Lung ultrasound allows for earlier diagnosis of bronchiolitis than auscultation: an animal experiment and human case series.

PURPOSE: Early diagnosis of bronchiolitis in infants allows for risk stratification for central apnea, and, when available, the timely initiation of antiviral treatment. An animal model could demonstrate if earlier diagnosis is possible with ultrasound than with clinical exam. Even if possible, translating this to pediatrics would require observations from undifferentiated human infants. METHODS: We used serial daily clinical and lung ultrasound exams in a bovine calf model (Bos taurus) of respiratory syncytial virus bronchiolitis. Ultrasound and clinical examiners were blinded to each other's findings and the treatments used in 24 calves. Time to diagnosis was compared using Kaplan-Meier curves. A case series of human infants with upper respiratory tract infections, without clinical signs of bronchiolitis, and in whom lung ultrasound was performed, was extracted from hospital records. RESULTS: In the bovine model, lung ultrasound findings emerged earlier and lasted later than auscultatory findings. Relying on auscultation, 5/24 (21%) of animals were diagnosed by post-inoculation day 5 whereas 24/24 (100%) were diagnosed by ultrasound. We identified seven infants in whom lung ultrasound was used to diagnose bronchiolitis before adventitial lung sounds emerged. Three of these subsequently developed typical clinical findings of bronchiolitis in the hospital. Two had alternative explanations for their abnormal lung ultrasounds (both required surgical intervention). Two were discharged and required no further medical attention. CONCLUSION: Lung ultrasound allowed earlier diagnosis of bronchiolitis than clinical exam in the bovine model. In the human case series this was also true, but alternative causes of abnormal ultrasound were frequent.

Validating a bovine model for lung ultrasound of bronchiolitis.

PURPOSE: Bronchiolitis is a very common acute lung disease in infants caused commonly by respiratory syncytial virus (RSV). Point-of-care lung ultrasound is increasingly used in clinical care but proof that ultrasound reflects histological disease is lacking. Bovine calves are a good model for RSV bronchiolitis. We answered the following two questions: (1) does point-of-care lung ultrasound reflect lung pathology at the histological level in a bovine calf model of bronchiolitis? and (2) are point-of-care lung ultrasound images in human infants similar to those obtained in calves? METHODS: We experimentally infected 24 five to six-week-old bovine calves with RSV and compared six window lung ultrasound with lung histology10 days after inoculation. The calves were treated with antivirals and antipyretics leading to variable severity of illness. We used canonical discriminant analysis to determine if abnormal lung ultrasound findings reflected different histological findings. We compared the ultrasounds obtained from the calves with ultrasounds obtained from 10 human infants who were diagnosed clinically with bronchiolitis. RESULTS: Canonical discriminant analysis generally demonstrated good class separation based on the maximal severity of ultrasound finding in each acoustic window. Lung ultrasound performed poorly at detecting bronchopneumonia. Bovine ultrasounds looked similar to human infant lung ultrasounds. CONCLUSION: Point-of-care lung ultrasound abnormalities reflect lung pathology at the histological level in a bovine calf model of bronchiolitis. Point-of-care lung ultrasound images in human infants are similar to those obtained in calves.

Neutrophil extracellular traps (NETs) in a randomized controlled trial of a combination of antiviral and nonsteroidal anti-inflammatory treatment in a bovine model of respiratory syncytial virus infection.

The function of neutrophils in viral infections has long been established and studies have been done to examine the role of neutrophil extracellular traps (NETs). Further study and analysis of NETs in viral infections may reveal a new therapeutic target. Administration of ibuprofen and GS-561937, a fusion protein inhibitor (FPI), have been experimentally shown to decrease the severity of bovine respiratory syncytial virus (BRSV) infection. Our aims were to determine the effect of ibuprofen and FPI on NETs after BRSV infection as a monotherapy or combined therapy. METHODS: We conducted a randomized placebo-controlled trial of ibuprofen, FPI, or as a dual therapy initiated at 3 or 5 days after experimental infection with BRSV in 36 five to six-week-old Holstein calves (Bos Taurus). Lung tissue samples were collected and stained with antibodies conjugated with fluorescence dyes to visualize and quantify the NETs in situ. We estimated the average NETs in the sample lung tissue slides and compared the areas occupied by NETS within and between the treatment groups. RESULTS: There were significantly fewer NETs in the lung tissue from calves that were given ibuprofen and both ibuprofen and fusion protein inhibitor from day 3 post infection compared to the placebo group. Calves administered with ibuprofen, fusion protein inhibitor or both from day five had visually fewer NETs than the placebo but the difference was not significant. CONCLUSION: BRSV can induce NET formation in vitro and in vivo. A combination of both drugs (Ibuprofen and FPI) resulted in less NETs observed in lung tissue of BRSV infected calves compared to the placebo or monotherapy groups.

A randomized controlled trial comparing non-steroidal anti-inflammatory and fusion protein inhibitors singly and in combination on the histopathology of bovine respiratory syncytial virus infection.

Bovine respiratory syncytial virus (RSV) has substantial morbidity in young calves, and closely parallels human RSV in infants. We performed a randomized controlled trial in five to six-week-old Holstein calves (Bos taurus). comparing fusion protein inhibitor (FPI) and non-steroidal anti-inflammatory drug (NSAID) singly and in combination at three and five days after experimental BRSV infection. Thirty-six calves received one of six treatments; Ibuprofen started on day 3, Ibuprofen started on day 5, FPI started on day 5, FPI and Ibuprofen started on day 3, FPI and Ibuprofen started on day 5, or placebo. We have previously reported significant clinical benefits when combined FPI and NSAID treatment was started at three and five days after bovine RSV infection. Necropsy was performed on Day 10 following infection and hematoxylin and eosin staining was performed on sections from each lobe. Histology was described using a four-point scale. We performed canonical discrimination analysis (CDA) to determine the structural level where differences between treatments occurred and mixed effects regression to estimate effect sizes. Separation from placebo was maximal for dual therapy at the levels of the alveolus, septum, and bronchus in CDA. We found that the clinical benefits of combined FPI and NSAID treatment of BRSV extend at least partially from histopathological changes in the lung when treatment was started three days after infection. We found decreased lung injury when ibuprofen was started as monotherapy on day 3, but not day 5 following infection. Combined therapy with both an FPI and ibuprofen was always better than ibuprofen alone. We did not prove that the clinical benefits seen starting FPI and ibuprofen five days after infection can be solely explained by histopathological differences as identified on H&E staining.

Correction: A randomized controlled trial of a combination of antiviral and nonsteroidal anti-inflammatory treatment in a bovine model of respiratory syncytial virus infection.

[This corrects the article DOI: 10.1371/journal.pone.0230245.].

Analysis of lung transcriptome in calves infected with Bovine Respiratory Syncytial Virus and treated with antiviral and/or cyclooxygenase inhibitor.

Bovine Respiratory Syncytial virus (BRSV) is one of the major infectious agents in the etiology of the bovine respiratory disease complex. BRSV causes a respiratory syndrome in calves, which is associated with severe bronchiolitis. In this study we describe the effect of treatment with antiviral fusion protein inhibitor (FPI) and ibuprofen, on gene expression in lung tissue of calves infected with BRSV. Calves infected with BRSV are an excellent model of human RSV in infants: we hypothesized that FPI in combination with ibuprofen would provide the best therapeutic intervention for both species. The following experimental treatment groups of BRSV infected calves were used: 1) ibuprofen day 3-10, 2) ibuprofen day 5-10, 3) placebo, 4) FPI day 5-10, 5) FPI and ibuprofen day 5-10, 6) FPI and ibuprofen day 3-10. All calves were infected with BRSV on day 0. Daily clinical evaluation with monitoring of virus shedding by qRT-PCR was conducted. On day10 lung tissue with lesions (LL) and non-lesional (LN) was collected at necropsy, total RNA extracted, and RNA sequencing performed. Differential gene expression analysis was conducted with Gene ontology (GO) and KEGG pathway enrichment analysis. The most significant differential gene expression in BRSV infected lung tissues was observed in the comparison of LL with LN; oxidative stress and cell damage was especially noticeable. Innate and adaptive immune functions were reduced in LL. As expected, combined treatment with FPI and Ibuprofen, when started early, made the most difference in gene expression patterns in comparison with placebo, especially in pathways related to the innate and adaptive immune response in both LL and LN. Ibuprofen, when used alone, negatively affected the antiviral response and caused higher virus loads as shown by increased viral shedding. In contrast, when used with FPI Ibuprofen enhanced the specific antiviral effect of FPI, due to its ability to reduce the damaging effect of prostanoids and oxidative stress.

A Review of Neutrophil Extracellular Traps (NETs) in Disease: Potential Anti-NETs Therapeutics.

Activated neutrophils release neutrophil extracellular traps (NETs) in response to a variety of stimuli. NETosis is driven by protein-arginine deiminase type 4, with the release of intracellular granule components that function by capturing and destroying microbes, including viral, fungal, bacterial, and protozoal pathogens. The positive effects of pathogen control are countered by pro-inflammatory effects as demonstrated in a variety of diseases. Components of NETS are non-specific, and other than controlling microbes, they cause injury to surrounding tissue by themselves or by increasing the pro-inflammatory response. NETs can play a role in enhancement of the inflammation seen in autoimmune diseases including psoriasis, rheumatoid arthritis, and systemic lupus erythematosis. In addition, autoinflammatory diseases such as gout have been associated with NETosis. Inhibition of NETs may decrease the severity of many diseases improving survival. Herein, we describe NETosis in different diseases focusing on the detrimental effect of NETs and outline possible therapeutics that can be used to mitigate netosis. There is a need for more studies and clinical trials on these and other compounds that could prevent or destroy NETs, thereby decreasing damage to patients.

A randomized controlled trial of a combination of antiviral and nonsteroidal anti-inflammatory treatment in a bovine model of respiratory syncytial virus infection.

INTRODUCTION: Bovine respiratory syncytial virus (RSV) is a valid model for human RSV and an important bovine pathogen. Very early administration of ibuprofen and GS-561937, a fusion protein inhibitor (FPI), have separately been shown to decrease the severity of bovine RSV. Our aims were to determine how long after RSV inoculation ibuprofen and GS-561937 can be administered with clinical benefit and whether using both was better than monotherapy. MATERIALS AND METHODS: We conducted a blinded randomized placebo controlled trial of ibuprofen, GS-561937 (FPI), or combinations of the two initiated at 3 or 5 days after artificial infection with bovine RSV in 36 five to six-week-old Holstein calves (Bos taurus). We measured clinical scores, respiratory rate, and viral shedding daily for 10 days following inoculation. We estimated the average effect for each drug and compared treatment arms using mixed effects models. RESULTS: We found a significant decrease in clinical scores only in the combined treatment arms. This benefit was greater when treatment was initiated at 3 days rather than 5 days post infection with decreased clinical scores and lower respiratory rates at both time points. Ibuprofen alone started on day 3 increased, and FPI with ibuprofen started on day 3 decreased, viral shedding. CONCLUSION: Dual therapy with Ibuprofen and FPI, on average, decrease clinical severity of illness in a bovine model of RSV when started at 3 and 5 days after infection.