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Background; Emphysema is a common phenotype of chronic obstructive pulmonary disease (COPD). Although resection of emphysematous tissue can improve lung mechanics, it is invasive and fraught with adverse effects. Meanwhile, radiofrequency (RF) treatment is an extracorporeal method that leads to tissue destruction and remodeling, resulting in “volume reduction” and overall improvement in lung compliance of emphysematous lungs. Whether these changes lead to improved exercise tolerance is unknown. Here, we investigated the effectiveness of RF treatment to improve the exercise capacity of mice with emphysema.Methods;Fifty-two mice (7 weeks of age) were used in this experiment. A bilateral emphysema model was created by intratracheally instilling porcine pancreatic elastase (PPE) (1.5U/100g body weight). RF treatment (0.5W/ g body weight) was performed extracorporeally 14 days later and mice were sacrificed after another 21 days. The exercise capacity of mice was measured using a treadmill. Treadmill runs were performed just before PPE instillation (baseline), before RF treatment and before sacrifice. Following sacrifice, lung compliance and mean linear intercept (Lm) were measured and fibrosis was assessed using a modified Ashcroft score.Results; There were 3 experimental groups: controls (instilled with saline, n=12), emphysema (instilled with porcine pancreatic elastase, PPE, n=11) and emphysema + treatment (instilled with PPE and given RF, n=9). At endpoint, the maximum velocity of the emphysema + treatment group was significantly higher than that of the emphysema group, indicating improved exercise tolerance (86.29% of baseline vs 61.69% of baseline, p=0.011). Histological analysis revealed a significant reduction in emphysema as denoted by Lm between the two groups (median 29.60 µm vs 35.68 µm, p=0.033). The emphysema + treatment group also demonstrated a higher prevalence of lung fibrosis (≧Grade 3) compared with the emphysema group (11.7% vs 5.4%, p

The associations between airway eosinophilia, measured in sputum or peripheral blood, and acute exacerbations of chronic obstructive pulmonary disease (AECOPD) are inconsistent. We therefore aimed to determine the association between eosinophilia in bronchoalveolar lavage (BAL) fluid and AECOPD in a clinical cohort. We analyzed differential cell counts from baseline BAL fluid in participants in the DISARM clinical trial (Clinicaltrials.gov #NCT02833480) and classified participants by the presence or absence of BAL eosinophilia (>1% of total leukocytes). We determined the association between BAL eosinophilia and AECOPD over 1 year of follow-up using negative binomial regression and Cox proportional hazards test. N = 63 participants were randomized, and N = 57 had BAL differential cell counts available. Participants with BAL eosinophilia (N = 21) had a significantly increased rate of acute exacerbations (unadjusted incidence rate ratio (IRR) 2.0, p = 0.048; adjusted IRR 2.24, p = 0.04) and a trend toward greater probability of acute exacerbation (unadjusted hazard ratio (HR) 1.74, p = 0.13; adjusted HR 2.3, p = 0.1) in the year of follow-up compared to participants without BAL eosinophilia (N = 36). These associations were not observed for BAL neutrophilia (N = 41 participants), BAL lymphocytosis (N = 27 participants) or peripheral blood eosinophilia at various threshold definitions (2%, N = 37; 3%, N = 27; 4%, N = 16). BAL may therefore be a sensitive marker of eosinophilic inflammation in the distal lung and may be of benefit for risk stratification or biomarker-guided therapy in COPD.

The classical M1/M2 polarity of macrophages may not be applicable to inflammatory lung diseases including chronic obstructive pulmonary disease (COPD) due to the complex microenvironment in lungs and the plasticity of macrophages. We examined macrophage sub-phenotypes in bronchoalveolar lavage (BAL) fluid in 25 participants with CD40 (a M1 marker) and CD163 (a M2 marker). Of these, we performed RNA-sequencing on each subtype in 10 patients using the Illumina NextSeq 500. Approximately 25% of the macrophages did not harbor classical M1 or M2 surface markers (double negative, DN), and these cells were significantly enriched in COPD patients compared with non-COPD patients (46.7% vs. 14.5%, p

COPD is characterised by the destruction of lung tissue resulting in alveolar tissue destruction, enlargement of alveolar spaces, poor gas exchange and airway collapse due to the loss of elastic recoil [1]. Lung volume reduction surgery (LVRS) is effective in reducing long term morbidity and mortality of patients with severe emphysema who have a predominance of upper lobe disease and are able to tolerate the surgical procedure [2, 3]. However, the financial cost and the peri-operative morbidity and mortality of the procedure have limited its application in clinical practice [4, 5]. Here, we investigated the possibility of using external radiofrequency (RF) as a novel extracorporeal treatment for emphysema in a rat model of unilateral emphysema. Footnotes This manuscript has recently been accepted for publication in the European Respiratory Journal . It is published here in its accepted form prior to copyediting and typesetting by our production team. After these production processes are complete and the authors have approved the resulting proofs, the article will move to the latest issue of the ERJ online. Please open or download the PDF to view this article. Conflict of interest: Dr. Wada reports grants from IKOMED, grants from MITACS, during the conduct of the study. Conflict of interest: Dr. Jaw reports grants from Mitacs (joint application with iKomed), during the conduct of the study. Conflict of interest: Dr. Tsuruta reports grants from IKOMED, grants from MITACS, during the conduct of the study. Conflict of interest: Dr. Moritani reports grants from IKOMED, grants from MITACS, during the conduct of the study. Conflict of interest: Dr. Tsutsui reports grants from IKOMED, grants from MITACS, during the conduct of the study. Conflict of interest: Dr. Tam has nothing to disclose. Conflict of interest: Dr. Vasilescu has nothing to disclose. Conflict of interest: Dr. CHEUNG reports grants from Ikomed, grants from Mitac, during the conduct of the study. Conflict of interest: Dr. Yamasaki reports grants from IKOMED, grants from MITACS, during the conduct of the study. Conflict of interest: Dr. Lichtenstein reports In addition, Dr. Lichtenstein has a patent Method for selectively heating tissue issued to Ikomed Technologies Inc and Founder and Investor in Ikomed Technologies Inc.. Conflict of interest: Dr. Machan reports he is an Investor in Ikomed Technologies. Conflict of interest: Dr. Gelbart reports In addition, Dr. Gelbart has a patent 8444635 licensed to Ikomed. Conflict of interest: Dr. Man has nothing to disclose. Conflict of interest: Dr. Sin reports grants from IKOMED, during the conduct of the study; grants from Merck, personal fees from Sanofi-Aventis, personal fees from Regeneron, grants and personal fees from Boehringer Ingelheim, grants and personal fees from AstraZeneca, personal fees from Novartis, outside the submitted work.

H5N1 influenza viruses, which cause disease in humans, have unusually high pathogenicity. The temporal response of primary human monocyte-derived macrophages infected with highly pathogenic H5N1 and seasonal H1N1 influenza viruses was evaluated using mass spectrometry-based quantitative proteomic profiling. This was done in order to demonstrate significant perturbation of the host proteome upon viral infection, as early as 1 hour after infection. This early host response distinguished H5N1 infection from H1N1 infection, the latter inducing less of a response. The most pronounced effect was observed on the translational machinery, suggesting that H5N1 might gain advantage in replication by using the cell protein synthesis machinery early in the infection.

Avian influenza A H5N1 remains unusual in its virulence for humans. While infection of humans remains inefficient, many of those with H5N1 disease have a rapidly progressing viral pneumonia leading to acute respiratory distress syndrome and death but its pathogenesis remains an enigma. Comparisons in the virology and pathogenesis of human seasonal influenza viruses (H3N2 and H1N1) and H5N1 in patients, animal models and in relevant primary human cell cultures remains instructive. While the direct effects of viral replication and differences in the tropism of the virus for cells in the lower respiratory tract clearly contribute to the pathogenesis, we focus here on the possible contribution of the host innate immune response in the pathogenesis of this disease.

Theorf3a(also called X1 or U274) gene is the largest unique open reading frame in the severe acute respiratory syndrome coronavirus genome and has been proposed to encode a protein with three transmembrane domains and a large cytoplasmic domain. Recent work has suggested that the 3a protein may play a structural role in the viral life cycle, although the mechanisms for this remain uncharacterized. Here, the expression of the 3a protein in variousin vitrosystems is shown, it has been localized to the Golgi region and its membrane topology in transfected cells has been confirmed. Three potential caveolin-1-binding sites were reported to be present in the 3a protein. By using various biochemical, biophysical and genetic techniques, interaction of the 3a protein with caveolin-1 is demonstrated. Any one of the potential sites in the 3a protein was sufficient for this interaction. These results are discussed with respect to the possible roles of the 3a protein in the viral life cycle.

In 1997, avian influenza virus H5N1 was transmitted directly from chicken to human and resulted in a severe disease that had a higher mortality rate in adults than in children. The characteristic mononuclear leukocyte infiltration in the lung and the high inflammatory response in H5N1 infection prompted us to compare the chemokine responses between influenza virus–infected adult and neonatal monocyte-derived macrophages (MDMs). The effects of avian influenza virus A/Hong Kong/483/97 (H5N1) (H5N1/97), its precursor A/Quail/Hong Kong/G1/97 (H9N2) (H9N2/G1), and human influenza virus A/Hong Kong/54/98 (H1N1) (H1N1/98) were compared. Significantly higher expression of CCL2, CCL3, CCL5, and CXCL10 was induced by avian influenza viruses than by human influenza virus. Moreover, the increase in CCL3 expression in H5N1/97-infected adult MDMs was significantly higher than that in neonatal MDMs. Enhanced expression of CCR1 and CCR5 was found in avian virus–infected adult MDMs. The strong induction of chemokines and their receptors by avian influenza viruses, particularly in adult MDMs, may account for the severity of H5N1 disease

Severe acute respiratory syndrome (SARS) is a new infectious disease with a global impact. Understanding its pathogenesis and developing specific diagnostic methods for its early diagnosis are crucial for the effective management and control of this disease. By using proteomic technology, truncated forms of alpha(1)-antitrypsin (TF-alpha(1)-AT) were found to increase significantly and consistently in sera of SARS patients compared to control subjects. The result showed a sensitivity of 100% for SARS patients and a specificity of 92.8% for controls. Furthermore, the levels of these proteins significantly correlated with certain clinico-pathological parameters. The dramatic increase in TF-alpha(1)-AT may be the result of degradation of alpha(1)-AT. As alpha(1)-AT plays an important role in the protection of lung function, its degradation may be an important factor in the pathogenesis of SARS. These findings indicate that increased TF-alpha(1)-AT may be therapeutically relevant, and may also be a useful biological marker for the diagnosis of SARS.

Background Parvovirus B19 (B19) infection is known to cause chronic infection leading to anemia in immunocompromised patients. Although nosocomial B19 infections in immunocompetent patients have been documented, no outbreaks in immunocompromised patients have been previously reported. Whether transmission can occur from a patient with chronic infection is also unknown. Methods An outbreak of B19 infection in a renal transplant unit was investigated by molecular analysis of the virus strains and a case-control study. Results Three patients had genetically identical virus strains suggesting the occurrence of nosocomial transmission. The index case transmitted infection many weeks after the onset of her clinical symptoms. Other patients at risk of acquiring infection were those most intensively immunosuppressed. Viral load in the serum correlated with the hematological response. A rebound in the viral load was associated with clinical relapse and the failure of i.v. immunoglobulin therapy. Conclusion Nosocomial transmission of B19 can occur from immunocompromised patients even when they are in the chronic stage of the infection. The clinical and virological response to i.v. immunoglobulin therapy is variable and depends on the overall level of immunosuppression of the patient.

Public health risks associated to infection by human coronaviruses remain considerable and vaccination is a key option for preventing the resurgence of severe acute respiratory syndrome coronavirus (SARS-CoV). We have previously reported that antibodies elicited by a SARS-CoV vaccine candidate based on recombinant, full-length SARS-CoV Spike-protein trimers, trigger infection of immune cell lines. These observations prompted us to investigate the molecular mechanisms and responses to antibody-mediated infection in human macrophages., published_or_final_version

© 2012 Millet et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Background: Entry of Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) and its envelope fusion with host cell membrane are controlled by a series of complex molecular mechanisms, largely dependent on the viral envelope glycoprotein Spike (S). There are still many unknowns on the implication of cellular factors that regulate the entry process. Methodology/Principal Findings: We performed a yeast two-hybrid screen using as bait the carboxy-terminal endodomain of S, which faces the cytosol during and after opening of the fusion pore at early stages of the virus life cycle. Here we show that the ezrin membrane-actin linker interacts with S endodomain through the F1 lobe of its FERM domain and that both the eight carboxy-terminal amino-acids and a membrane-proximal cysteine cluster of S endodomain are important for this interaction in vitro. Interestingly, we found that ezrin is present at the site of entry of S-pseudotyped lentiviral particles in Vero E6 cells. Targeting ezrin function by small interfering RNA increased S-mediated entry of pseudotyped particles in epithelial cells. Furthermore, deletion of the eight carboxy-terminal amino acids of S enhanced S-pseudotyped particles infection. Expression of the ezrin dominant negative FERM domain enhanced cell susceptibility to infection by SARS-CoV and S pseudotyped particles and potentiated S-dependent membrane fusion. Conclusions/Significance: Ezrin interacts with SARS-CoV S endodomain and limits virus entry and fusion. Our data present a novel mechanism involving a cellular factor in the regulation of S-dependent early events of infection. This work was supported by the Research Grant Council of Hong Kong (RGC#760208)and the RESPARI project of the International Network of Pasteur Institutes.

Background: The SARS outbreak in 2003 provides a unique opportunity for the study of human responses to a novel virus. We have previously reported that dendritic cells (DCs) might be involved in the immune escape mechanisms for SARS-CoV. In this study, we focussed on the gene expression of toll-like receptors (TLRs), chemokine receptors (CCRs) and death receptor ligands in SARS-CoV infected DCs. We also compared adult and cord blood (CB) DCs to find a possible explanation for the age-dependent severity of SARS. Results: Our results demonstrates that SARS-CoV did not modulate TLR-1 to TLR-10 gene expression but significantly induced the expression of CCR-1, CCR-3, and CCR-5. There was also strong induction of TNF-related apoptosis-inducing ligand (TRAIL), but not Fas ligand gene expression in SARS-CoV infected DCs. Interestingly, the expressions of most genes studied were higher in CB DCs than adult DCs. Conclusion: The upregulation of chemokines and CCRs may facilitate DC migration from the infection site to the lymph nodes, whereas the increase of TRAIL may induce lymphocyte apoptosis. These findings may explain the increased lung infiltrations and lymphoid depletion in SARS patients. Further explorations of the biological significance of these findings are warranted. © 2009 Law et al; licensee BioMed Central Ltd., published_or_final_version

Severe human disease associated with influenza A H5N1 virus was first detected in Hong Kong in 1997. Its recent reemergence in Asia and high associated mortality highlight the need to understand its pathogenesis. We investigated the roles of death receptor ligands (DRLs) in H5N1 infection. Significant up-regulation of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and TNF-alpha, but not Fas ligand (FasL) mRNA, was detected in human monocyte-derived macrophages (MDMs) infected with avian influenza viruses A/Hong Kong/483/97 (H5N1/97) or its precursor, A/Quail/Hong Kong/G1/97. H5N1/97-infected MDMs exhibited the strongest induction of apoptosis in Jurkat T cells, and it could be reduced by TRAIL-receptor 2 blocking antibody. Furthermore, influenza virus infection enhanced the sensitivity of Jurkat T cells to apoptosis induced by TNF-alpha, TRAIL, and FasL. Our data suggested that functional TRAIL produced by influenza virus-infected MDMs was related to their cytotoxicity and that the enhanced sensitization to DRL-induced apoptosis detected in avian influenza may contribute to disease pathogenesis.

Lymphopenia and increasing viral load in the first 10 days of severe acute respiratory syndrome (SARS) suggested immune evasion by SARS-coronavirus (CoV). In this study, we focused on dendritic cells (DCs) which play important roles in linking the innate and adaptive immunity. SARS-CoV was shown to infect both immature and mature human monocyte-derived DCs by electron microscopy and immunofluorescence. The detection of negative strands of SARS-CoV RNA in DCs suggested viral replication. However, no increase in viral RNA was observed. Using cytopathic assays, no increase in virus titer was detected in infected DCs and cell-culture supernatant, confirming that virus replication was incomplete. No induction of apoptosis or maturation was detected in SARS-CoV-infected DCs. The SARS-CoV-infected DCs showed low expression of antiviral cytokines (interferon α [IFN-α], IFN-β, IFN-γ, and interleukin 12p40 [IL-12p40]), moderate up-regulation of proinflammatory cytokines (tumor necrosis factor α [TNF-α] and IL-6) but significant up-regulation of inflammatory chemokines (macrophage inflammatory protein 1α [MIP-1α], regulated on activation normal T cell expressed and secreted [RANTES]), interferon-inducible protein of 10 kDa [IP-10], and monocyte chemoattractant protein 1 [MCP-1]). The lack of antiviral cytokine response against a background of intense chemokine upregulation could represent a mechanism of immune evasion by SARS-CoV. © 2005 by The American Society of Hematology., postprint

Host response to infection with pathogens such as influenza viruses serves to primarily generate immune responses in an attempt to counter the invading pathogens. Consequently, how the host interacts with the virus not only has important influence on its pathogenesis, but also its transmission in a population and dissemination within the infected host. On the other hand, an overly active immune response may actually lead to excessive inflammation, which could be detrimental to the host as many lines of evidence have suggested for human H5N1 infection and the 1918 H1N1 pandemic virus. Macrophages are key orchestrator of the immune response and being one of the most abundant cell types in the respiratory system that can be infected with influenza viruses. Therefore, this study aims utilize high-throughput mass spectrometry to compare time series global proteomic profiles of primary human monocyte-derived macrophages infected with highly pathogenic H5N1 and seasonal H1N1 influenza viruses, in order to allow combinational analysis of proteomic datasets with existing body of transcriptomic datasets to provide further insight into the cellular and molecular host response to influenza virus infection. Global proteomic profiling of influenza virus infected macrophages revealed that many of the proteome changes could not be accounted for by transcriptomic profiling with microarrays. The low concordance of the global proteome profiling results with transcriptomic data indicates that high throughput quantitative proteomic analysis can provide a significant additional dimension to enable combinational analysis with existing datasets from studies using high-throughput genomic platforms. Results from this study suggest proteome changes in infected macrophages that were common to both viruses could be involved in processes that are similar, such as viral replication as both viruses replicate equally well in our macrophage model. On the other hand, pathways derived from analysis of differentially affected proteomes may contribute to the high pathogenic nature of H5N1 viruses. Therefore systematic integrative analysis of datasets from different sources could significantly contribute to detecting differentially expressed genes or pathways that are of relevance during virus infection and allows assessment of heterogeneity. This is likely to contribute to target identification for host-directed treatment of disease caused by influenza viruses.