblet cells hyperplasia and collagen deposition were shown in the airways of acute and chronic Tp-exposed mice. Conclusion Our data suggest that the intranasal exposure to Tp extract, with no systemic sensitization and no use of adjuvants, induces a robust allergic inflammation in the lungs of mice, in both acute and chronic models. Our Tp extract seems to be a potent allergen extract which may be used in asthma model studies.

Clinical characteristics of children and adolescents with severe therapy-resistant asthma in Brazil.

Abstract Objective: To describe the clinical characteristics, lung function, radiological findings, and the inflammatory cell profile in induced sputum in children and adolescents with severe therapy-resistant asthma (STRA) treated at a referral center in southern Brazil. Methods: We retrospectively analyzed children and adolescents (3-18 years of age) with uncontrolled STRA treated with high-dose inhaled corticosteroids and long-acting ß2 agonists. We prospectively collected data on disease control, lung function, skin test reactivity to allergens, the inflammatory cell profile in induced sputum, chest CT findings, and esophageal pH monitoring results. Results: We analyzed 21 patients (mean age, 9.2 ± 2.98 years). Of those, 18 (86%) were atopic. Most had uncontrolled asthma and near-normal baseline lung function. In 4 and 7, induced sputum was found to be eosinophilic and neutrophilic, respectively; the inflammatory cell profile in induced sputum having changed in 67% of those in whom induced sputum analysis was repeated. Of the 8 patients receiving treatment with omalizumab (an anti-IgE antibody), 7 (87.5%) showed significant improvement in quality of life, as well as significant reductions in the numbers of exacerbations and hospitalizations. Conclusions: Children with STRA present with near-normal lung function and a variable airway inflammatory pattern during clinical follow-up, showing a significant clinical response to omalizumab. In children, STRA differs from that seen in adults, further studies being required in order to gain a better understanding of the disease mechanisms.

Protective effect of early prenatal stress on the induction of asthma in adult mice: Sex-specific differences.

Adversities faced during the prenatal period can be related to the onset of diseases in adulthood. However, little is known about the effects on the respiratory system. This study aimed to evaluate the effects of prenatal stress in two different time-points during pregnancy on pulmonary function and on the inflammatory profile of mice exposed to an asthma model. Male and female BALB/c mice were divided into 3 groups: control (CON), prenatal stress from the second week of pregnancy (PNS1) and prenatal stress on the last week of pregnancy (PNS2). Both PNS1 and PNS2 pregnant females were submitted to restraint stress. As adults, fear/anxiety behaviors were assessed, and animals were subjected to an asthma model induced by ovalbumin. Pulmonary function, inflammatory parameters in bronchoalveolar lavage (BAL) and histology were evaluated. There was a significant decrease in the number of entries and time spent in the central quadrant on the open field test for the PNS1 animals. Females (PNS1) showed improved pulmonary function (airway resistance, tissue damping and pulmonary elastance), significant increase in the percentage of neutrophils and lymphocytes and a decrease in eosinophils when compared to controls. There was a significant decrease in inflammatory cytokines in BAL of both males (IL-5 and IL-13) and females (IL-4, IL-5 and IL-13) from PNS1 and PNS2 when compared to the CON group. Prenatal stress starting from the beginning of pregnancy reduces the impact of asthma development in adult female mice, showing an improved pulmonary function and a lower inflammatory response in the lungs.

Impact of omalizumab in children from a middle-income country with severe therapy-resistant asthma: A real-life study

Severe asthma in children is a global health problem. Severe therapy‐resistant asthma (STRA) in children is a major clinical challenge due to persistent symptoms despite high doses of corticosteroids and results in high public health costs. Omalizumab (anti‐IgE monoclonal antibody) has been described as an effective add‐on therapy in these patients. The characteristics of children with STRA from low‐ and middle‐income countries have scarcely been reported, and no real‐life study has been published on the effects of omalizumab in this group of patients. The aim of our study is to report the first clinical real‐life experiences with omalizumab in Brazilian children with STRA.

Revisão sistemática sobre modelos experimentais de asma aguda e crônica induzidos com extrato de ácaro da poeira doméstica

Introducao: Asma e uma doenca cronica das vias aereas inferiores com elevada prevalencia. Pesquisadores no mundo todo tem desenvolvido varios estudos experimentais em camundongos com o objetivo de entender melhor os mecanismos da doenca e testar novas terapias. Acaros estao presentes de forma abundante na poeira domestica, sendo considerados os alergenos mais comuns desencadeantes de asma alergica. Este estudo objetiva apresentar e discutir desfechos inflamatorios no tecido pulmonar dos camundongos, verificar a diferenca entre os modelos agudo e cronico de asma alergica, tempo de exposicao ao alergeno, dose administrada e seu impacto nas pesquisas em modelos experimentais com asma. Metodos: A revisao da literatura foi realizada em quatro bancos de dados (PubMed, Scielo, Scopus e ScienceDirect). Os artigos selecionados foram avaliados primeiramente por dois pesquisadores de forma independente, de acordo com os criterios de inclusao. Resultados: Foram separados 126 artigos. Aplicados os criterios de inclusao e exclusao, somente 15 foram selecionados. Sao artigos que apresentaram diferentes protocolos de exposicao ao HDM. A dose de HDM mais encontrada foi 100µg seguida por 25µg, e o tipo de modelo foi agudo. Conclusao: No modelo agudo, observa-se um elevado nivel de inflamacao das vias aereas. Ja o modelo cronico reproduz melhor as caracteristicas da asma em humanos, hiper-responsividade bronquica e remodelamento das vias aereas. Palavras-chave: Asma; acaro; alergeno; camundongos

Falta de associação entre carga viral e gravidade da bronquiolite aguda em lactentes

Objetivo: Investigar a correlacao entre a carga viral do virus sincicial respiratorio e o tempo de internacao hospitalar em lactentes com episodios de sibilância aguda. Metodos: Este foi um estudo transversal de dois anos envolvendo lactentes de ate 12 meses de idade com bronquiolite no momento da internacao em um hospital terciario. Para a identificacao dos virus respiratorios foram coletadas secrecoes nasofaringeas. As amostras foram analisadas (por todo o periodo do estudo) por imunofluorescencia direta e (no segundo ano do estudo) por PCR quantitativa em tempo real para tres virus humanos (rinovirus, virus sincicial respiratorio e metapneumovirus). Resultados: Das 110 amostras avaliadas por imunofluorescencia direta, 56 (50,9%) foram positivas para um unico virus, e 16 (14,5%) foram positivas para dois ou mais virus. Nessas 72 amostras, o virus mais prevalente foi o virus sincicial respiratorio, seguido por influenza. Das 56 amostras avaliadas por PCR quantitativa em tempo real, 24 (42,8%) foram positivas para um unico virus, e 1 (1,7%) foi positiva para dois virus. Nessas 25 amostras, o virus mais prevalente foi o virus sincicial respiratorio, seguido por rinovirus humano. A coinfeccao nao influenciou o tempo de internacao ou outros desfechos. Alem disso, nao houve associacao entre a carga viral de virus sincicial respiratorio e o tempo de internacao. Conclusoes: A coinfeccao e a carga viral do virus sincicial respiratorio nao parecem influenciar os desfechos em lactentes com bronquiolite aguda.

Lack of association between viral load and severity of acute bronchiolitis in infants

ABSTRACT Objective: To investigate the correlation between respiratory syncytial viral load and length of hospitalization in infants with acute wheezing episodes. Methods: This was a two-year, cross-sectional study of infants ≤ 12 months of age with bronchiolitis at the time of admission to a tertiary hospital. For the identification of respiratory viruses, nasopharyngeal secretions were collected. Samples were analyzed (throughout the study period) by direct immunofluorescence and (in the second year of the study) by quantitative real-time PCR. We screened for three human viruses: rhinovirus, respiratory syncytial virus, and metapneumovirus. Results: Of 110 samples evaluated by direct immunofluorescence, 56 (50.9%) were positive for a single virus, and 16 (14.5%) were positive for two or more viruses. Among those 72 samples, the most prevalent virus was respiratory syncytial virus, followed by influenza. Of 56 samples evaluated by quantitative real-time PCR, 24 (42.8%) were positive for a single virus, and 1 (1.7%) was positive for two viruses. Among those 25 samples, the most prevalent virus was again respiratory syncytial virus, followed by human rhinovirus. Coinfection did not influence the length of the hospital stay or other outcome s. In addition, there was no association between respiratory syncytial virus load and the length of hospitalization. Conclusions: Neither coinfection nor respiratory syncytial viral load appears to influence the outcomes of acute bronchiolitis in infants.