In Vivo Evidence of Respiratory Syncytial Virus Persistence in a Subset of Pulmonary Dendritic Cells Following a Primary Infection.

Respiratory syncytial virus (RSV) causes annual epidemics of infections affecting the whole population. In vitro, it has been shown to infect and persist in human dendritic cells (DCs) for prolonged periods. Initially persistence is associated with low levels of replication before the virus becomes dormant. Reactivation of viral replication can be triggered many months later. Infection of DCs is likely to influence the host's ability to generate effective long-term memory responses. A well-established animal was utilized to confirm that RSV both infects and persists in pulmonary DCs in vivo. Mice were infected with a modified strain of RSV expressing red fluorescent protein (RSV-RFP) when replicating. Clinical symptoms of infection were monitored using weight change and inflammatory cell counts from bronchoalveolar lavage, which correlated with the RSV viral titer (quantitative polymerase chain reaction). Lung tissues were collected at 3, 5, 7, and 21 days postinfection (dpi) to assess leukocyte populations by flow cytometry. Clinical symptoms and RSV viral load peaked at 5 dpi. RSV-RFP was most prevalent in macrophages at 3 dpi and also observed in B cells and DCs. At 21 dpi, RSV-RFP remained evident in a subset of conventional DCs (CD103+CD11b+) even though both clinical symptoms and pulmonary inflammation had resolved. These results confirm that in this well-established mouse model, RSV persists in lung conventional DCs following resolution of the acute infection. Further work is required to explore whether the virus continues with low-level replication before becoming dormant in vivo, as has been described in vitro.

Accumulation mode particles and LPS exposure induce TLR-4 dependent and independent inflammatory responses in the lung.

BACKGROUND: Accumulation 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). METHODS: The 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. RESULTS: The 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. CONCLUSIONS: These findings suggest AMP-LPS as a stronger stimulus for allergic inflammation in the airways then LPS alone.

The potential of phage therapy in cystic fibrosis: Essential human-bacterial-phage interactions and delivery considerations for use in Pseudomonas aeruginosa-infected airways.

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.

Infective respiratory syncytial virus is present in human cord blood samples and most prevalent during winter months.

BACKGROUND: Human 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. METHODS: Droplet 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. RESULTS: RSV 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. CONCLUSIONS: Demonstrating 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.

The impact of the H1N1 influenza pandemic on clinical presentations and viral epidemiology of acute respiratory infection in preschool children in Brazil.

We assessed the impact of the H1N1 influenza pandemic on acute respiratory infection in young children from low-income families in Brazil. Influenza (specifically H1N1) detection in acute respiratory infection quintupled during the pandemic and, during its peak, it was associated with 30% of all acute respiratory infection visits to the emergency department. H1N1 was also associated with increased risk of hospitalization and coinfection.

Respiratory syncytial virus binds and undergoes transcription in neutrophils from the blood and airways of infants with severe bronchiolitis.

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.

Viral and atypical bacterial detection in acute respiratory infection in children under five years.

BACKGROUND: Acute 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. METHODS: Clinical/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. FINDINGS: 407 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. INTERPRETATION: This 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.

Enhanced recombinant adeno-associated virus-mediated vascular endothelial growth factor expression in the adult mouse retina: a potential model for diabetic retinopathy.

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.GFP-injected 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 < or = 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.