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1.  Lymphocyte subsets in experimental rhinovirus infection in chronic obstructive pulmonary disease☆ 
Respiratory Medicine  2014;108(1):78-85.
COPD is associated with increased numbers of T cells in the lungs, particularly CD8+ T cells. The mechanisms of increased T cells are unknown but may be related to repeated virus infections in COPD patients. We analysed lymphocyte subsets in blood and bronchoalveolar lavage in smokers and COPD subjects during experimental rhinovirus infections.
Lymphocytes were isolated from blood and bronchoalveolar lavage from COPD subjects and non-obstructed smokers prior to, and following experimental rhinovirus infection. Lymphocyte surface markers and intracellular cytokines were analysed using flow cytometry.
Following rhinovirus infection CD4+ and CD8+ T cell numbers in the COPD subjects were significantly reduced in blood and CD3+ and CD8+ T cells increased in bronchoalveolar lavage compared to baseline. T cells did not increase in BAL in the control subjects. CD3+ T cells correlated with virus load.
Following rhinovirus infection T cells move from the circulation to the lung. Repeated virus infections may contribute to T cell accumulation in COPD patients.
PMCID: PMC3969590  PMID: 24099891
Chronic obstructive pulmonary disease; Acute exacerbations of COPD; Respiratory viruses; T lymphocytes
2.  Reprogramming of lysosomal gene expression by interleukin-4 and Stat6 
BMC Genomics  2013;14:853.
Lysosomes play important roles in multiple aspects of physiology, but the problem of how the transcription of lysosomal genes is coordinated remains incompletely understood. The goal of this study was to illuminate the physiological contexts in which lysosomal genes are coordinately regulated and to identify transcription factors involved in this control.
As transcription factors and their target genes are often co-regulated, we performed meta-analyses of array-based expression data to identify regulators whose mRNA profiles are highly correlated with those of a core set of lysosomal genes. Among the ~50 transcription factors that rank highest by this measure, 65% are involved in differentiation or development, and 22% have been implicated in interferon signaling. The most strongly correlated candidate was Stat6, a factor commonly activated by interleukin-4 (IL-4) or IL-13. Publicly available chromatin immunoprecipitation (ChIP) data from alternatively activated mouse macrophages show that lysosomal genes are overrepresented among Stat6-bound targets. Quantification of RNA from wild-type and Stat6-deficient cells indicates that Stat6 promotes the expression of over 100 lysosomal genes, including hydrolases, subunits of the vacuolar H+ ATPase and trafficking factors. While IL-4 inhibits and activates different sets of lysosomal genes, Stat6 mediates only the activating effects of IL-4, by promoting increased expression and by neutralizing undefined inhibitory signals induced by IL-4.
The current data establish Stat6 as a broadly acting regulator of lysosomal gene expression in mouse macrophages. Other regulators whose expression correlates with lysosomal genes suggest that lysosome function is frequently re-programmed during differentiation, development and interferon signaling.
PMCID: PMC3880092  PMID: 24314139
3.  Inhibiting AKT Phosphorylation Employing Non-Cytotoxic Anthraquinones Ameliorates TH2 Mediated Allergic Airways Disease and Rhinovirus Exacerbation  
PLoS ONE  2013;8(11):e79565.
Severe asthma is associated with T helper (TH) 2 and 17 cell activation, airway neutrophilia and phosphoinositide-3-kinase (PI3K) activation. Asthma exacerbations are commonly caused by rhinovirus (RV) and also associated with PI3K-driven inflammation. Anthraquinone derivatives have been shown to reduce PI3K-mediated AKT phosphorylation in-vitro.
To determine the anti-inflammatory potential of anthraquinones in-vivo.
BALB/c mice were sensitized and challenged with crude house dust mite extract to induce allergic airways disease and treated with mitoxantrone and a novel non-cytotoxic anthraquinone derivative. Allergic mice were also infected with RV1B to induce an exacerbation.
Anthraquinone treatment reduced AKT phosphorylation, hypoxia-inducible factor-1α and vascular endothelial growth factor expression, and ameliorated allergen- and RV-induced airways hyprereactivity, neutrophilic and eosinophilic inflammation, cytokine/chemokine expression, mucus hypersecretion, and expression of TH2 proteins in the airways. Anthraquinones also boosted type 1 interferon responses and limited RV replication in the lung.
Non-cytotoxic anthraquinone derivatives may be of therapeutic benefit for the treatment of severe and RV-induced asthma by blocking pro-inflammatory pathways regulated by PI3K/AKT.
PMCID: PMC3818233  PMID: 24223970
4.  Influenza infection and COPD 
Influenza is a disease with global impact that causes enormous morbidity and mortality on an annual basis. It primarily infects the respiratory tract and causes a broad range of illness ranging from symptomless infection to fulminant primary viral and secondary bacterial pneumonia. The severity of infection depends on both the virus strain and a number of host factors, primarily age and the presence of comorbid conditions such as cardiopulmonary disease. The mortality and utilization of healthcare resources associated with influenza is concentrated in the elderly and those with coexisting disease such as chronic obstructive pulmonary disease (COPD). Increasing use of vaccination and the development of new antiviral drugs hold out hope that the burden of disease associated with influenza can be reduced. However the constant emergence of new influenza strains and the current risk of avian influenza pandemic serve as warnings that influenza will remain a serious pathogen for the foreseeable future.
PMCID: PMC2692119  PMID: 18044066
COPD; influenza; exacerbations
5.  Cross-Serotype Immunity Induced by Immunization with a Conserved Rhinovirus Capsid Protein 
PLoS Pathogens  2013;9(9):e1003669.
Human rhinovirus (RV) infections are the principle cause of common colds and precipitate asthma and COPD exacerbations. There is currently no RV vaccine, largely due to the existence of ∼150 strains. We aimed to define highly conserved areas of the RV proteome and test their usefulness as candidate antigens for a broadly cross-reactive vaccine, using a mouse infection model. Regions of the VP0 (VP4+VP2) capsid protein were identified as having high homology across RVs. Immunization with a recombinant VP0 combined with a Th1 promoting adjuvant induced systemic, antigen specific, cross-serotype, cellular and humoral immune responses. Similar cross-reactive responses were observed in the lungs of immunized mice after infection with heterologous RV strains. Immunization enhanced the generation of heterosubtypic neutralizing antibodies and lung memory T cells, and caused more rapid virus clearance. Conserved domains of the RV capsid therefore induce cross-reactive immune responses and represent candidates for a subunit RV vaccine.
Author Summary
Human rhinovirus infections cause the majority of common colds as well as asthma and chronic obstructive pulmonary disease (COPD) exacerbations. The disease burden attributable to rhinoviruses is therefore huge. Despite this and the fact that human rhinoviruses were discovered over 50 years ago, there are currently no specific antiviral therapies or vaccine available. The lack of a rhinovirus vaccine can at least in part be attributed to the fact that rhinoviruses like other pathogens have high variability in surface antibody binding regions, resulting in >100 serotypically distinct strains. We have defined areas of the rhinovirus polyprotein which are highly conserved across strains and which may therefore induce cross-reactive immune responses capable of providing broader protection. Using a mouse model, we show that immunization with a recombinant rhinovirus capsid protein induces cross-reactive cellular and humoral immune responses. After subsequent infection, immunization enhances both neutralising antibody and lung effector and memory T cell responses, expediting virus clearance. Importantly these effects were evident upon challenge with multiple heterologous rhinovirus serotypes, indicating that immunization with conserved rhinovirus capsid proteins may represent a viable strategy for producing a broadly cross-reactive vaccine.
PMCID: PMC3784482  PMID: 24086140
6.  Blood Eosinophils to Direct Corticosteroid Treatment of Exacerbations of Chronic Obstructive Pulmonary Disease 
Rationale: Exacerbations of chronic obstructive pulmonary disease (COPD) and responses to treatment are heterogeneous.
Objectives: Investigate the usefulness of blood eosinophils to direct corticosteroid therapy during exacerbations.
Methods: Subjects with COPD exacerbations were entered into a randomized biomarker-directed double-blind corticosteroid versus standard therapy study. Subjects in the standard arm received prednisolone for 2 weeks, whereas in the biomarker-directed arm, prednisolone or matching placebo was given according to the blood eosinophil count biomarker. Both study groups received antibiotics. Blood eosinophils were measured in the biomarker-directed and standard therapy arms to define biomarker-positive and -negative exacerbations (blood eosinophil count > and ≤ 2%, respectively). The primary outcome was to determine noninferiority in health status using the chronic respiratory questionnaire (CRQ) and in the proportion of exacerbations associated with a treatment failure between subjects allocated to the biomarker-directed and standard therapy arms.
Measurements and Main Results: There were 86 and 80 exacerbations in the biomarker-directed and standard treatment groups, respectively. In the biomarker-directed group, 49% of the exacerbations were not treated with prednisolone. CRQ improvement after treatment in the standard and biomarker-directed therapy groups was similar (0.8 vs. 1.1; mean difference, 0.3; 95% confidence interval, 0.0–0.6; P = 0.05). There was a greater improvement in CRQ in biomarker-negative exacerbations given placebo compared with those given prednisolone (mean difference, 0.45; 95% confidence interval, 0.01–0.90; P = 0.04). In biomarker-negative exacerbations, treatment failures occurred in 15% given prednisolone and 2% of those given placebo (P = 0.04).
Conclusions: The peripheral blood eosinophil count is a promising biomarker to direct corticosteroid therapy during COPD exacerbations, but larger studies are required.
Clinical trial registered with (ISRCTN92422949).
PMCID: PMC3400995  PMID: 22447964
chronic obstructive pulmonary disease; exacerbations; prednisolone; infection; eosinophils
7.  Lung microbiology and exacerbations in COPD 
Chronic obstructive pulmonary disease (COPD) is the most common chronic respiratory condition in adults and is characterized by progressive airflow limitation that is not fully reversible. The main etiological agents linked with COPD are cigarette smoking and biomass exposure but respiratory infection is believed to play a major role in the pathogenesis of both stable COPD and in acute exacerbations. Acute exacerbations are associated with more rapid decline in lung function and impaired quality of life and are the major causes of morbidity and mortality in COPD. Preventing exacerbations is a major therapeutic goal but currently available treatments for exacerbations are not very effective. Historically, bacteria were considered the main infective cause of exacerbations but with the development of new diagnostic techniques, respiratory viruses are also frequently detected in COPD exacerbations. This article aims to provide a state-of-the art review of current knowledge regarding the role of infection in COPD, highlight the areas of ongoing debate and controversy, and outline emerging technologies and therapies that will influence future diagnostic and therapeutic pathways in COPD.
PMCID: PMC3437812  PMID: 22969296
COPD; exacerbations; bacteria; viruses
8.  Correction: The Role of IL-15 Deficiency in the Pathogenesis of Virus-Induced Asthma Exacerbations 
PLoS Pathogens  2012;8(4):10.1371/annotation/43a4a197-1739-4561-8b8d-b13cd6d7009f.
PMCID: PMC3321056
9.  Viruses exacerbating chronic pulmonary disease: the role of immune modulation 
BMC Medicine  2012;10:27.
Chronic pulmonary diseases are a major cause of morbidity and mortality and their impact is expected to increase in the future. Respiratory viruses are the most common cause of acute respiratory infections and it is increasingly recognized that respiratory viruses are a major cause of acute exacerbations of chronic pulmonary diseases such as asthma, chronic obstructive pulmonary disease and cystic fibrosis. There is now increasing evidence that the host response to virus infection is dysregulated in these diseases and a better understanding of the mechanisms of abnormal immune responses has the potential to lead to the development of new therapies for virus-induced exacerbations. The aim of this article is to review the current knowledge regarding the role of viruses and immune modulation in chronic pulmonary diseases and discuss avenues for future research and therapeutic implications.
PMCID: PMC3353868  PMID: 22420941
Asthma; cystic fibrosis; chronic obstructive pulmonary disease; respiratory viruses; rhinovirus; interferon
10.  Role of Interleukin-1 and MyD88-Dependent Signaling in Rhinovirus Infection▿† 
Journal of Virology  2011;85(15):7912-7921.
Rhinoviral infection is an important trigger of acute inflammatory exacerbations in patients with underlying airway disease. We have previously established that interleukin-1β (IL-1β) is central in the communication between epithelial cells and monocytes during the initiation of inflammation. In this study we explored the roles of IL-1β and its signaling pathways in the responses of airway cells to rhinovirus-1B (RV-1B) and further determined how responses to RV-1B were modified in a model of bacterial coinfection. Our results revealed that IL-1β dramatically potentiated RV-1B-induced proinflammatory responses, and while monocytes did not directly amplify responses to RV-1B alone, they played an important role in the responses observed with our coinfection model. MyD88 is the essential signaling adapter for IL-1β and most Toll-like receptors. To examine the role of MyD88 in more detail, we created stable MyD88 knockdown epithelial cells using short hairpin RNA (shRNA) targeted to MyD88. We determined that IL-1β/MyD88 plays a role in regulating RV-1B replication and the inflammatory response to viral infection of airway cells. These results identify central roles for IL-1β and its signaling pathways in the production of CXCL8, a potent neutrophil chemoattractant, in viral infection. Thus, IL-1β is a viable target for controlling the neutrophilia that is often found in inflammatory airway disease and is exacerbated by viral infection of the airways.
PMCID: PMC3147909  PMID: 21593174
11.  RSV-Induced Bronchial Epithelial Cell PD-L1 Expression Inhibits CD8+ T Cell Nonspecific Antiviral Activity 
Respiratory syncytial virus (RSV) is a major cause of bronchiolitis in infants. It is also responsible for high morbidity and mortality in the elderly. Programmed death ligands (PD-Ls) on antigen-presenting cells interact with receptors on T cells to regulate immune responses. The programmed death receptor-ligand 1/programmed death receptor 1 (PD-L1-PD-1) pathway is inhibitory in chronic viral infections, but its role in acute viral infections is unclear. We hypothesized that bronchial epithelial cell (BEC) expression of PD-Ls would inhibit local effector CD8+ T cell function. We report that RSV infection of primary human BECs strongly induces PD-L1 expression. In a co-culture system of BECs with purified CD8+ T cells, we demonstrated that RSV-infected BECs increased CD8+ T cell activation, proliferation, and antiviral function. Blocking PD-L1 on RSV-infected BECs co-cultured with CD8+ T cells enhanced CD8+ T cell IFN-γ, IL-2, and granzyme B production. It also decreased the virus load of the BECs. Based on our findings, we believe therapeutic strategies that target the PD-L1-PD-1 pathway might increase antiviral immune responses to RSV and other acute virus infections.
PMCID: PMC3086441  PMID: 21148500
12.  Experimental Rhinovirus Infection as a Human Model of Chronic Obstructive Pulmonary Disease Exacerbation 
Rationale: Respiratory virus infections are associated with chronic obstructive pulmonary disease (COPD) exacerbations, but a causative relationship has not been proven. Studies of naturally occurring exacerbations are difficult and the mechanisms linking virus infection to exacerbations are poorly understood. We hypothesized that experimental rhinovirus infection in subjects with COPD would reproduce the features of naturally occurring COPD exacerbations and is a valid model of COPD exacerbations.
Objectives: To evaluate experimental rhinovirus infection as a model of COPD exacerbation and to investigate the mechanisms of virus-induced exacerbations.
Methods: We used experimental rhinovirus infection in 13 subjects with COPD and 13 nonobstructed control subjects to investigate clinical, physiologic, pathologic, and antiviral responses and relationships between virus load and these outcomes.
Measurements and Main Results: Clinical data; inflammatory mediators in blood, sputum, and bronchoalveolar lavage; and viral load in nasal lavage, sputum, and bronchoalveolar lavage were measured at baseline and after infection with rhinovirus 16. After rhinovirus infection subjects with COPD developed lower respiratory symptoms, airflow obstruction, and systemic and airway inflammation that were greater and more prolonged compared with the control group. Neutrophil markers in sputum related to clinical outcomes and virus load correlated with inflammatory markers. Virus load was higher and IFN production by bronchoalveolar lavage cells was impaired in the subjects with COPD.
Conclusions: We have developed a new model of COPD exacerbation that strongly supports a causal relationship between rhinovirus infection and COPD exacerbations. Impaired IFN production and neutrophilic inflammation may be important mechanisms in virus-induced COPD exacerbations.
PMCID: PMC3081284  PMID: 20889904
disease exacerbation; respiratory tract infections; COPD; rhinovirus
13.  The Role of IL-15 Deficiency in the Pathogenesis of Virus-Induced Asthma Exacerbations 
PLoS Pathogens  2011;7(7):e1002114.
Rhinovirus infections are the major cause of asthma exacerbations. We hypothesised that IL-15, a cytokine implicated in innate and acquired antiviral immunity, may be deficient in asthma and important in the pathogenesis of asthma exacerbations. We investigated regulation of IL-15 induction by rhinovirus in human macrophages in vitro, IL-15 levels in bronchoalveolar lavage (BAL) fluid and IL-15 induction by rhinovirus in BAL macrophages from asthmatic and control subjects, and related these to outcomes of infection in vivo. Rhinovirus induced IL-15 in macrophages was replication-, NF-κB- and α/β interferon-dependent. BAL macrophage IL-15 induction by rhinovirus was impaired in asthmatics and inversely related to lower respiratory symptom severity during experimental rhinovirus infection. IL-15 levels in BAL fluid were also decreased in asthmatics and inversely related with airway hyperresponsiveness and with virus load during in vivo rhinovirus infection. Deficient IL-15 production in asthma may be important in the pathogenesis of asthma exacerbations.
Author Summary
We previously reported deficiency in interferon production in asthma, which correlated with disease severity and viral load during experimental rhinovirus infection. Here we show that macrophages produce IL-15 upon rhinovirus infection and that IFN-β plays an important role in IL-15 production. In asthmatic subjects, there is a deficiency in rhinovirus-induced production of IL-15 by macrophages, which indicates immunodeficiency in asthma is surprisingly broad, also involving IL-15, an important cytokine that bridges innate and acquired immunity. These results show that IFN-β therapy in asthma exacerbations could be effective not only due to direct anti-viral effects of IFN-β, but also by inducing IL-15 production. We also show induction of IFN-β and IL-15 to be NF-kB dependent, an important finding which has implications for NF-kB inhibitor drug development programmes as these drugs have potential to worsen rather than improve asthma exacerbation severity, by further enhancing deficiencies of IL-15 and IFN-β. This study investigating the role of IL-15 in rhinovirus infection and asthma has also major implications in other diseases, for example pandemic influenza, where asthma is a major risk factor for severe disease and death, and COPD and cystic fibrosis where IFN-β deficiency is also present.
PMCID: PMC3136447  PMID: 21779162
14.  Interferon-lambda as a new approach for treatment of allergic asthma? 
EMBO Molecular Medicine  2011;3(6):306-308.
PMCID: PMC3377083  PMID: 21634009
allergy; asthma; interferon; mouse model; Th1/Th2
15.  Association of bacteria and viruses with wheezy episodes in young children: prospective birth cohort study 
Objective To study the association between wheezy symptoms in young children and the presence of bacteria in the airways.
Design Birth cohort study.
Setting Clinical research unit in Copenhagen.
Participants Children of asthmatic mothers, from age 4 weeks to 3 years, with planned visits and acute admissions to the research clinic.
Main outcome measure Frequency of bacteria and virus carriage in airway aspirates during wheezy episodes and at planned visits without respiratory symptoms.
Results 984 samples (361 children) were analysed for bacteria, 844 (299 children) for viruses, and 696 (277 children) for both viruses and bacteria. Wheezy episodes were associated with both bacterial infection (odds ratio 2.9, 95% confidence interval 1.9 to 4.3; P<0.001) and virus infection (2.8, 1.7 to 4.4; P<0.001). The associations of bacteria and viruses were independent of each other.
Conclusion Acute wheezy episodes in young children were significantly associated with bacterial infections similar to but independent of the association with virus infections.
PMCID: PMC2950260  PMID: 20921080
16.  Human Rhinovirus 1B Exposure Induces Phosphatidylinositol 3-Kinase–dependent Airway Inflammation in Mice 
Rationale: Infection with rhinovirus (RV) triggers exacerbations of asthma and chronic obstructive lung disease.
Objectives: We sought to develop a mouse model of RV employing RV1B, a minor group serotype that binds to the low-density lipoprotein receptor.
Methods: C57BL/6 mice were inoculated intranasally with RV1B, replication-deficient ultraviolet (UV)-irradiated RV1B, or RV39, a major group virus.
Measurements and Main Results: Viral RNA was present in the lungs of RV1B-treated mice, but not in those exposed to UV-irradiated RV1B or RV39. Lung homogenates of RV-treated mice contained infectious RV 4 days after inoculation. RV1B exposure induced neutrophilic and lymphocytic airway inflammation, as well as increased lung expression of KC, macrophage-inflammatory protein-2, and IFN-α and IFN-β. RV1B-exposed mice showed airway hyperresponsiveness 1 and 4 days after inoculation. UV-irradiated RV1B induced modest neutrophilic airway inflammation and hyperresponsiveness 1 day after exposure. Both RV1B and UV-irradiated RV1B, but not RV39, increased lung phosphorylation of Akt. Confocal immunofluorescence showed colocalization of RV1B and phospho-Akt in the airway epithelium. Finally, pretreatment with the phosphatidylinositol 3-kinase inhibitor LY294002 attenuated chemokine production and neutrophil infiltration.
Conclusions: We conclude that RV1B induces airway inflammation in vivo. Evidence is presented that viral replication occurs in vivo and is required for maximal responses. On the other hand, viral replication was not required for a subset of RV-induced responses, including neutrophilic inflammation, airway hyperresponsiveness, and Akt phosphorylation. Finally, phosphatidylinositol 3-kinase/Akt signaling is required for maximal RV1B-induced airway neutrophilic inflammation, likely via its essential role in virus internalization.
PMCID: PMC2383993  PMID: 18276942
asthma; chronic obstructive pulmonary disease; Akt; low-density lipoprotein receptor
17.  Pathophysiology of viral-induced exacerbations of COPD 
Inflammation of the lower airways is a central feature of chronic obstructive pulmonary disease (COPD). Inflammatory responses are associated with an increased expression of a cascade of proteins including cytokines, chemokines, growth factors, enzymes, adhesion molecules and receptors. In most cases the increased expression of these proteins is the result of enhanced gene transcription: many of these genes are not expressed in normal cells under resting conditions but they are induced in the inflammatory process in a cell-specific manner. Transcription factors regulate the expression of many pro-inflammatory genes and play a key role in the pathogenesis of airway inflammation.
Many studies have suggested a role for viral infections as a causative agent of COPD exacerbations. In this review we will focus our attention on the relationship between common respiratory viral infections and the molecular and inflammatory mechanisms that lead to COPD exacerbation.
PMCID: PMC2699953  PMID: 18268922
18.  Rhinovirus Replication in Human Macrophages Induces NF-κB-Dependent Tumor Necrosis Factor Alpha Production 
Journal of Virology  2006;80(16):8248-8258.
Rhinoviruses (RV) are the major cause of acute exacerbations of asthma and chronic obstructive pulmonary disease (COPD). Rhinoviruses have been shown to activate macrophages, but rhinovirus replication in macrophages has not been reported. Tumor necrosis factor alpha (TNF-α) is implicated in the pathogenesis of acute exacerbations, but its cellular source and mechanisms of induction by virus infection are unclear. We hypothesized that rhinovirus replication in human macrophages causes activation and nuclear translocation of NF-κB, leading to TNF-α production. Using macrophages derived from the human monocytic cell line THP-1 and from primary human monocytes, we demonstrated that rhinovirus replication was productive in THP-1 macrophages, leading to release of infectious virus into supernatants, but was limited in monocyte-derived macrophages, likely due to type I interferon production, which was robust in monocyte-derived but deficient in THP-1-derived macrophages. Similar to bronchial epithelial cells, only small numbers of cells supported complete virus replication. We demonstrated RV-induced activation of NF-κB and colocalization of p65/NF-κB nuclear translocation with virus replication in both macrophage types. The infection induced TNF-α release in a time- and dose-dependent, RV serotype- and receptor-independent manner and was largely (THP-1 derived) or completely (monocyte derived) dependent upon virus replication. Finally, we established the requirement for NF-κB but not p38 mitogen-activated protein kinase in induction of TNF-α. These data suggest RV infection of macrophages may be an important source of proinflammatory cytokines implicated in the pathogenesis of exacerbations of asthma and COPD. They also confirm inhibition of NF-κB as a promising target for development of new therapeutic intervention strategies.
PMCID: PMC1563804  PMID: 16873280
19.  An experimental model of rhinovirus induced chronic obstructive pulmonary disease exacerbations: a pilot study 
Respiratory Research  2006;7(1):116.
Acute exacerbations of COPD are a major cause of morbidity, mortality and hospitalisation. Respiratory viruses are associated with the majority of exacerbations but a causal relationship has not been demonstrated and the mechanisms of virus-induced exacerbations are poorly understood. Development of a human experimental model would provide evidence of causation and would greatly facilitate understanding mechanisms, but no such model exists.
We aimed to evaluate the feasibility of developing an experimental model of rhinovirus induced COPD exacerbations and to assess safety of rhinovirus infection in COPD patients. We carried out a pilot virus dose escalating study to assess the minimum dose of rhinovirus 16 required to induce experimental rhinovirus infection in subjects with COPD (GOLD stage II). Outcomes were assessed by monitoring of upper and lower respiratory tract symptoms, lung function, and virus replication and inflammatory responses in nasal lavage.
All 4 subjects developed symptomatic colds with the lowest dose of virus tested, associated with evidence of viral replication and increased pro-inflammatory cytokines in nasal lavage. These were accompanied by significant increases in lower respiratory tract symptoms and reductions in PEF and FEV1. There were no severe exacerbations or other adverse events.
Low dose experimental rhinovirus infection in patients with COPD induces symptoms and lung function changes typical of an acute exacerbation of COPD, appears safe, and provides preliminary evidence of causation.
PMCID: PMC1578567  PMID: 16956406
20.  Increased proinflammatory responses from asthmatic human airway smooth muscle cells in response to rhinovirus infection 
Respiratory Research  2006;7(1):71.
Exacerbations of asthma are associated with viral respiratory tract infections, of which rhinoviruses (RV) are the predominant virus type. Airway smooth muscle is important in asthma pathogenesis, however little is known about the potential interaction of RV and human airway smooth muscle cells (HASM). We hypothesised that rhinovirus induction of inflammatory cytokine release from airway smooth muscle is augmented and differentially regulated in asthmatic compared to normal HASM cells.
HASM cells, isolated from either asthmatic or non-asthmatic subjects, were infected with rhinovirus. Cytokine production was assayed by ELISA, ICAM-1 cell surface expression was assessed by FACS, and the transcription regulation of IL-6 was measured by luciferase activity.
RV-induced IL-6 release was significantly greater in HASM cells derived from asthmatic subjects compared to non-asthmatic subjects. This response was RV specific, as 5% serum- induced IL-6 release was not different in the two cell types. Whilst serum stimulated IL-8 production in cells from both subject groups, RV induced IL-8 production in only asthmatic derived HASM cells. The transcriptional induction of IL-6 was differentially regulated via C/EBP in the asthmatic and NF-κB + AP-1 in the non-asthmatic HASM cells.
This study demonstrates augmentation and differential transcriptional regulation of RV specific innate immune response in HASM cells derived from asthmatic and non-asthmatics, and may give valuable insight into the mechanisms of RV-induced asthma exacerbations.
PMCID: PMC1534024  PMID: 16670028
21.  Toll-Like Receptor 3 Is Induced by and Mediates Antiviral Activity against Rhinovirus Infection of Human Bronchial Epithelial Cells 
Journal of Virology  2005;79(19):12273-12279.
Rhinoviruses (RV) are the major cause of the common cold and acute exacerbations of asthma and chronic obstructive pulmonary disease. Toll-like receptors (TLRs) are a conserved family of receptors that recognize and respond to a variety of pathogen-associated molecular patterns. TLR3 recognizes double-stranded RNA, an important intermediate of many viral life cycles (including RV). The importance of TLR3 in host responses to virus infection is not known. Using BEAS-2B (a human bronchial epithelial cell-line), we demonstrated that RV replication increased the expression of TLR3 mRNA and TLR3 protein on the cell surface. We observed that blocking TLR3 led to a decrease in interleukin-6, CXCL8, and CCL5 in response to poly(IC) but an increase following RV infection. Finally, we demonstrated that TLR3 mediated the antiviral response. This study demonstrates an important functional requirement for TLR3 in the host response against live virus infection and indicates that poly(IC) is not always a good model for studying the biology of live virus infection.
PMCID: PMC1211516  PMID: 16160153
22.  Asthmatic bronchial epithelial cells have a deficient innate immune response to infection with rhinovirus 
Rhinoviruses are the major trigger of acute asthma exacerbations and asthmatic subjects are more susceptible to these infections. To investigate the underlying mechanisms of this increased susceptibility, we examined virus replication and innate responses to rhinovirus (RV)-16 infection of primary bronchial epithelial cells from asthmatic and healthy control subjects.
Viral RNA expression and late virus release into supernatant was increased 50- and 7-fold, respectively in asthmatic cells compared with healthy controls. Virus infection induced late cell lysis in asthmatic cells but not in normal cells. Examination of the early cellular response to infection revealed impairment of virus induced caspase 3/7 activity and of apoptotic responses in the asthmatic cultures. Inhibition of apoptosis in normal cultures resulted in enhanced viral yield, comparable to that seen in infected asthmatic cultures. Examination of early innate immune responses revealed profound impairment of virus-induced interferon-β mRNA expression in asthmatic cultures and they produced >2.5 times less interferon-β protein. In infected asthmatic cells, exogenous interferon-β induced apoptosis and reduced virus replication, demonstrating a causal link between deficient interferon-β, impaired apoptosis and increased virus replication. These data suggest a novel use for type I interferons in the treatment or prevention of virus-induced asthma exacerbations.
PMCID: PMC2213100  PMID: 15781584
23.  IL-4 increases type 2, but not type 1, cytokine production in CD8+ T cells from mild atopic asthmatics 
Respiratory Research  2005;6(1):67.
Virus infections are the major cause of asthma exacerbations. CD8+ T cells have an important role in antiviral immune responses and animal studies suggest a role for CD8+ T cells in the pathogenesis of virus-induced asthma exacerbations. We have previously shown that the presence of IL-4 during stimulation increases the frequency of IL-5-positive cells and CD30 surface staining in CD8+ T cells from healthy, normal subjects. In this study, we investigated whether excess IL-4 during repeated TCR/CD3 stimulation of CD8+ T cells from atopic asthmatic subjects alters the balance of type 1/type 2 cytokine production in favour of the latter.
Peripheral blood CD8+ T cells from mild atopic asthmatic subjects were stimulated in vitro with anti-CD3 and IL-2 ± excess IL-4 and the expression of activation and adhesion molecules and type 1 and type 2 cytokine production were assessed.
Surface expression of very late antigen-4 [VLA-4] and LFA-1 was decreased and the production of the type 2 cytokines IL-5 and IL-13 was augmented by the presence of IL-4 during stimulation of CD8+ T cells from mild atopic asthmatics.
These data suggest that during a respiratory virus infection activated CD8+ T cells from asthmatic subjects may produce excess type 2 cytokines and may contribute to asthma exacerbation by augmenting allergic inflammation.
PMCID: PMC1198257  PMID: 16001979
24.  Airborne rhinovirus detection and effect of ultraviolet irradiation on detection by a semi-nested RT-PCR assay 
BMC Public Health  2003;3:5.
Rhinovirus, the most common cause of upper respiratory tract infections, has been implicated in asthma exacerbations and possibly asthma deaths. Although the method of transmission of rhinoviruses is disputed, several studies have demonstrated that aerosol transmission is a likely method of transmission among adults. As a first step in studies of possible airborne rhinovirus transmission, we developed methods to detect aerosolized rhinovirus by extending existing technology for detecting infectious agents in nasal specimens.
We aerosolized rhinovirus in a small aerosol chamber. Experiments were conducted with decreasing concentrations of rhinovirus. To determine the effect of UV irradiation on detection of rhinoviral aerosols, we also conducted experiments in which we exposed aerosols to a UV dose of 684 mJ/m2. Aerosols were collected on Teflon filters and rhinovirus recovered in Qiagen AVL buffer using the Qiagen QIAamp Viral RNA Kit (Qiagen Corp., Valencia, California) followed by semi-nested RT-PCR and detection by gel electrophoresis.
We obtained positive results from filter samples that had collected at least 1.3 TCID50 of aerosolized rhinovirus. Ultraviolet irradiation of airborne virus at doses much greater than those used in upper-room UV germicidal irradiation applications did not inhibit subsequent detection with the RT-PCR assay.
The air sampling and extraction methodology developed in this study should be applicable to the detection of rhinovirus and other airborne viruses in the indoor air of offices and schools. This method, however, cannot distinguish UV inactivated virus from infectious viral particles.
PMCID: PMC140314  PMID: 12525263
25.  Synergism between allergens and viruses and risk of hospital admission with asthma: case-control study 
BMJ : British Medical Journal  2002;324(7340):763.
To investigate the importance of sensitisation and exposure to allergens and viral infection in precipitating acute asthma in adults resulting in admission to hospital.
Case-control study.
Large district general hospital.
60 patients aged 17-50 admitted to hospital over a year with acute asthma, matched with two controls: patients with stable asthma recruited from the outpatient department and patients admitted to hospital with non-respiratory conditions (inpatient controls).
Main outcome measures
Atopic status (skin testing and total and specific IgE), presence of common respiratory viruses and atypical bacteria (polymerase chain reaction), dust samples from homes, and exposure to allergens (enzyme linked immunosorbent assay (ELISA): Der p 1, Fel d 1, Can f 1, and Bla g 2).
Viruses were detected in 31 of 177 patients. The difference in the frequency of viruses detected between the groups was significant (admitted with asthma 26%, stable asthma 18%, inpatient controls 9%; P=0.04). A significantly higher proportion of patients admitted with asthma (66%) were sensitised and exposed to either mite, cat, or dog allergen than patients with stable asthma (37%) and inpatient controls (15%; P<0.001). Being sensitised and exposed to allergens was an independent associate of the group admitted to hospital (odds ratio 2.3, 95% confidence interval 1.0 to 5.4; P=0.05), whereas the combination of sensitisation, high exposure to one or more allergens, and viral detection considerably increased the risk of being admitted with asthma (8.4, 2.1 to 32.8; P=0.002).
Allergens and viruses may act together to exacerbate asthma.
What is already known on this topicStudies on segmental allergen challenge of the lung and experimental rhinovirus infection show synergistic effects between allergens and respiratory virus infectionNo studies have investigated an interaction between sensitisation, exposure to allergens, and virus infections in real life exacerbations of asthmaWhat this study addsAllergens and viruses may act together to exacerbate asthma, indicating that domestic exposure to allergens acts synergistically with viruses in sensitised patients, increasing the risk of hospital admissionStrategies to reduce the impact of asthma exacerbations in adults should include interventions directed at both viruses and reducing exposure to allergens
PMCID: PMC100316  PMID: 11923159

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