Impact of elexacaftor/tezacaftor/ivacaftor on Pseudomonas aeruginosa acquisition and chronic infection at a single pediatric cystic fibrosis center

Background: As cystic fibrosis (CF) lung disease progresses, the airways become colonized with opportunistic pathogens such as Pseudomonas aeruginosa secondary to airway surface liquid depletion. Acquisition of P. aeruginosa is associated with decline in lung function and increase in treatment burden and mortality. In October 2019, the Food and Drug Administration approved elexacaftor/tezacaftor/ivacaftor (ELX/TEZ/IVA), a highly effective modulator therapy (HEMT) for individuals aged 12 and older with one copy of the F508del CFTR mutation. ELX/TEZ/IVA increases the amount of and function of CF transmembrane conductance regulator (CFTR) in the respiratory epithelium, increasing mucociliary clearance (MCC) and reducing static airway mucous, a major trigger for chronic infection and inflammation. Method(s): A retrospective analysis of inhaled tobramycin (iTOB) prescriptions prescribed between January 1, 2016, and December 31, 2021, was performed. This captured data before and after ELX/TEZ/IVA approval at Children's Mercy Kansas City (CMKC). The number of individuals with new P. aeruginosa acquisition was determined by identifying electronic prescriptions for iTOB eradication courses. An eradication course was defined as a first lifetime prescription for iTOB or a new prescription for iTOB submitted at least 1 year after a previous prescription. The number of individuals considered chronically infected with P. aeruginosa was determined by identifying individuals receiving chronic iTOB prescriptions and confirmed by respiratory cultures indicating chronic infection based on the Leeds criteria (P. aeruginosa recovered in >=50% of airway cultures in the previous 12 months). Result(s): Eradication courseswere prescribed to 34 individuals in 2016 (15% of people receiving care at CMKC). The number of eradication prescriptions declined in 2020 and 2021, with only 15 (7%) individuals prescribed eradication therapy in 2020 and 12 (5%) in 2021. A similar pattern was observed for prescriptions for chronic infection. In 2016, 57 individuals (25% of our patient population) were receiving iTOB for chronic P. aeruginosa infection. Reductions were seen in 2020 and 2021, with 28 (13%) and 20 (9%) individuals prescribed chronic therapy, respectively. The number of individuals prescribed iTOB for P. aeruginosa eradication and chronic infection per year is represented in Figure 1.(Figure Presented)Conclusions: CMKC experienced a decrease in the number of courses of iTOB prescribed over the last 6 years. HEMT use is associated with greater MCC and anti-inflammatory effects affecting the airway microbiome. The decrease in respiratory cultures growing P. aeruginosa likely reflects these phenomena. A confounding factor is the SARS-CoV-2 pandemic and widespread use of HEMT. Clinic closures and implementation of telemedicine limited in-person patient visits during 2020 and 2021. Despite limited in-person visits, the average number of respiratory cultures per individual at CMKC in 2020 was 3.5, which is consistent with previous years.Wewere able to obtain frequent surveillance cultures through implementation of a drive-through respiratory specimen collection process. Hence, the decrease in number of iTOB courses cannot be attributed to a decrease in frequency of respiratory cultures, although we cannot assess the impact of school closures and a decrease in social gatherings on new P. aeruginosa acquisition or chronic infection. Looking at all these variables, the widespread use of HEMT likely played a significant role in reducing new P. aeruginosa acquisition and chronic P. aeruginosa infection.Copyright © 2022, European Cystic Fibrosis Society. All rights reserved

Investigating neutrophil function during SARS-CoV2 infection in human airway epithelial cells from elderly and young individuals

The COVID-19 pandemic caused by the SARS-CoV2 virus poses a global health threat with over 5 million deaths recorded. There is little understanding regarding SARS-CoV2 pathogenesis in the human airways and disease severity increases with age. Neutrophils are white blood cells found in large numbers in the airways of the lungs in severe COVID-19 patients. It is not known whether this influx of neutrophils into the airway has a protective or detrimental effect. We aim to understand the role of neutrophils during COVID-19 pathology, using an experimental infection model of the airway epithelium from the eldelry and children. To do this, we collect nasal airway cells from healthy elderly and children and grow them at air-liquid interface. Once differentiation and ciliation of these cells is reached, we infect the cells with SARS-CoV2 virus and allow neutrophils to migrate from the basolateral (blood) to the apical (air) side of the epithelium, similar to a physiological airway. Using flow cytometric analyses, we measure the expression of activation markers and the number of neutrophils that migrate across the epithelium of different ages in response to SARS-CoV2 infection. Preliminary work shows less viable neutrophils recovered from the elderly epithelium, more activated neutrophils when migrating through the elderly epithelium, as well as increased numbers of neutrophils remaining on the basolateral (blood) side of the elderly epithelium. These findings point to an inflammatory neutrophil phenotype influenced by the damaged elderly epithelium and supports the hypothesis that neutrophils are responsible for the severity of disease.

Role of vitamin D in modulating the immune response to SARS-CoV-2 and other coronavirus infections.

In recent years, new data have been obtained on the significant prevalence of vitamin D (VD) deficiency in the population, and knowledge about the role of vitamin D in the regulation of many physiological processes in the body, including the functioning of the immune system, has increased. The SARS-CoV-2 pandemic has further highlighted the issue of an adequate immune response in vitamin D deficiency. Objective of the review. To present and summarize the evidence on the role of VD in different parts of the immune response in COVID-19, to analyze available studies of the VD status effect on the course and outcome of COVID-19 in patients from different population groups. Material and methods. A search of domestic and foreign literature on the role of VD in the immune response in respiratory viral infections and SARS-CoV-2, as well as practical measures of VD-status correction in COVID-19, was performed. We used Scopus, Web of Science, PubMed, Google Scholar, eLibrary, and Cyberleninka databases. Results. Numerous clinical and observational studies have found an association between 25-hydroxyvitamin D levels, COVID-19 severity, and mortality. This association can be explained by the multifaceted role of vitamin D in the physiology of the human immune and endocrine systems. On the immunological side, the active form of VD promotes the secretion of antimicrobial peptides responsible for inhibiting viral replication and stimulates autophagy by increasing the level of Beclin1 protein and decreasing the level of mTOR protein regulating cellular homeostasis. It leads to the presentation of antigens followed by activation of the antiviral pathway of type I interferons. VD also stabilizes intercellular junctions, including those in the airway epithelium, reducing their permeability to pathogens, stimulates the activity of angiotensin-converting enzyme-2, whose receptors are a conduit for SARS-CoV-2 into cells, and several pathophysiological responses associated with the disease symptoms and acute lung injury. Adequate vitamin D status can provide significant benefits during the pandemic. Conclusion. To date, ideas about the role of vitamin D in regulating the immune response in respiratory infections have significantly expanded. However, its use in the complex preventive measures and adjuvant therapy of viral infections, including COVID-19, should be the subject of further scientific research.Copyright © 2023, Media Sphera Publishing Group. All rights reserved.

Simultaneous Primary Sinonasal Adenocarcinoma and Breast Adenocarcinoma: A Case Report

Sinonasal cancer accounts for roughly only 3% of upper respiratory tract malignancies and generally presents as a primary malignancy. Although extremely rare, the sinonasal cavity is also a known location for metastasis, with 8% of these cases originating from primary breast cancer. When attempting to differentiate primary disease from metastasis, immunohistochemical analyses play a crucial role in reaching the correct diagnosis. To date, there are a handful of reports describing metastasis involving the paranasal sinuses but even fewer reporting primary sinonasal cancer with coexisting primary malignancy. Here we present a case of primary sinonasal adenocarcinoma in the setting of a long-standing history of breast cancer. The patient, a 73-year-old female, was diagnosed with T1cN1aM0, progesterone receptor positive and estrogen receptor negative ductal carcinoma in situ of the left breast in November 2019. She subsequently underwent bilateral mastectomy and treated with 3 cycles of chemotherapy and anastrozole, which were both discontinued due to intolerance. Of note, in March 2019, MRI of the head incidentally found a 3 x 2 cm mass in right nasal cavity extending into ethmoid sinus. One year later, she presented with mild right sided nasal obstruction and drainage, and biopsy revealed squamous and respiratory mucosa with chronic inflammation. The patient elected to cancel initial surgical resection of the mass due to the COVID-19 pandemic. The patient returned in March 2022 with complaints of eye pressure, double vision, headaches, and worsening nasal obstruction. PET/CT scan was negative for distant metastasis but demonstrated increased uptake in sinus cavity. MRI showed a larger 5 x 3.7 cm mass impressing on medial inferior margins of orbit. Imaging also suggested evidence of dehiscence of lamina and irregular neo-osteogenesis of the skull base. She underwent approach and resection of the mass with histology demonstrating a well differentiated, low grade non-intestinal mucinous adenocarcinoma. Immunohistochemistry was positive for pankeratin and CK7, favoring a primary sinonasal origin. It was estrogen receptor negative and negative for GATA3, a sensitive and fairly specific stain in mammary carcinoma. Adjuvant radiation was recommended postoperatively, however the patient declined this therapy. This case highlights the role of immunohistochemistry to discriminate a new primary cancer from metastasis in patients with a history of breast cancer. Clinically, patients with sinonasal metastasis can present with symptoms ranging from unilateral nasal obstruction, facial pain, diplopia, and decreased vision. On imaging, suspicion of malignancy is raised when there is evidence of destruction of bony boundaries and invasion of surrounding tissues such as the orbit and anterior skull base, as found in our patient. Notably, metastasis to the paranasal sinuses can mimic a primary cancer of the nasal cavity, with both tumors showing epithelial differentiation. However, primary tumors often show neoplastic changes in the overlying respiratory epithelium and do not express estrogen receptor, progesterone receptor, or HER2 positivity, which are known to be correlated with breast cancer. In this setting, GATA3 and estrogen receptor negativity allowed us to diagnose primary nasal cancer more confidently. Therefore, clinicians should be aware of metastatic disease and expand immunohistochemistry panels when appropriate.

The effects of acute hydrogen peroxide exposure on respiratory cilia motility and viability.

COVID-19 has seen the propagation of alternative remedies to treat respiratory disease, such as nebulization of hydrogen peroxide (H2O2). As H2O2 has known cytotoxicity, it was hypothesised that H2O2 inhalation would negatively impact respiratory cilia function. To test this hypothesis, mouse tracheal samples were incubated with different H2O2 concentrations (0.1-1%) then cilia motility, cilia generated flow, and cell death was assessed 0-120 min following H2O2 treatment. 0.1-0.2% H2O2 caused immediate depression of cilia motility and complete cessation of cilia generated flow. Higher H2O2 concentrations (≥0.5%) caused immediate complete cessation of cilia motility and cilia generated flow. Cilia motility and flow was restored 30 min after 0.1% H2O2 treatment. Cilia motility and flow remained depressed 120 min after 0.2-0.5% H2O2 treatment. No recovery was seen 120 min after treatment with ≥1% H2O2. Live/dead staining revealed that H2O2 treatment caused preferential cell death of ciliated respiratory epithelia over non-ciliated epithelia, with 1% H2O2 causing 35.3 ± 7.0% of the ciliated epithelia cells to die 120 min following initial treatment. This study shows that H2O2 treatment significantly impacts respiratory cilia motility and cilia generated flow, characterised by a significant impairment in cilia motility even at low concentrations, the complete cessation of cilia motility at higher doses, and a significant cytotoxic effect on ciliated respiratory epithelial cells by promoting cell death. While this data needs further study using in vivo models, it suggests that extreme care should be taken when considering treating respiratory diseases with nebulised H2O2.

Role of vitamin D in modulating the immune response to SARS-CoV-2 and other coronavirus infections

In recent years, new data have been obtained on the significant prevalence of vitamin D (VD) deficiency in the population, and knowledge about the role of vitamin D in the regulation of many physiological processes in the body, including the functioning of the immune system, has increased. The SARS-CoV-2 pandemic has further highlighted the issue of an adequate immune response in vitamin D deficiency. Objective of the review. To present and summarize the evidence on the role of VD in different parts of the immune response in COVID-19, to analyze available studies of the VD status effect on the course and outcome of COVID-19 in patients from different population groups. Material and methods. A search of domestic and foreign literature on the role of VD in the immune response in respiratory viral infections and SARS-CoV-2, as well as practical measures of VD-status correction in COVID-19, was performed. We used Scopus, Web of Science, PubMed, Google Scholar, eLibrary, and Cyberleninka databases. Results. Numerous clinical and observational studies have found an association between 25-hydroxyvitamin D levels, COVID-19 severity, and mortality. This association can be explained by the multifaceted role of vitamin D in the physiology of the human immune and endocrine systems. On the immunological side, the active form of VD promotes the secretion of antimicrobial peptides responsible for inhibiting viral replication and stimulates autophagy by increasing the level of Beclin1 protein and decreasing the level of mTOR protein regulating cellular homeostasis. It leads to the presentation of antigens followed by activation of the antiviral pathway of type I interferons. VD also stabilizes intercellular junctions, including those in the airway epithelium, reducing their permeability to pathogens, stimulates the activity of angiotensin-converting enzyme-2, whose receptors are a conduit for SARS-CoV-2 into cells, and several pathophysiological responses associated with the disease symptoms and acute lung injury. Adequate vitamin D status can provide significant benefits during the pandemic. Conclusion. To date, ideas about the role of vitamin D in regulating the immune response in respiratory infections have significantly expanded. However, its use in the complex preventive measures and adjuvant therapy of viral infections, including COVID-19, should be the subject of further scientific research.

Covid-19: All facts, no myth

On December 31, 2019, the China Health Authority alerted WHO about 27 cases of pneumonia of unknown etiology in Wuhan City. It was subsequently named Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) and the disease as Coronavirus Disease 2019 (COVID-19). The disease has now become pandemic. Current review was done to summarize information on COVID-19 published in various scientific works. Electronic databases containing medical articles viz., MEDLINE/PubMed, Google Scholar etc were searched using the Medical Subject Headings 'COVID-19', '2019-nCoV', 'coronavirus' and 'SARS-CoV-2' during antecedent one year. All study designs were incorporated to harvest clinical, laboratory, imaging, and hospital course data. The intermediate host of the virus is still unknown. Respiratory droplets produced by the patient is main source of transmission. SARS-CoV-2 invades the airway epithelium by binding to angiotensin-converting enzyme-2 (ACE2) receptor with Coronavirus spike (S) protein. Most common symptoms are fever (98%), dry cough (77%), and dyspnea (63.5%). Later, complications like acute respiratory distress syndrome, septic shock etc may occur. Advanced age and co-morbidities like Diabetes have higher mortality otherwise Case Fatality Rate is 2-3%. RT-PCR is the diagnosis of choice. Since no universally accepted registered drug or FDA approved vaccine has come by now, prevention is the key. Hands should be regularly cleaned with soap or alcohol based sanitizer and in public, Nose and Mouth should be covered with face-mask and social distance of one meter should be maintained. While Vaccines are expected by early 2021, we should not forget to take comprehensive measures to prevent future outbreaks of zoonotic origin. Copyright © 2020, Kathmandu University. All rights reserved.

EXTREMELY RARE CASE OF CARTILAGINOUS TUMOR IN THE POSTERIOR WALL OF THE TRACHEA

SESSION TITLE: Lung Cancer Case Report Posters 3 SESSION TYPE: Case Report Posters PRESENTED ON: 10/17/2022 12:15 pm - 01:15 pm INTRODUCTION: Tracheal tumor accounts for 0.4% of all tumors and only 10% of them are benign (1). We present, to our knowledge, the first case of a primary benign tracheal tumor with features of chondroid metaplasia arising from the posterior wall of the trachea. CASE PRESENTATION: 58-year-old male non—smoker with non-significant past medical history, presented to the Emergency department for COVID-19 pneumonia. CTA chest was done showing bilateral pulmonary embolism and a 12 mm polypoid tracheal mass arising from the posterior wall of the trachea extending into the lumen (Figure#1). The patient was asymptomatic prior to his COVID 19 infection;he denied any chest pain, hemoptysis, trauma, or prior intubation. After recovering from COVID-19, the patient was scheduled for an outpatient rigid bronchoscopy which revealed a tracheal polyp arising from the mid-distal posterior membranous trachea. (Figure#2). An electrocautery snare was used to simultaneously cut and cauterize the stalk using a lasso technique. The polyp was removed in its entirety without complication. Histopathology examination demonstrated a respiratory epithelium lined cyst with cartilaginous tissue, favoring chondroid metaplasia. DISCUSSION: Primary benign tracheal tumors with cartilaginous features are uncommon, especially in the posterior membrane of the trachea, which lacks cartilaginous support. Diagnosis of any benign tracheal tumor is usually delayed since most patients are asymptomatic. The majority of such tumors are found incidentally, as in this case. One of the most common benign tracheal tumors is hamartoma, which can have respiratory epithelium and cartilaginous tissue, however they do not have features of chondroid metaplasia, and are generally found in the lateral or anterior wall of the trachea. Furthermore, endobronchial lesions only account for 3% of all pulmonary hamartomas. (2) Reports of airway chondroid metaplasia are usually described in the larynx and are commonly associated with prior trauma or inflammation in the area which is not known to have occurred in this case (3). The histopathologic findings and unusual location of this tumor makes this case unique. CONCLUSIONS: The tracheal origin of this benign tumor, arising from the posterior membrane with cartilaginous features is extremely rare, and has not previously been described in the literature. Reference #1: Park CM, Goo JM, Lee HJ, Kim MA, Lee CH, Kang MJ. Tumors in the tracheobronchial tree: CT and FDG PET features. Radiographics. 2009 Jan-Feb;29(1):55-71. doi: 10.1148/rg.291085126. PMID: 19168836. Reference #2: Hurst IJ Jr, Nelson KG. Tracheal hamartoma. Chest. 1977 Nov;72(5):661-2. doi: 10.1378/chest.72.5.661. PMID: 913152. Reference #3: Orlandi A, Fratoni S, Hermann I, Spagnoli LG. Symptomatic laryngeal nodular chondrometaplasia: a clinicopathological study. J Clin Pathol. 2003 Dec;56(12):976-7. doi: 10.1136/jcp.56.12.976. PMID: 14645364;PMCID: PMC1770148. DISCLOSURES: No relevant relationships by Jorge Cedano Consultant relationship with Olympus America Please note: 8/1/21-present Added 04/18/2022 by Lucas Pitts, value=Consulting fee

THE PANDEMIC'S FUNGAL REVENGE: COVID-19 ASSOCIATED PULMONARY ASPERGILLOSIS (CAPA)

SESSION TITLE: Pathologies of the Post-COVID-19 World SESSION TYPE: Rapid Fire Case Reports PRESENTED ON: 10/18/2022 10:15 am - 11:10 am INTRODUCTION: Since the start of the COVID-19 pandemic, COVID-19 Associated Pulmonary Aspergillosis (CAPA) has been on the rise. This superinfection, if not properly identified and treated, has shown to increase mortality up to 67% in COVID-19 patients. We are presenting a late presentation of CAPA after 4-month of COVID-19 infection and treated successfully. CASE PRESENTATION: A 57-year-old female patient with past medical history type 2 diabetes mellitus, hypertension and cardiomyopathy in addition to COVID-19 pneumonia treated for months ago with azithromycin, Bamlanivimab/Etesevimab, and Dexamethasone who presents to the hospital with massive hemoptysis and shortness of breath requiring intubation and mechanical ventilation. There was no reported history of recent travel, smoking, alcohol, or illicit drug use. Physical exam showed diminished lung sounds at the right lower lobe. Her labs showed mild leukocytosis, lactic acidosis and negative COVID-19 PCR. CT scan showed dense consolidation on right lower lobe consistent with lobar pneumonia and centrilobular ground glass opacities in the right upper lobe. Bronchoscopy showed complete obstruction of right bronchus intermedius and minimal blood clots in LLL. BAL respiratory culture, fungal smear, acid fast bacilli were non-diagnostic and negative for malignancy. Patient continued to have hemoptysis and bronchoscopy was repeated with negative cytology and cultures. The patient continued to have hemoptysis and she was transferred to tertiary center were bronchoscopy was repeated and confirmed right bronchus intermedius stenosis, blood clots, and suspicious right mainstem nodules with mucosal lesion. Biopsy results from bronchoscopy came back positive for the morphologic features of Aspergillus species. The patient was started on voriconazole with significant improvement in her symptoms. DISCUSSION: The recent literature of COVID-19 suggest association between COVID infection and invasive pulmonary Aspergillosis. COVID-19 virus causes damage in the airway epithelium and enable aspergillus to invade the pulmonary tract leading to serious infections with Aspergillus. It has also been known that Aspergillus infections are associated with diabetes mellitus and immune suppression which can be precipitated by steroid use and other treatments for COVID-19 infection like IL-6 inhibitors. Here in our patient with help of tissue biopsy we diagnosed CAPA, started treatment early and treated successfully. CONCLUSIONS: CAPA can be difficult to diagnose and needs high index of suspicion in the appropriate clinical scenario when dealing with post COVID respiratory complaints like hemoptysis. Bronchoalveolar lavage alone without tissue biopsy might miss the diagnosis in the context of invasive aspergillosis like the scenario we observed in our case. Doing tissue biopsy through bronchoscopy might add more clinical benefit when Aspergillus infections are suspected. Reference #1: Chih-Cheng Lai, Weng-Liang Yu, COVID-19 associated with pulmonary aspergillosis: A literature review, https://doi.org/10.1016/j.jmii.2020.09.004 DISCLOSURES: No relevant relationships by Haytham Adada No relevant relationships by Mahmoud Amarna No relevant relationships by Rishika Bajaj No relevant relationships by Camelia Chirculescu No relevant relationships by Sonia Dogra No relevant relationships by Azad Patel

Abstracts of the Annual Meeting of French Society of Pharmacology and Therapeutics

The proceedings contain 286 papers. The topics discussed include: is there still a place for methotrexate in severe psoriatic arthritis?;Improving availability of naloxone: an emergency;national pharmacovigilance surveillance of Astrazeneca Covid-19 vaccine (ChAdOx1-S vaccine);recommendations of the French Society of Rheumatology and the French Society of Physical Medicine and Rehabilitation on the non-pharmacological management of knee osteoarthritis;management of drug?drug interactions with nirmatrelvir/ritonavir in patients treated for Covid-19: guidelines from the French Society of Pharmacology and Therapeutics (SFPT);adverse events associated with JAK inhibitors;impact of the new CFTR modulators treatment on the respiratory epithelium;pharmacokinetics study showed increased brain delivery of anti-PD-1 after ultrasound-mediated blood-brain barrier in glioblastoma mouse models;and modulation of HCN channel activity in oxaliplatin-induced peripheral neuropathy.

Hypercapnia Increases ACE2 Protein Expression and Pseudo-SARSCoV- 2 Virus Entry in Airway Epithelial Cells by Augmenting Cellular Cholesterol

Rationale: Individuals with COPD who develop COVID-19 are at increased risk of hospitalization, ICU admission and death. COPD is associated with increased airway epithelial expression of ACE2, the receptor mediating SARS-CoV-2 entry into cells. Hypercapnia commonly develops in advanced COPD and is associated with frequent and potentially fatal pulmonary infections. We previously reported that hypercapnia increases viral replication, lung injury and mortality in mice infected with influenza A virus. Also, global gene expression profiling of primary human bronchial epithelial (HBE) cells showed that elevated CO2 upregulates expression of cholesterol biosynthesis genes, including HMGCS1, and downregulates ATP-binding cassette (ABC) transporters that promote cholesterol efflux. Given that cellular cholesterol is important for entry of viruses into cells, in the current study we assessed the impact of hypercapnia on regulation of cellular cholesterol levels, and resultant effects on expression of ACE2 and entry of Pseudo-SARS-CoV-2 in cultured HBE, BEAS-2B and VERO cells, and airway epithelium of mice. Methods: Differentiated HBE, BEAS-2B or VERO cells were pre-incubated in normocapnia (5% CO2, PCO2 36 mmHg) or hypercapnia (15% CO2, PCO2 108 mmHg), both with normoxia, for 4 days. Expression of ACE2 and sterol regulatory element binding protein 2 (SREPB2), the master regulator of cholesterol synthesis, was assessed by immunoblot or immunofluorescence. Cholesterol was measured in cell lysates by Amplex red assay. Cells cultured in normocapnia or hypercapnia were also infected with Pseudo SARS-CoV-2, a Neon Green reporter baculovirus. For in vivo studies, C57BL/6 mice were exposed to normoxic hypercapnia (10% CO2/21% O2) for 7 days, or air as control, and airway epithelial expression of ACE2, SREBP2, ABCA1, ABCG1 and HMGCS1 was assessed by immunofluorescence. SREBP2 was blocked using the small molecules betulin or AM580, and cellular cholesterol was disrupted using MβCD. Results: Hypercapnia increased expression and activation of SREBP2 and decreased expression of ABC transporters, thereby augmenting epithelial cholesterol levels. Elevated CO2 also augmented ACE2 expression and Pseudo-SARSCoV- 2 entry into epithelial cells in vitro and in vivo. These effects were all reversed by blocking SREBP2 or disrupting cellular cholesterol. Conclusion: Hypercapnia augments cellular cholesterol levels by altering expression of cholesterol biosynthetic enzymes and efflux transporters, leading to increased epithelial expression of ACE2 and entry of Pseudo-SARS-CoV-2 into cells. These findings suggest that ventilatory support to limit hypercapnia or pharmacologic interventions to decrease cellular cholesterol might reduce viral burden and improve clinical outcomes of SARSCoV- 2 infection in advanced COPD and other severe lung diseases.

Induced Pluripotent Stem Cell Derived Airway to Assess Cystic Fibrosis Intrinsic Epithelial Response to SARS-CoV-2

Rationale There is a lack of knowledge of how CFTR-deficient airway epithelium intrinsically responds to SARS-CoV-2. Though prior work has demonstrated altered CF airway expression of viral entry factors, it is unknown whether these alterations are protective and whether they reflect host genetic variation or secondary response of chronic inflammation. We address this gap by infecting induced pluripotent stem cell (iPSC)-derived airways from CF patients and syngeneic CFTR-corrected controls with SARS-CoV-2 and assessing differential susceptibility to infection and inflammatory and anti-viral response. MethodsCF (F508del homozygous) and syngeneic CFTR-corrected (CRISPR-Cas9) iPSC- were differentiated into airway epithelium cultured at airliquid interface (ALI) by a directed differentiation protocol that generates a pure population of major and rare airway cell-types. After 21 days in ALI culture, the iPSC-airway were infected with either mock or SARS-CoV-2 (isolate USA-WA1/2020) with MOI of 4, and harvested at 0, 1, 3 days post infection (dpi) for RT-PCR and immune-stainingResultsBoth CF and CFTR-corrected iPSC-airway express viral entry factors of ACE2 and TMPRSS2, and are permissive to SARS-CoV-2 infection. CF iPSC-airway exhibited significantly increase in SARS-CoV-2 nucleocapsid protein (N) transcript at 1 dpi, accompanied by increases in IFN2, RSAD2, and CXCL10 at 3 dpi, compared to its CFTR-corrected counter-part. There are no baseline significant differences in ACE2, TMPRSS2, TP63, NGFR, MUC5B, MUC5AC, SCGB1A1, FOXJ1, FOXI1 expression between CF and CFTR-corrected iPSC-airway before SARS-CoV-2 infection. ConclusionsOur preliminary studies indicate increased early SARS-CoV-2 infection in CFTR-deficient epithelium with accompanied subsequent rise in anti-viral and inflammatory response compared to its genetically controlled CFTR-corrected counterpart. Future studies are aimed at assessing differential CF epithelial kinetics of SARS-CoV-2 viral entry and replication, morphological changes, global transcriptomic response, and how treatment with CFTRmodulator would alter the epithelial response. Ultimately, we aim to establish a reductionist, physiologically relevant model system that is coupled with gene-editing technology to study intrinsic CF epithelial response to SARS-CoV-2, which would generate insights to aid practice guidelines for CF patients, and open future directions to evaluate gene-specific mechanisms of airway response to pathogens. (Figure Presented).

IL-25 Inhibits Airway Anti-Viral Immunity and Promotes Virus Exacerbation of Allergic Airways Disease

Rationale We have previously reported blocking the IL-25 receptor (IL-17RB) prevented viral increased allergic airways inflammation and this was associated with reduced lung viral load. To investigate IL-25 regulation of airway anti-viral immunity we hypothesised that IL-25 directly inhibits airway epithelial cell (AEC) type I/III interferon expression and antibody blockade of IL-25 in vivo boosts lung interferon expression and reduces lung viral load in parallel with reduced type 2 airway inflammation. Methods In vitro Immunofluorescence was used to visualise epithelial IL-25 and IL- 17RB proteins in endobronchial biopsies from patients with asthma and healthy subjects and in AEC differentiated at ALI. AEC from n = 14 donors with asthma were differentiated at the air-liquid interface (ALI) and infected with RV-A1, MOI=0.1. A subset of AECs was treated with anti-IL-25 mAb (LNR125) before infecting with RV-A1 or human coronavirus 229E. Differentiated AEC from healthy donors were treated with recombinant IL-25 protein and infected with RV-A1. Nanostring immune transcriptomic data expressed as digital mRNA counts for exact copy number or was expressed as log2 fold change ratio against -log10 Bejamini-Yekutieli-corrected p-values. In vivo 6- 8-week-old, BALB/c mice sensitised and intranasally challenged daily for 3 days with ovalbumin to induced allergic airways disease. A single subcutaneous injection of 250 μg LNR125 was administered during ovalbumin challenge. Mice were then infected i.n. with RV-A1, 6 hours after final allergen challenge. On day 1 and day 7 post-infection, BAL were collected, lung lobe tissue was collected for viral RNA and cytokine expression. Results IL-25 and IL-17RB were constitutively expressed at the apical surface of airway epithelium in biopsies and AEC cultures. RV infection increased IL-25 expression by AEC from asthmatic donors. LNR125 treatment reduced IL-25 mRNA and significantly increased RV induced IFN-β a and IFN-λ protein expression and this was confirmed by Nanostring transcriptomic analyses which also identified down-regulated type-2 immune genes CCL26 (eotaxin 3) and IL1RL1(IL-33 receptor). LN125 treatment also increased IFN-λ expression by 229E-infected differentiated AECs. IL-25 treatment increased viral load associated with 50% reduced expression of IFN-β and CXCL10 and 75% reduced IFN-λ. Allergen challenged, RV-infected mice treated with LNR125 had significantly increased BAL IFN-β protein and 60% reduction in lung viral load associated with reduced IL-25, IL-4, IL-5 and IL-13 BAL proteins compared to controls. Conclusion IL-25-induced inflammation combined with suppression of AEC anti-viral immunity identify IL-25 as a central mediator of viral asthma exacerbations and therefore a target for mAb-based treatment.

Rational development of tools to appropriately respond to SARSCoV- 2 needs

For many years, our laboratory has been developing cellular models for the study of human pathogenic viruses with RNA genomes, in order to study the replication of these pathogens, to propose new therapeutic pathways, to screen and test inhibitors. In response to the COVID-19 outbreak, we have set up the tools for the study of SARS-CoV-2 replication. First, clinical and reference SARS-CoV-2 strains have been successfully isolated and amplified using Vero E6 cells in the BSL3 facility of Bordeaux University (UB'L3, www.mfp.cnrs.fr/wp/larecherche/ andevir/ubl3/). We set up the monitoring of SARS-CoV-2 replication using conventional RT-qPCR quantification as well as evaluation of the cytopathic effect by microscopic observation or content analysis. Using VERO cells, we are now able to precisely titer viral supernatant (determination of the TCID50) and screen for potential antiviral molecule (determination of EC50 and CC50). We have developed a full-length Spike sequencing based on a Sanger approach1 as well as whole genome sequencing by nanopore technology, allowing the tracking of emerging variants. In parallel, we developed various other models to study SARS-CoV-2 replication including Calu-3 cells, modified human cells expressing Ace2 (e.g. 293T, U2OS) or even more complex cellular models (reconstituted human airway epithelium, vessels) according to the biological question to resolve. As an example, bronchial epithelia reconstituted from biopsies of adult or child donors were used to evaluate the inflammatory response upon SARS-CoV-2 infection in an age-dependent manner [2] (see poster G. Beucher). Similarly reconstituted blood vessels were used to study the impact of SARS-CoV- 2 infection on the vascular system and determine whether clinical observations (blood brain barrier damages, myocarditis) are due to direct infection of cells or indirect effects. Finally, we evaluate the efficacy of different chemical or physical processes for viral inactivation in air or on surfaces.

Atmospheric Reactive Oxygen Species and Some Aspects of the Antiviral Protection at the Respiratory Epithelium.

The review summarizes literature data on molecular and biochemical mechanisms of nonspecific protection of respiratory epithelium. The special attention is paid to comprehensive analysis of up-to-date data on the activity of the lactoperoxidase system expressed on the surface of the respiratory epithelium which provides the generation of hypothiocyanate and hypoiodite in the presence of locally produced or inhaled hydrogen peroxide. Molecular mechanisms of production of active compounds with antiviral and antibacterial effects, expression profiles of enzymes, transporters and ion channels involved in the generation of hypothiocyanite and hypoiodite in the mucous membrane of the respiratory system in physiological and pathological conditions (inflammation) are discussed. A hypothesis about the effect of atmospheric air composition on the efficiency of hypothiocyanate and hypoiodite generation in the respiratory epithelium in the context of its antibacterial and antiviral protection is presented. The causes and consequences of insufficiency of the lactoperoxidase system caused by the action of atmospheric factors are discussed in the context of controlling the sensitivity of the epithelium to the action of bacterial agents and viruses. Good evidence exists that restoration of the lactoperoxidase system activity can be achieved by application of pharmacological agents aimed to compensate for the deficit of halides in tissues, and by the control of chemical composition of the inhaled air.

COVID-19 associated mixed mold infection: a rare case report of aspergillosis and mucormycosis

Background: Fungal epidemic was announced amid COVID pandemic with several cases of COVID-associated Mucormycosis and Aspergillosis being reported however there is not sufficient data regarding mixed fungal infection. Case Study: A 54-year-old male patient diagnosed with severe COVID 19 pneumonia and diabetes 1-month back presented to OPD with C/O of chest pain and breathlessness for two days associated with haemoptysis, heaviness and congestion of right nostril but no fever. O/E patient was tachypnoeic, hypoxic and in shock, Neutrophil count 87%, RBS-530 mg/dl, urine ketone body was absent. Chest x-ray showed opacity over the left upper and mid-zone, HRCTthorax showed a bird-nest-sign noted in the left upper lobe S/O invasive fungal infection. MRI PNS showed mucosal thickening S/O sinusitis, Fungal infection. Sino-nasal mucosa KHO-mount and fungal culture showed mixed infection of Rhizopus species and aspergillus flavus. Right nasal HP study showed mixed invasive moulds infection. Initially, the patient was treated conservatively later on inj. amphotericin-B was started. The patient's condition worsened on day-18 and succumbed a day later. Discussion: Uncontrolled diabetes-mellitus, and corticosteroids leading to hyperglycaemia, extensive use of broad-spectrum antibiotics increases the risk of invasive Moulds. In our case study, patients suffered from COVIDpneumonitis and had uncontrolled diabetes leading to damage of airway epithelium inviting an invasion of tissues by moulds. Conclusion: Mixed fungal infections as COVID-19 sequelae may be an emerging issue and seen particularly in post- COVID patients with uncontrolled diabetes, and on steroids. The focus should be on prompt management: hit hard approach with both medical and surgical treatment.

The Paradoxical Relationship Between Nicotine and SARS-CoV-2 Infection: A Systematic Review

Across the board, smoking is considered to be negative toward our health. While this information has been known for a relatively long time, the COVID19 pandemic has stirred up a controversial idea: that smokers are protected from severe COVID-19 relative to non-smokers. This suggests that smoking is a helpful agent in the evolving fight against SARS-CoV-2, and impressionable individuals are at risk of starting to smoke as a means of protecting themselves from the virus. To address the validity of this claim, a systematic review was done according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. PubMed was searched for relevant articles and the results were screened according to inclusion criteria. Our search yielded a total of 81 results and after removal of duplicates, non-English papers, and a quality appraisal a total of 16 papers were included in this review. We found that while smokers were more likely present with a less severe disease due to downregulation of severe cytokine storm, they were overall more likely to contract COVID-19 due to upregulation of ACE-2 receptors which SARS-CoV-2 uses to enter the cells of the respiratory epithelium. Also, long time smokers who develop COPD are more likely to have fatal outcomes from COVID-19 infection. Further, these results were due to the effects of nicotine and not cigarettes themselves. Since cigarettes contain numerous carcinogens, they are not recommended as a prophylaxis for COVID-19. However, we recommend that nicotine should be a topic for further research as potential therapy.

SARS-CoV-2 Infection Dysregulates Cilia and Basal Cell Homeostasis in the Respiratory Epithelium of Hamsters.

Similar to many other respiratory viruses, SARS-CoV-2 targets the ciliated cells of the respiratory epithelium and compromises mucociliary clearance, thereby facilitating spread to the lungs and paving the way for secondary infections. A detailed understanding of mechanism involved in ciliary loss and subsequent regeneration is crucial to assess the possible long-term consequences of COVID-19. The aim of this study was to characterize the sequence of histological and ultrastructural changes observed in the ciliated epithelium during and after SARS-CoV-2 infection in the golden Syrian hamster model. We show that acute infection induces a severe, transient loss of cilia, which is, at least in part, caused by cilia internalization. Internalized cilia colocalize with membrane invaginations, facilitating virus entry into the cell. Infection also results in a progressive decline in cells expressing the regulator of ciliogenesis FOXJ1, which persists beyond virus clearance and the termination of inflammatory changes. Ciliary loss triggers the mobilization of p73+ and CK14+ basal cells, which ceases after regeneration of the cilia. Although ciliation is restored after two weeks despite the lack of FOXJ1, an increased frequency of cilia with ultrastructural alterations indicative of secondary ciliary dyskinesia is observed. In summary, the work provides new insights into SARS-CoV-2 pathogenesis and expands our understanding of virally induced damage to defense mechanisms in the conducting airways.

Alopecia areata in a patient with coronavirus disease of 2019

Pandemic coronavirus disease (COVID-19) caused by coronavirus 2 (SARS-CoV-2) causes a severe acute respiratory syndrome affecting primarily the airway epithelium. With the increasing involvement of dermatologists in managing this crisis, skin symptoms are receiving more and more attention. In this case, we will describe alopecia areata in a 7-year-old girl associated with COVID-19

Human Organotypic Airway and Lung Organoid Cells of Bronchiolar and Alveolar Differentiation Are Permissive to Infection by Influenza and SARS-CoV-2 Respiratory Virus.

The ongoing coronavirus disease 2019 (COVID-19) pandemic has led to the initiation of unprecedented research efforts to understand the pathogenesis mediated by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). More knowledge is needed regarding the cell type-specific cytopathology and its impact on cellular tropism. Furthermore, the impact of novel SARS-CoV-2 mutations on cellular tropism, alternative routes of entry, the impact of co-infections, and virus replication kinetics along the respiratory tract remains to be explored in improved models. Most applied virology models are not well suited to address the remaining questions, as they do not recapitulate the histoarchitecture and cellular composition of human respiratory tissues. The overall aim of this work was to establish from single biopsy specimens, a human adult stem cell-derived organoid model representing the upper respiratory airways and lungs and explore the applicability of this model to study respiratory virus infection. First, we characterized the organoid model with respect to growth pattern and histoarchitecture, cellular composition, and functional characteristics. Next, in situ expression of viral entry receptors, including influenza virus-relevant sialic acids and SARS-CoV-2 entry receptor ACE2 and TMPRSS2, were confirmed in organoids of bronchiolar and alveolar differentiation. We further showed successful infection by pseudotype influenza A H7N1 and H5N1 virus, and the ability of the model to support viral replication of influenza A H7N1 virus. Finally, successful infection and replication of a clinical isolate of SARS-CoV-2 were confirmed in the organoids by TCID50 assay and immunostaining to detect intracellular SARS-CoV-2 specific nucleocapsid and dsRNA. The prominent syncytia formation in organoid tissues following SARS-CoV-2 infection mimics the findings from infected human tissues in situ. We conclude that the human organotypic model described here may be particularly useful for virology studies to evaluate regional differences in the host response to infection. The model contains the various cell types along the respiratory tract, expresses respiratory virus entry factors, and supports successful infection and replication of influenza virus and SARS-CoV-2. Thus, the model may serve as a relevant and reliable tool in virology and aid in pandemic preparedness, and efficient evaluation of antiviral strategies.