Project description:Background:Interleukin-6 signal blockade showed preliminary beneficial effects in treating inflammatory response against SARS-CoV-2 leading to severe respiratory distress. Herein we describe the outcomes of off-label intravenous use of Sarilumab in severe SARS-CoV-2-related pneumonia. Methods:53 patients with SARS-CoV-2 severe pneumonia received intravenous Sarilumab; pulmonary function improvement or Intensive Care Unit (ICU) admission rate in medical wards, live discharge rate in ICU treated patients and safety profile were recorded. Sarilumab 400 mg was administered intravenously on day 1, with eventual additional infusion based on clinical judgement, and patients were followed for at least 14 days, unless previously discharged or dead. Findings:Of the 53 SARS-CoV-2pos patients receiving Sarilumab, 39(73·6%) were treated in medical wards [66·7% with a single infusion; median PaO2/FiO2:146(IQR:120-212)] while 14(26·4%) in ICU [92·6% with a second infusion; median PaO2/FiO2: 112(IQR:100-141.5)].Within the medical wards, 7(17·9%) required ICU admission, 4 of whom were re-admitted to the ward within 5-8 days. At 19 days median follow-up, 89·7% of medical inpatients significantly improved (46·1% after 24 h, 61·5% after 3 days), 70·6% were discharged from the hospital and 85·7% no longer needed oxygen therapy. Within patients receiving Sarilumab in ICU, 64·2% were discharged from ICU to the ward and 35·8% were still alive at the last follow-up. Overall mortality rate was 5·7%. Interpretation:IL-6R inhibition appears to be a potential treatment strategy for severe SARS-CoV-2 pneumonia and intravenous Sarilumab seems a promising treatment approach showing, in the short term, an important clinical outcome and good safety.
Project description:Coronavirus disease 2019 (COVID-19) is a disease that causes fatal disorders including severe pneumonia. To develop a therapeutic drug for COVID-19, a model that can reproduce the viral life cycle and can evaluate the drug efficacy of anti-viral drugs is essential. In this study, we established a method to generate human bronchial organoids (hBO) from commercially available cryopreserved primary human bronchial epithelial cells (hBEpC) and examined whether they could be used as a model for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) research. The hBO were found to contain basal, club, ciliated, and goblet cells. Also, angiotensin-converting enzyme 2 (ACE2), which is a receptor for SARS-CoV-2, and transmembrane serine proteinase 2 (TMPRSS2), which is an essential serine protease for priming spike protein of SARS-CoV-2, were highly expressed. After hBO were infected with SARS-CoV-2, remarkable amplification of the viral genome and the expression of spike protein of the virus was confirmed. In addition, cytotoxicity and pyknosis cells were observed due to the virus infection. Furthermore, treatment with camostat, an inhibitor of TMPRSS2, reduced the viral copy number to 2% of the control group. RNA-seq analyses revealed genes whose expression was altered by SARS-CoV-2 infection and camostat treatment. These results suggest that our hBO are acceptable for SARS-CoV-2 infection and replication, but also can be used as a model for COVID-19 drug discovery. Overall design: Total RNA was isolated from human bronchial organoids, primary human bronchial epithelial cells, and A549 cell, and then RNA-seq was performed. The human bronchial organoids were infected with SARS-CoV-2 in the presence of absence of camostat. At 5 days after the infection, total RNA was collected and RNA-seq was performed.
Project description:Background:There are numerous biologics for treating patients with severe asthma. A cost-effective method for selecting the most appropriate biologic therapy for a patient is thus important. Bronchoscopy-guided bronchial epithelium sampling may provide information for determining the type of inflammation in the airways of severe asthma patients through immunochemical analysis and thus help clinicians select the correct biologics. Case presentation:We report the case of a female with severe asthma and eosinophilia who initially responded to omalizumab treatment. She developed an allergic reaction after four injections of omalizumab. Omalizumab desensitization was successfully conducted. To select an appropriate biologic agent after this hypersensitivity episode, we performed bronchoscopy-guided bronchial epithelium sampling. Omalizumab treatment was resumed based on the findings of immunohistochemical staining after a successful desensitization procedure, leading to long-term control of her severe asthma. Conclusions:Selecting an adequate biologic agent for severe, uncontrolled asthma is a challenge in clinical medical practice. Although phenotypes, blood eosinophils, and serum IgE levels have been proposed for use as a reference, there is a dissociation between the blood immune-cell level and the airway epithelium immune reaction, as confirmed in previous studies. Airway epithelium immunohistochemistry staining for targeted immune cells has been used to determine various types of airway inflammation; however, this technique is rarely used in a clinical setting. Previous studies have revealed the relative safety of performing bronchoscopy biopsies for patients with severe asthma. Among the sampling techniques used for tissue diagnosis, including nasal biopsies, nasal or bronchial brushing, and bronchoalveolar lavage, bronchoscopy-guided bronchial epithelium sampling provides more accurate information about the epithelial and inflammatory cells in the tissue context. It is thus a powerful tool for selecting the most suitable biologics in difficult clinical conditions.
Project description:Severe equine asthma is a naturally occurring lung inflammatory disease of mature animals characterized by neutrophilic inflammation, bronchoconstriction, mucus hypersecretion and airway remodeling. Exacerbations are triggered by inhalation of dust and microbial components. Affected animals eventually are unable of aerobic performance. In this study transcriptomic differences between asthmatic and non-asthmatic animals in the response of the bronchial epithelium to an inhaled challenge were determined.Paired endobronchial biopsies were obtained pre- and post-challenge from asthmatic and non-asthmatic animals. The transcriptome, determined by RNA-seq and analyzed with edgeR, contained 111 genes differentially expressed (DE) after challenge between horses with and without asthma, and 81 of these were upregulated. Genes involved in neutrophil migration and activation were in central location in interaction networks, and related gene ontology terms were significantly overrepresented. Relative abundance of specific gene products as determined by immunohistochemistry was correlated with differential gene expression. Gene sets involved in neutrophil chemotaxis, immune and inflammatory response, secretion, blood coagulation and apoptosis were overrepresented among up-regulated genes, while the rhythmic process gene set was overrepresented among down-regulated genes. MMP1, IL8, TLR4 and MMP9 appeared to be the most important proteins in connecting the STRING protein network of DE genes.Several differentially expressed genes and networks in horses with asthma also contribute to human asthma, highlighting similarities between severe human adult and equine asthma. Neutrophil activation by the bronchial epithelium is suggested as the trigger of the inflammatory cascade in equine asthma, followed by epithelial injury and impaired repair and differentiation. Circadian rhythm dysregulation and the sonic Hedgehog pathway were identified as potential novel contributory factors in equine asthma.
Project description:Recurrent lung infections and pneumonia are emerging as significant comorbidities in the HIV-infected population in the era of combination antiretroviral therapy (cART). HIV infection has been reported to suppress nasal mucociliary clearance (MCC). Since the primary components driving nasal MCC and bronchial MCC are identical, it is possible that bronchial MCC is affected as well. Effective MCC requires optimal ciliary beating which depends on the maintenance of the airway surface liquid (ASL), a function of cystic fibrosis transmembrane conductance regulator (CFTR) activity and the integrity of the signaling mechanism that regulates ciliary beating and fluid secretion. Impairment of either component of the MCC apparatus can compromise its efficacy and promote microbial colonization. We demonstrate that primary bronchial epithelium expresses HIV receptor CD4 and co-receptors CCR5 and CXCR4 and can be infected by both R5 and X4 tropic strains of HIV. We show that HIV Tat suppresses CFTR biogenesis and function in primary bronchial epithelial cells by a pathway involving TGF-? signaling. HIV infection also interferes with bronchial epithelial cell differentiation and suppresses ciliogenesis. These findings suggest that HIV infection suppresses tracheobronchial mucociliary clearance and this may predispose HIV-infected patients to recurrent lung infections, pneumonia and chronic bronchitis.
Project description:Influenza A viruses recruit components of the nuclear import pathway to enter the host cell nucleus and promote viral replication. Here, we analyzed the role of the nuclear import factor importin-?7 in H1N1 influenza virus pulmonary tropism by using various ex vivo imaging techniques (magnetic resonance imaging, confocal laser scanning microscopy, and correlative light-electron microscopy). We infected importin-?7 gene-deficient (?7(-/-)) mice with a recombinant H1N1 influenza virus and compared the in vivo viral kinetics with those in wild-type (WT) mice. In WT mice, influenza virus replication in the bronchial and alveolar epithelium already occurred a few days after infection. Accordingly, extensive mononuclear infiltration and alveolar destruction were present in the lungs of infected WT mice, followed by 100% lethality. Conversely, in ?7(-/-) mice, virus replication was restricted mostly to the bronchial epithelium with marginal alveolar infection, resulting in significantly reduced lung damage and enhanced animal survival. To investigate the host immune response during alveolar virus replication, we studied the role of primary macrophages in virus propagation and clearance. The ability of macrophages to support or clear the virus infection, as well as the host cellular immune responses, did not significantly differ between WT and ?7(-/-) mice. However, cytokine and chemokine responses were generally elevated in WT mice, likely reflective of increased viral replication in the lung. In summary, these data show that a cellular factor, importin-?7, is required for enhanced virus replication in the alveolar epithelium, resulting in elevated cytokine and chemokine levels, extensive mononuclear infiltration, and thus, severe pneumonia and enhanced virulence in mice. Importance: Influenza A viruses are respiratory pathogens that may cause pneumonia in humans. Viral infection and replication in the alveoli of the respiratory tract are believed to be crucial for the development of the acute respiratory distress syndrome associated with fatal outcomes in influenza virus-infected patients. Here, we report the requirement of a cellular factor, importin-?7, for efficient virus replication in the alveolar epithelium of mice. Using complementary ex vivo imaging approaches, we show that influenza virus replication is restricted to the bronchial epithelium, followed by enhanced survival in importin-?7-deficient mice. In contrast, the presence of this gene results in enhanced virus replication in the alveoli, elevated cytokine and chemokine responses, mononuclear infiltration, alveolar destruction, and 100% lethality in wild-type mice. Taken together, our results show that importin-?7 is particularly required for virus replication in the alveolar epithelium in association with severe pneumonia and death in mice.
Project description:Recurrent lung infections are a common cause of morbidity and mortality in people living with HIV and this is exacerbated in smokers even when administered combination antiretroviral therapy (cART). The incidence of pneumonia is increased with smoking and treatment interruption and is directly dependent on viral load in patients when adjusted for CD4 counts. CFTR dysfunction plays an important role in aberrant airway innate immunity as it is pivotal in regulating mucociliary clearance (MCC) rates and other antibacterial mechanisms of the airway. In our earlier work, we have demonstrated that bronchial epithelium expresses canonical HIV receptors CD4, CCR5 and CXCR4 and can be infected with HIV. HIV Tat suppresses CFTR mRNA and function via TGF-? signaling. In the present study, we demonstrate that cigarette smoke (CS) potentiates HIV infection of bronchial epithelial cells by upregulating CD4 and CCR5 expression. HIV and CS individually and additively suppress CFTR biogenesis and function, possibly explaining the increased incidence of lung infections in HIV patients and its exacerbation in HIV smokers.
Project description:Previous studies have shown that physiological responses to cigarette smoke can be detected via bronchial airway epithelium gene expression profiling and that heterogeneity in this gene expression response to smoking is associated with lung cancer. In this study, we sought to determine the similarity of the effects of tobacco smoke throughout the respiratory tract by determining patterns of smoking-related gene expression in paired nasal and bronchial epithelial brushings collected from 14 healthy nonsmokers and 13 healthy current smokers. Using whole genome expression arrays, we identified 119 genes whose expression was affected by smoking similarly in both bronchial and nasal epithelium, including genes related to detoxification, oxidative stress, and wound healing. While the vast majority of smoking-related gene expression changes occur in both bronchial and nasal epithelium, we also identified 27 genes whose expression was affected by smoking more dramatically in bronchial epithelium than nasal epithelium. Both common and site-specific smoking-related gene expression profiles were validated using independent microarray datasets. Differential expression of select genes was also confirmed by RT-PCR. That smoking induces largely similar gene expression changes in both nasal and bronchial epithelium suggests that the consequences of cigarette smoke exposure can be measured in tissues throughout the respiratory tract. Our findings suggest that nasal epithelial gene expression may serve as a relatively noninvasive surrogate to measure physiological responses to cigarette smoke and/or other inhaled exposures in large-scale epidemiological studies.
Project description:BACKGROUND:Data on the prevalence of bacterial and viral co-infections among patients admitted to the ICU for acute respiratory failure related to SARS-CoV-2 pneumonia are lacking. We aimed to assess the rate of bacterial and viral co-infections, as well as to report the most common micro-organisms involved in patients admitted to the ICU for severe SARS-CoV-2 pneumonia. PATIENTS AND METHODS:In this monocenter retrospective study, we reviewed all the respiratory microbiological investigations performed within the first 48 h of ICU admission of COVID-19 patients (RT-PCR positive for SARS-CoV-2) admitted for acute respiratory failure. RESULTS:From March 13th to April 16th 2020, a total of 92 adult patients (median age: 61 years, 1st-3rd quartiles [55-70]; males: n?=?73/92, 79%; baseline SOFA: 4 [3-7] and SAPS II: 31 [21-40]; invasive mechanical ventilation: n?=?83/92, 90%; ICU mortality: n?=?45/92, 49%) were admitted to our 40-bed ICU for acute respiratory failure due to SARS-CoV-2 pneumonia. Among them, 26 (28%) were considered as co-infected with a pathogenic bacterium at ICU admission with no co-infection related to atypical bacteria or viruses. The distribution of the 32 bacteria isolated from culture and/or respiratory PCRs was as follows: methicillin-sensitive Staphylococcus aureus (n?=?10/32, 31%), Haemophilus influenzae (n?=?7/32, 22%), Streptococcus pneumoniae (n?=?6/32, 19%), Enterobacteriaceae (n?=?5/32, 16%), Pseudomonas aeruginosa (n?=?2/32, 6%), Moraxella catarrhalis (n?=?1/32, 3%) and Acinetobacter baumannii (n?=?1/32, 3%). Among the 24 pathogenic bacteria isolated from culture, 2 (8%) and 5 (21%) were resistant to 3rd generation cephalosporin and to amoxicillin-clavulanate combination, respectively. CONCLUSIONS:We report on a 28% rate of bacterial co-infection at ICU admission of patients with severe SARSCoV-2 pneumonia, mostly related to Staphylococcus aureus, Haemophilus influenzae, Streptococcus pneumoniae and Enterobacteriaceae. In French patients with confirmed severe SARSCoV-2 pneumonia requiring ICU admission, our results encourage the systematic administration of an empiric antibiotic monotherapy with a 3rd generation cephalosporin, with a prompt de-escalation as soon as possible. Further larger studies are needed to assess the real prevalence and the predictors of co-infection together with its prognostic impact on critically ill patients with severe SARS-CoV-2 pneumonia.
Project description:Although sex differences in asthma severity are recognized, the mechanisms by which sex steroids such as estrogen influence the airway are still under investigation. Airway tone, a key aspect of asthma, represents a balance between bronchoconstriction and dilation. Nitric oxide (NO) from the bronchial epithelium is an endogenous bronchodilator. We hypothesized that estrogens facilitate bronchodilation by generating NO in bronchial epithelium. In acutely dissociated human bronchial epithelial cells from female patients exposure to 17?-estradiol (E(2); 10 pM-100 nM) resulted in rapid increase of diaminofluorescein fluorescence (NO indicator) within minutes, comparable with that induced by ATP (20 ?M). Estrogen receptor (ER) isoform-specific agonists (R,R)-5,11-diethyl-5,6,11,12-tetrahydro-2,8-chrysenediol (THC) (ER?) and diaryl-propionitrile (DPN) (ER?) stimulated NO production to comparable levels and at comparable rates, whereas the ER antagonist 7?,17?-[9-[(4,4,5,5,5-pentafluoropentyl)sulfinyl]nonyl]estra-1,3,5(10)-triene-3,17-diol (ICI 182,780) (1 ?M) was inhibitory. Estrogen effects on NO were mediated via caveolin-1 (blocked using the caveolin-1 scaffolding domain peptide) and by increased intracellular calcium concentration [prevented by 20 ?M 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester but not by blocking Ca(2+) influx using LaCl(3)]. Estrogen increased endothelial NO synthase activation (inhibited by 100 ?M N(G)-nitro-l-arginine methyl ester) and phosphorylated Akt. In epithelium-intact human bronchial rings contracted with acetylcholine (1 ?M), E(2), THC, and DPN all produced acute bronchodilation in a dose-dependent fashion. Such bronchodilatory effects were substantially reduced by epithelial denudation. Overall, these data indicate that estrogens, acting via ER? or ER?, can acutely produce NO in airway epithelium (akin to vascular endothelium). Estrogen-induced NO and its impairment may contribute to altered bronchodilation in women with asthma.