BackgroundAcute respiratory infection (ARI) is a leading cause of morbidity and mortality in children worldwide. This study aimed to determine the viral and atypical bacterial causes of different severities and clinical manifestations of ARI in preschool children from low-income families in North-East Brazil.MethodsClinical/demographic data and nasopharyngeal aspirates (NPA) were prospectively collected from children <5 years presenting with ARI over one year to a paediatric A&E department. Disease severity was grouped according to presence of lower respiratory tract signs, need for hospital admission and need for oxygen. Clinical manifestation of ARI was based on discharge diagnosis from hospital with four conditions predominating: bronchiolitis, pneumonia, episodic viral wheeze/asthma and upper respiratory tract infection. Multiplex PCR was used to detect 17 common respiratory viral and atypical bacterial pathogens in NPA.Findings407 children with a median age of eight months were recruited. Pathogens were detected in 85·5% samples with co-infection being particularly common (39·5%). Respiratory Syncytial Virus (RSV; 37%), Adenoviruses (AdV; 25%), Rhinoviruses (hRV; 19%), Bocavirus (hBoV; 19%), human Meta-pneumovirus (hMPV; 10%) and Mycoplasma pneumoniae (Mpp; 10%) were most prevalent. Detection and co-infection rates were similar in all severities and clinical manifestations of ARI apart from RSV, which was associated with more severe disease and specifically more severe cases of bronchiolitis, and Mpp, which was associated with more severe cases of pneumonia. Mpp was detected in 17% of children admitted to hospital with pneumonia.InterpretationThis study underlines the importance of viral and atypical bacterial pathogens in ARI in pre-school children and highlights the complex epidemiology of these pathogens in this age group. Generally, viruses and atypical bacteria were detected in all severities and clinical manifestations of ARI but RSV and Mpp were associated with more severe cases of bronchiolitis and pneumonia respectively.

Background. Neutrophils are the predominant cell in the lung inflammatory infiltrate of infants with respiratory syncytial virus (RSV) bronchiolitis. Although it has previously been shown that neutrophils from both blood and bronchoalveolar lavage (BAL) are activated, little is understood about their role in response to RSV infection. This study investigated whether RSV proteins and mRNA are present in neutrophils from blood and BAL of infected infants.Methods. We obtained blood and BAL samples from 20 infants with severe RSV bronchiolitis and 8 healthy control infants. Neutrophil RSV F, G, and N proteins, RSV N genomic RNA, and messenger RNA (mRNA) were quantified.Results. RSV proteins were found in BAL and blood neutrophils in infants with RSV disease but not in neutrophils from healthy infants. BAL and blood neutrophils from infants with RSV disease, but not those from healthy infants, expressed RSV N genomic RNA, indicating uptake of whole virus; 17 of 20 BAL and 8 of 9 blood neutrophils from patients expressed RSV N mRNA.Conclusions. This work shows, for the first time, the presence of RSV proteins and mRNA transcripts within BAL and blood neutrophils from infants with severe RSV bronchiolitis.

BackgroundThe mechanisms regulating antibody expression within the human lung during airway infection are largely unknown. In this study, our objectives were to determine if infection with respiratory syncytial virus (RSV) upregulates expression of the B cell differentiation factors A proliferation inducing ligand (APRIL) and B cell activating factor of the TNF family (BAFF), if this is a common feature of viral airway infection, and how this is regulated in human airway epithelial cells. Methods We measured BAFF and APRIL protein expression in bronchoalveolar lavage (BAL) fluid from infants with severe RSV disease, and healthy control children, and in nasopharyngeal aspirates from preschool children with other single respiratory viral infections. We also measured mRNA expression in bronchial brushings from RSV-infected infants, and in RSV-infected paediatric primary airway epithelial cell cultures ( pAEC). Beas-2B cell cultures were used to examine mechanisms regulating BAFF expression.

As antimicrobial-resistant microbes become increasingly common and a significant global issue, novel approaches to treating these infections particularly in those at high risk are required. This is evident in people with cystic fibrosis (CF), who suffer from chronic airway infection caused by antibiotic resistant bacteria, typically Pseudomonas aeruginosa. One option is bacteriophage (phage) therapy, which utilises the natural predation of phage viruses upon their host bacteria. This review summarises the essential and unique aspects of the phage-microbe-human lung interactions in CF that must be addressed to successfully develop and deliver phage to CF airways. The current evidence regarding phage biology, phage-bacterial interactions, potential airway immune responses to phages, previous use of phages in humans and method of phage delivery to the lung are also summarised.

Background Respiratory syncytial virus (RSV) infection of airway epithelial cells (AECs) is an important initial event in RSV bronchiolitis. AEC immunological responses are thought to be critical in driving the subsequent inflammation in the airway. This study examined viral replication, cytotoxicity and cytokine production in cultures of primary AECs from children compared with responses to RSV infection in an immortalised epithelial cell line and to those from infants with RSV bronchiolitis. Methods RSV replication, proinflammatory cytokine responses and cytotoxicity in RSV-infected primary AEC cultures derived from bronchial brushings from the lungs of children were compared with those seen in BEAS-2B cultures, as well as AECs and bronchoalveolar lavage fluid collected from children with and without RSV bronchiolitis. Results Viral replication, cytotoxicity and inflammatory cytokine production were greater in primary AEC cultures than in BEAS-2B cells. Different response patterns were observed, with RSV infection of primary AEC cultures causing distinct peaks of viral replication and matched cytotoxic responses. Some primary AEC culture immunological responses, such as interleukin 8, were similar in magnitude to those seen in clinical samples from the lungs of children with RSV bronchiolitis. Although variable amounts of RSV were detected by PCR in freshly isolated primary AECs, RSV was not detected by immunocytochemistry. Conclusion This is one of the first studies to examine comprehensively the responses to RSV infection in primary AEC cultures from children and shows marked differences from those of a commercially available immortalised human cell line but reassuring similarities to results found in vivo. This suggests that future work investigating responses of AECs to RSV infection should use primary AEC cultures.

Diabetic retinopathy, one of the most serious complications of long-term diabetes, could clinically be divided into two stages: 1) background retinopathy that does not cause visual impairment and 2) proliferative retinopathy, which is a potentially blinding condition. This study aims to investigate the correlation between enhancement of vascular endothelial growth factor (VEGF) expression and neovascular changes. A binary recombinant adeno-associated virus construct producing green fluorescent protein (GFP) and VEGF under the control of the human cytomegalovirus promoter, recombinant adeno-associated virus (rAAV).VEGF.GFP, was produced and injected into the subretinal space of C57BL mice. GFP expression was tracked by fluorescence fundus photography, and VEGF expression was confirmed by immunohistochemistry and enzyme-linked immunoassay. Neovascular changes were monitored by fluorescein angiography and histology and by quantifying the number of inner retinal vessels. GFP expression was found in 100% of injected eyes, and vascular changes were detected in 9 of 10 rAAV.VEGF.GFPinjected eyes. Of these, four demonstrated microaneurysms and five showed moderate to severe leakage. There was a statistically significant increase in blood vessel number in the inner nuclear layer (P < 0.03) and dilatation of retinal veins (P < 0.05). This work has demonstrated that the development of different stages of diabetic retinopathy is closely correlated with an increased VEGF level in the retina. Diabetes 52: 857-863, 2003

BackgroundHuman respiratory syncytial virus (RSV) remains the most common cause of severe lower respiratory tract disease amongst infants, and continues to cause annual epidemics of respiratory disease every winter worldwide. Demonstrating placental transmission of viable RSV in human samples is a major paradigm shift in respiratory routes considered likely for RSV transmission.MethodsDroplet digital PCR (ddPCR) was used to identify RSV present in cord blood mononucleocytes (CBM). CBMs testing positive for RSV were treated with phytohemagglutinin (PHA), PHA and nitric oxide (NO) or PHA, NO and palivizumab, and co-cultured with HeLa cell monolayers. Subsequent immuno-staining for RSV was used to visualize infective viral plaques.ResultsRSV was detected in 26 of 45 samples (57.7%) by ddPCR. CBM’s collected in winter were more likely to test positive for RSV (17/21 samples, risk = 80%, OR = 7.08; 95% CI 1.80–27.80; p = 0.005) compared to non-winter months (9/24 samples, 37.5%). RSV plaques were observed in non-treated and treated co-cultured HeLa monolayers.ConclusionsDemonstrating active RSV in CBMs suggests in utero transmission of infective virus to the fetus without causing overt disease. This is likely to have an important impact on immune development as well as future virus-host interactions, thereby warranting further investigation.

BackgroundAccumulation mode particles (AMP) are formed from engine combustion and make up the inhalable vapour cloud of ambient particulate matter pollution. Their small size facilitates dispersal and subsequent exposure far from their original source, as well as the ability to penetrate alveolar spaces and capillary walls of the lung when inhaled. A significant immuno-stimulatory component of AMP is lipopolysaccharide (LPS), a product of Gram negative bacteria breakdown. As LPS is implicated in the onset and exacerbation of asthma, the presence or absence of LPS in ambient particulate matter (PM) may explain the onset of asthmatic exacerbations to PM exposure.This study aimed to delineate the effects of LPS and AMP on airway inflammation, and potential contribution to airways disease by measuring airway inflammatory responses induced via activation of the LPS cellular receptor, Toll-like receptor 4 (TLR-4).MethodsThe effects of nebulized AMP, LPS and AMP administered with LPS on lung function, cellular inflammatory infiltrate and cytokine responses were compared between wildtype mice and mice not expressing TLR-4.ResultsThe presence of LPS administered with AMP appeared to drive elevated airway resistance and sensitivity via TLR-4. Augmented TLR4 driven eosinophilia and greater TNF-α responses observed in AMP-LPS treated mice independent of TLR-4 expression, suggests activation of allergic responses by TLR4 and non-TLR4 pathways larger than those induced by LPS administered alone. Treatment with AMP induced macrophage recruitment independent of TLR-4 expression.ConclusionsThese findings suggest AMP-LPS as a stronger stimulus for allergic inflammation in the airways then LPS alone.Electronic supplementary materialThe online version of this article (10.1186/s12931-017-0701-z) contains supplementary material, which is available to authorized users.