Activation of Inflammasome complex in nasopharyngeal epithelial cells from patients with Coronavirus disease 2019 contributes to inflammatory state and worse disease outcomes.

Pathogenesis of Coronavirus disease 2019 (COVID-19) has been associated with dysregulation of both adaptive and innate immune systems. Hence, we determined the contribution of inflammasome in the nasopharyngeal epithelial cells isolated from COVID-19 subjects to disease pathogenesis and outcomes. Epithelial cells from 150 COVID-19 patients and 150 healthy controls were yielded through nasopharyngeal swab sampling. Patients were categorized into three groups of those with clinical presentations/need hospitalization, with clinical presentations/no need hospitalization and cases without clinical symptoms/no need hospitalization. Finally, the transcriptional amount of inflammasome related genes were assessed in the nasopharyngeal epithelial cells using qPCR. There was a significant upregulation of nod-like receptor (NLR) family pyrin domain containing 1 (NLRP1), nod-like receptor (NLR) family pyrin domain containing 3 (NLRP3), Apoptosis-associated speck-like protein containing a CARD (ASC) and Caspase-1 mRNA expressions in patients compared to controls. NLRP1, NLRP3, ASC and Caspase-1 were upregulated in epithelial cells of patients with clinical symptoms/need hospitalization and cases with clinical symptoms/no need hospitalization when compared to controls. There was a correlation between expression of inflammasome-related genes and clinicopathological features. Abnormal expression of inflammasome-related genes in the nasopharyngeal epithelial cells obtained from COVID-19 patients may be of prognostic value to determine the intensity of the disease's outcomes and requirement for alternative supports in hospitals.

OMICRON SPIKE ENDOWS SARS-CoV-2 WITH ENHANCED INFECTIVITY IN NASAL EPITHELIA

Background: Omicron lineages, including BA.1 and BA.2, emerged following mass COVID-19 vaccination campaigns, displaced previous SARS-CoV-2 variants of concern worldwide, and gave rise to sublineages that continue to spread among humans. Previous research has shown that Omicron lineages exhibit a decreased propensity for lower respiratory tract (lung) infection compared to ancestral SARS-CoV-2, which may explain the decreased pathogenicity associated with Omicron infections. Nonetheless, Omicron lineages exhibit an unprecedented transmissibility in humans, which until now has been solely attributed to escape from vaccine-induced neutralizing antibodies. Method(s): We comprehensively analyzed BA1 and BA2 infection in primary human nasal epithelial cells cultured at the air-liquid interface, which recapitulates the physiological architecture of the nasal epithelium in vivo. Meanwhile we also took advantage of the VSV-based pseudovirus decorated with different Spike variants. Result(s): In primary human nasal epithelial cells cultured at the air-liquid interface, which recapitulates the physiological architecture of the nasal epithelium in vivo, BA.1 and BA.2 exhibited enhanced infectivity relative to ancestral SARS-CoV-2. Using VSV-based pseudovirus decorated with different Spike variants, we found that increased infectivity conferred by Omicron Spike is due to superior attachment and entry into nasal epithelial cells. In contrast to ancestral SARS-CoV-2, invasion of nasal epithelia by Omicron occurred via the cell surface and endosomal routes of entry and was accompanied by elevated induction of type-I interferons, indicative of a robust innate immune response. Furthermore, BA.1 was less sensitive to inhibition by the antiviral state elicited by type-I and type-III interferons, and this was recapitulated by pseudovirus bearing BA.1 and BA.2 Spike proteins. Conclusion(s): Our results suggest that the constellation of Spike mutations unique to Omicron allow for increased adherence to nasal epithelia, flexible usage of virus entry pathways, and interferon resistance. These findings inform our understanding of how Omicron evolved elevated transmissibility between humans despite a decreased propensity to infect the lower respiratory tract. Additionally, the interferon insensitivity of the Omicron Spike-mediated entry process may explain why Omicron lineages lost the capacity to antagonize interferon pathways compared to ancestral SARS-CoV-2.

RETHINKING REMDESIVIR: ENHANCING ANTI-SARS-CoV-2 ACTIVITY OF ORAL LIPID RVn PRODRUGS

Background: SARS-CoV-2 evolution has contributed to successive waves of infections and severely compromised the efficacy of available SARS-CoV-2 monoclonal antibodies. Decaying vaccine-induced immunity, vaccine hesitancy, and limited vaccine protection in older and immunocompromised populations further compromises vaccine efficacy at the population level. Early antiviral treatments, including intravenous remdesivir (RDV), reduce hospitalization and severe disease due to COVID-19. An orally bioavailable RDV analog could facilitate earlier widespread administration to non-hospitalized COVID-19 patients. Method(s): We synthesized monoalkyl glyceryl ether phosphodiesters of GS-441524 (RVn), lysophospholipid analogs which allow for oral bioavailability and stability in plasma. We evaluated the in vivo efficacy of our lead compound, 1-O-octadecyl-2-O-benzyl-sn-glyceryl-3-phospho-RVn (V2043), in an oral treatment model of murine SARS-CoV-2 infection. We then synthesized numerous phospholipid analogs of RVn and determined which modifications enhanced in vitro antiviral activity and selectivity. The most effective compounds against SARS-CoV-2 were then evaluated for antiviral activity against other RNA viruses. Result(s): Oral treatment of SARS-CoV-2 infected BALB/c mice with V2043 (60 mg/kg once daily for 5 days, starting 12 hrs after infection) reduced lung viral load by more than 100-fold versus vehicle at day 2 and to below the LOD at day 5. V2043 inhibited previous and contemporary SARS-CoV-2 Variants of concern to a similar degree, as measured by the half maximal effective concentration (EC50) in a human lung epithelial cell line (Calu-3). Evaluation of multiple RVn analogs with hydrophobic esters at the sn-2 of glycerol revealed that in vitro antiviral activity was improved by the introduction of a 3-fluoro-4-methoxysubstituted benzyl or a 3-or 4-cyano-substituted benzyl. These compounds showed a 2-to 6-fold improvement in antiviral activity compared to analogs having an unsubstituted benzyl, such as V2043, and were more active than RDV. These compounds also showed enhanced antiviral activity against multiple contemporary and emerging RNA viruses. Conclusion(s): Collectively, our data support the development of RVn phospholipid prodrugs as oral antiviral agents for prevention and treatment of SARS-CoV-2 infections and as preparation for future outbreaks of pandemic RNA viruses.

The Role of Histone Deacetylases in Acute Lung Injury-Friend or Foe.

Acute lung injury (ALI), caused by intrapulmonary or extrapulmonary factors such as pneumonia, shock, and sepsis, eventually disrupts the alveolar-capillary barrier, resulting in diffuse pulmonary oedema and microatasis, manifested by refractory hypoxemia, and respiratory distress. Not only is ALI highly lethal, but even if a patient survives, there are also multiple sequelae. Currently, there is no better treatment than supportive care, and we urgently need to find new targets to improve ALI. Histone deacetylases (HDACs) are epigenetically important enzymes that, together with histone acetylases (HATs), regulate the acetylation levels of histones and non-histones. While HDAC inhibitors (HDACis) play a therapeutic role in cancer, inflammatory, and neurodegenerative diseases, there is also a large body of evidence suggesting the potential of HDACs as therapeutic targets in ALI. This review explores the unique mechanisms of HDACs in different cell types of ALI, including macrophages, pulmonary vascular endothelial cells (VECs), alveolar epithelial cells (AECs), and neutrophils.

Neutrophil elastase decreases SARS-CoV-2 spike protein binding to human bronchial epithelia by clipping ACE-2 ectodomain from the epithelial surface.

Patients with cystic fibrosis (CF) have decreased severity of severe acute respiratory syndrome-like coronavirus-2 (SARS-CoV-2) infections, but the underlying cause is unknown. Patients with CF have high levels of neutrophil elastase (NE) in the airway. We examined whether respiratory epithelial angiotensin-converting enzyme 2 (ACE-2), the receptor for the SARS-CoV-2 spike protein, is a proteolytic target of NE. Soluble ACE-2 levels were quantified by ELISA in airway secretions and serum from patients with and without CF, the association between soluble ACE-2 and NE activity levels was evaluated in CF sputum. We determined that NE activity was directly correlated with increased ACE-2 in CF sputum. Additionally, primary human bronchial epithelial (HBE) cells, exposed to NE or control vehicle, were evaluated by Western analysis for the release of cleaved ACE-2 ectodomain fragment into conditioned media, flow cytometry for the loss of cell surface ACE-2, its impact on SARS-CoV-2 spike protein binding. We found that NE treatment released ACE-2 ectodomain fragment from HBE and decreased spike protein binding to HBE. Furthermore, we performed NE treatment of recombinant ACE-2-Fc-tagged protein in vitro to assess whether NE was sufficient to cleave recombinant ACE-2-Fc protein. Proteomic analysis identified specific NE cleavage sites in the ACE-2 ectodomain that would result in loss of the putative N-terminal spike-binding domain. Collectively, data support that NE plays a disruptive role in SARS-CoV-2 infection by catalyzing ACE-2 ectodomain shedding from the airway epithelia. This mechanism may reduce SARS-CoV-2 virus binding to respiratory epithelial cells and decrease the severity of COVID19 infection.

Pharmacoinvasive Therapy in Treating Acute STEMI Patients in COVID-19 Era-Thrombolysis by Tenecteplase (TNK) / Streptokinase (STK) Followed by Rescue PCI of the Culprit Vessel

Background: Treating acute STEMI patients by primary PCI has dramatically fallen globally in covid era as there is chances of potential threat of spreading Covid among the non-Covid patient. Thereby, thrombolysis of acute STEMI patient either by Streptokinase (STK) or Tenecteplase (TNK) in grey zone till Covid RT PCR report to come, was the mode of treatment of acute myocardial infarction patient in our hospital. Post thrombolysis, Covid positive cases were managed conservatively in a Covid dedicated unit. Covid negative cases were treated by rescue PCI of the culprit lesion. Exact data on benefit of thrombolysis either by TNK or STK of STEMI patients in Covid era, is not well addressed in our patient population. Thereby, we have carried out this prospective observational study to see the outcomes of thrombolysis and subsequent intervention. Method(s): STEMI Patient who represented to our ER with chest pain and ECG and hs-TROP-I evidenced acute ST segment elevated myocardial infarction (STEMI), were enrolled in the study. Total 139 patients enrolled (Male:120, Female :19);average age for Male: 54yrs., female was: 56yrs. All patients were admitted in the grey zone of CCU where thrombolysis done either by TNK or STK. Positive for COVID-19, were patients excluded from intervention and managed conservatively in Covid-19 dedicated ward. Covid Negative patients were kept transferred to CCU green zone. Result(s): COVID-19 test was carried out on all studied patients. Among them, Covid-19 positive were 7.9% (11) patients and managed conservatively in dedicated Covid ward, Covid-19 negative were 92.1% (128). Primary PCI was performed in 5.03% (7). Rest was managed by Pharmacoinvasive therapy either by TNK or STK. Thrombolysis by Tenecteplase in 64% (89), Streptokinase in 17.9% (25) patient, 12.9% (18) patient did not receive any thrombolysis due to late presentation and primary PCI done in 5.4% (7). On average 2.1 days after Fibrinolysis, elective PCI carried out. Data analysis from 48 patients;chest pain duration (3.71 +/-2.8 hr., Chest pain to contact time 3.3+/-2.8hr., Chest pain to needle time 7.2 +/-12.7hr., thrombolysis to balloon time 117.5+/-314.8hr., as many of the patient develop LVF post thrombolysis. More than 50% stenosis resolution observed in 41.6% (20) patients, chest pain resolution with one hour of thrombolysis observed in 43.8% (21) patients and development of LVF in 20.8% (10) patients. Door to needle time was 30 min. At presentation of STEMI;Ant Wall MI 46.8% (65), Inferior Wall MI 52.5% (73) and high Lateral 0.7% (1). Average Serum hs Trop-I was 16656 for male and 12109 for female. LVEF were 41% for male and 48% for female. HbA1C were in Male 8.34%: Female 8.05%, SBP for Male 120mmHg: Female 128 mmHg. Total, 88 stents were deployed in 83 territories. CABG recommended for 5.03% (7) patients, PCI in 58.3% (81), remaining were kept on medical management. Stented territory was LAD 45.7% (37) and RCA 39.5% (32) and LCX 14.8% (12). Common stent used;Everolimus 61.4% (54), Sirolimus 25% (22), Progenitor cell with sirolimus 2.3%(2) and Zotarolimus 11.4% (10) Conclusion(s): In the era of COVID-19, in this prospective cohort study, on acute STEMI patient management, we found that Pharmaco therapy by Tenecteplase and Streptokinase, reduced patient symptom and ST resolution partially. Therefore, coronary angiogram and subsequent Rescue PCI by Drug Eluting Stents (DES) are key goals of complete revascularization.Copyright © 2023

Racial, ethnic, and sex-based disparities among high-risk individuals undergoing pancreatic cancer surveillance

Background: The international, multi-center Pancreatic Cancer Early Detection (PRECEDE) Consortium enrolls high-risk individuals (HRIs) undergoing pancreatic ductal adenocarcinoma (PDAC) surveillance. Enrollment began in 2020, and despite challenges related to the COVID-19 pandemic, the PRECEDE Consortium rapidly accrued a large cohort of HRIs. The purpose of this study is to describe the characteristics of this cohort and assess racial, ethnic, and sex-based disparities. Method(s): The PRECEDE Consortium (NCT04970056) is a prospective, multicenter study focused on improving survival from PDAC through early detection. Data from all HRIs who met criteria for PDAC surveillance and enrolled between May 2020 - March 2022 were collected and included in the analysis. Result(s): During the study period, 1299 HRIs enrolled in PRECEDE at 32 centers. HRIs were excluded if enrollment data was incomplete or criteria for PDAC surveillance were not met. Of 1113 who were included, 47.2% met criteria for familial pancreatic cancer (FPC) and 45.4% had a family history of PDAC along with a PV in a PDAC-risk gene (BRCA1, BRCA2, PALB2, ATM, MLH1, MSH2, MSH6, PMS2, or EPCAM). The remainder had familial atypical mole melanoma syndrome (5.7%), Peutz- Jeghers syndrome (1.6%), or hereditary pancreatitis (0.2%). More females than males enrolled (65.9% vs. 33.5%). The distribution of HRIs by race and ethnicity is depicted;the majority identified as white (87.7%). Study participants were primarily from the US (82.7%), the median age was 61 (27-85) and 18.5% had Ashkenazi Jewish ancestry. Nearly all HRIs consented to allow access to imaging data (99.6%), collection of germline DNA (97.7%), and biosample collection (99.5%). There were no race, ethnicity, or sex-based differences in rates of consent for collection of imaging, DNA, or biosamples. Conclusion(s): Enrollment of HRIs in prospective studies of PDAC surveillance is essential for advancing early detection research in PDAC. A distinct advantage of the PRECEDE Consortium for examining enrollment disparities is that recruitment began in 2020, providing a unique and current snapshot of the international PDAC surveillance landscape. Despite the recent attention on addressing disparities in healthcare delivery, significant racial, ethnic, and sex-based disparities persisted in the cohort of HRIs enrolled in the PRECEDE Consortium. Ensuring that the diversity of participants in the PRECEDE Consortium mirrors the communities served by participating centers is crucial. Further examining and addressing the reasons for these disparities is a major focus of the PRECEDE Consortium moving forward.

Single cell mRNA sequencing of nasal swabs indicate an impaired ciliated cell function in COVID-19 convalescents with persisting dyspnea

COVID-19 convalescents often experience persistent symptoms such as fatigue, neurologic complications or dyspnea, often referred to as "long COVID". To elucidate molecular mechanisms underlying ongoing dyspnea in COVID-19 convalescents, we analyzed single-cell RNA sequencing data from nasal swabs collected during acute infection, and three, six and twelve months post infection together with matching healthy controls. Patients with and without persisting symptoms and with varying severity during the acute phase were included. Post infection, we observed a time-dependent decrease in immune cells. Transcriptional analysis of nasal epithelial cells provided evidence of an impaired cilia assembly, organization and function in COVID-19 convalescents with dyspnea compared to healthy controls and convalescents without ongoing respiratory symptoms. Moreover, differences in the differentiation trajectories of ciliated cells were evident between patients with and without dyspnea, with less diverse differentiation endpoints in the dyspnea patients than in healthy controls or convalescents without respiratory impairment. Overall, our analyses revealed a potential deficiency of ciliated cells in COVID-19 convalescents with dyspnea compared to convalescents without ongoing respiratory symptoms or compared with healthy controls. Ciliated cells clear the lung from particles and mucus. If these cells are functionally impaired, pathogens remain in the airways, causing respiratory problems and infections. Thus, it is reasonable to assume, that impaired ciliated cell function contributes to the persistent respiratory symptoms seen in COVID-19 convalescents.

The OM-85 bacterial lysate: a new tool against SARS-CoV-2?

The emergence of SARS-CoV-2, a novel coronavirus, caused the global Coronavirus disease of 2019 (COVID-19) pandemic. Because SARS-CoV-2 mutates rapidly, vaccines that induce immune responses against viral components critical for target cell infection strongly mitigate but do not abrogate viral spread, and disease rates remain high worldwide. Complementary treatments are therefore needed to reduce the frequency and/or severity of SARS-CoV-2 infections. OM-85, a standardized lysate of 21 bacterial strains often found in the human airways, has immuno-modulatory properties and is widely used empirically in Europe, South America and Asia for the prophylaxis of recurrent upper airway infections in adults and children, with excellent safety profiles. In vitro studies from our laboratory recently demonstrated that OM-85 inhibits SARS-CoV-2 epithelial cell infection by downregulating SARS-CoV-2 receptor expression, raising the possibility that this bacterial extract might eventually complement the current COVID-19 therapeutic toolkit. Here we discuss how our results and those from other groups are fostering progress in this emerging field of research.

Application of the PHENotype SIMulator for rapid identification of potential candidates in effective COVID-19 drug repurposing.

The current, rapidly diversifying pandemic has accelerated the need for efficient and effective identification of potential drug candidates for COVID-19. Knowledge on host-immune response to SARS-CoV-2 infection, however, remains limited with few drugs approved to date. Viable strategies and tools are rapidly arising to address this, especially with repurposing of existing drugs offering significant promise. Here we introduce a systems biology tool, the PHENotype SIMulator, which -by leveraging available transcriptomic and proteomic databases-allows modeling of SARS-CoV-2 infection in host cells in silico to i) determine with high sensitivity and specificity (both>96%) the viral effects on cellular host-immune response, resulting in specific cellular SARS-CoV-2 signatures and ii) utilize these cell-specific signatures to identify promising repurposable therapeutics. Powered by this tool, coupled with domain expertise, we identify several potential COVID-19 drugs including methylprednisolone and metformin, and further discern key cellular SARS-CoV-2-affected pathways as potential druggable targets in COVID-19 pathogenesis.

Brief Research Report: Serum clara cell 16 kDa protein levels are increased in patients hospitalized for severe SARS-CoV-2 or sepsis infection.

Background: Clara cell 16 kDa protein (CC16) is a secretory protein primarily expressed in epithelial cells in the lungs. Previous studies show that CC16 exerts anti-inflammatory and immune-modulatory properties in both acute and chronic pulmonary diseases. However, despite the evidence of CC16's high biomarker potential, evaluation of its role in infectious diseases is yet very limited. Methods: Serum CC16 concentrations were measured by ELISA and assessed in two different types of severe infections. Using a case-control study design, patients treated for either severe SARS-CoV-2 or severe non-pulmonary sepsis infection were compared to age- and sex-matched healthy human subjects. Results: Serum CC16 was significantly increased in both types of infection (SARS-CoV-2: 96.22 ± 129.01 ng/ml vs. healthy controls: 14.05 ± 7.48 ng/ml, p = 0.022; sepsis: 35.37 ± 28.10 ng/ml vs. healthy controls: 15.25 ± 7.51 ng/ml, p = 0.032) but there were no distinct differences between infections with and without pulmonary focus (p = 0.089). Furthermore, CC16 serum levels were positively correlated to disease duration and inversely to the platelet count in severe SARS-CoV-2 infection. Conclusions: Increased CC16 serum levels in both SARS-CoV-2 and sepsis reinforce the high potential as a biomarker for epithelial cell damage and bronchoalveolar-blood barrier leakage in pulmonary as well as non-pulmonary infectious diseases.

Nasopharyngeal epithelial cells from patients with coronavirus disease 2019 express abnormal levels of Toll-like receptors.

Aberrant activation of the immune system has been attributed with etiology and pathogenesis of coronavirus disease 2019 (COVID-19). Here, the transcript levels of toll-like receptors (TLRs) were measured in the nasopharyngeal epithelial cells obtained from COVID-19 patients to assess the involvement of these molecules in the clinical outcome of COVID-19 patients. Nasopharyngeal swab samples were used to obtain epithelial cells from 120 COVID-19 patients and 100 healthy controls. COVID-19 cases were classified into those having clinical symptoms/needing for hospitalization, having clinical symptoms/not needing for hospitalization, and those without clinical symptoms|. The mRNA expression levels of TLRs were measured in the nasopharyngeal epithelial cells. Overall, mRNA expression of TLR1, TLR2, TLR4, and TLR6 was significantly higher in COVID-19 cases compared to controls. The mRNA expression of TLRs were all higher significantly in the samples from COVID-19 patients having clinical symptoms and needing hospitalization as well as in those with clinical symptoms/not needing for hospitalization in comparison to controls. TLR expression was significantly higher in those with clinical symptoms/needing for hospitalization and those with clinical symptoms/not needing for hospitalization compared to COVID-19 cases without clinical symptoms. In cases with clinical symptoms/needing for hospitalization and those with clinical symptoms/not needing for hospitalization, there was a correlation between TLR expression and clinicopathological findings. In conclusion, aberrant expression of TLRs in the nasopharyngeal epithelial cells from COVID-19 cases may predict the severity of the diseases and necessity for supportive cares in the hospital.

Pseudorabies virus-induced expression and antiviral activity of type I or type III interferon depend on the type of infected epithelial cell.

Type I and III Interferons (IFNs) are the initial antiviral cytokines produced in response to virus infection. These IFNs in turn bind to their respective receptors, trigger JAK-STAT signaling and induce the expression of IFN-stimulated genes (ISGs) to engage antiviral functions. Unlike the receptor for type I IFNs, which is broadly expressed, the expression of the type III IFN receptor is mainly confined to epithelial cells that line mucosal surfaces. Accumulating evidence has shown that type III IFNs may play a unique role in protecting mucosal surfaces against viral challenges. The porcine alphaherpesvirus pseudorabies virus (PRV) causes huge economic losses to the pig industry worldwide. PRV first replicates in the respiratory tract, followed by spread via neurons and via lymph and blood vessels to the central nervous system and internal organs, e.g. the kidney, lungs and intestinal tract. In this study, we investigate whether PRV triggers the expression of type I and III IFNs and whether these IFNs exert antiviral activity against PRV in different porcine epithelial cells: porcine kidney epithelial cells (PK-15), primary respiratory epithelial cells (PoREC) and intestinal porcine epithelial cells (IPEC-J2). We show that PRV triggers a multiplicity of infection-dependent type I IFN response and a prominent III IFN response in PK-15 cells, a multiplicity of infection-dependent expression of both types of IFN in IPEC-J2 cells and virtually no expression of either IFN in PoREC. Pretreatment of the different cell types with equal amounts of porcine IFN-λ3 (type III IFN) or porcine IFN-α (type I IFN) showed that IFN-α, but not IFN-λ3, suppressed PRV replication and spread in PK-15 cells, whereas the opposite was observed in IPEC-J2 cells and both types of IFN showed anti-PRV activity in PoREC cells, although the antiviral activity of IFN-α was more potent than that of IFN-λ3 in the latter cell type. In conclusion, the current data show that PRV-induced type I and III IFN responses and their antiviral activity depend to a large extent on the epithelial cell type used, and for the first time show that type III IFN displays antiviral activity against PRV in epithelial cells from the respiratory and particularly the intestinal tract.

Global Dynamics of SARS-CoV-2 Infection with Antibody Response and the Impact of Impulsive Drug Therapy.

Mathematical modeling is crucial to investigating tthe ongoing coronavirus disease 2019 (COVID-19) pandemic. The primary target area of the SARS-CoV-2 virus is epithelial cells in the human lower respiratory tract. During this viral infection, infected cells can activate innate and adaptive immune responses to viral infection. Immune response in COVID-19 infection can lead to longer recovery time and more severe secondary complications. We formulate a micro-level mathematical model by incorporating a saturation term for SARS-CoV-2-infected epithelial cell loss reliant on infected cell levels. Forward and backward bifurcation between disease-free and endemic equilibrium points have been analyzed. Global stability of both disease-free and endemic equilibrium is provided. We have seen that the disease-free equilibrium is globally stable for R0<1, and endemic equilibrium exists and is globally stable for R0>1. Impulsive application of drug dosing has been applied for the treatment of COVID-19 patients. Additionally, the dynamics of the impulsive system are discussed when a patient takes drug holidays. Numerical simulations support the analytical findings and the dynamical regimes in the systems.

PCL- And Gelatin-based Electrospun Biological Scaffolds For In Vitro Lung Tissue Engineering

Purpose/Objectives: Acute and chronic respiratory diseases constitute a substantial socioeconomic burden on a global scale, as made abundantly clear in the last two years with the rampant coronavirus disease 2019 (COVID-19) pandemic. Alas, the development of new therapies for pathological respiratory conditions has been hindered by the inadequacy of current preclinical models, which often fail to provide reliable predictions on drug safety and efficacy in humans. In particular, considerable anatomical and physiological differences between the respiratory systems of commonly used animal models and humans are one of the main issues leading to high drug attrition and clinical failure rates. Accordingly, the generation of physiologically relevant preclinical lung models for early drug development and pharmaceutical research is urgently needed. In this work, poly(ϵ-caprolactone) (PCL) and gelatin were used as raw materials to produce electrospun scaffolds for in vitro lung tissue engineering, in order to generate human biomimetic platforms for preclinical drug safety and efficacy testing. Methodology: PCL and gelatin were mixed at varying volume ratios: 1:0 (PP), 6:1 (PPG61), 4:1 (PPG41), and 2:1 (PPG21), so as to determine the optimal gelatin concentration for cell adhesion and growth. Poly(vinylpyrrolidone) (PVP) was added to every polymer mixture to facilitate the electrospinning process, and electrospun fibrous matrices were fabricated using a needleless electrospinning technique. Scaffold morphology, chemical composition, and wettability were characterized with scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopy, and water contact angle analysis, respectively. Biocompatibility testing was performed using human bronchial (16HBE) and alveolar (A549) epithelial cell lines, consisting of cell metabolic activity, proliferation, and adhesion evaluation over two weeks of in vitro culture. Results: All polymer blends resulted in the formation of electrospun scaffolds with a nanofibrous structure. The addition of gelatin in PPG61 scaffolds improved fiber morphology compared to PP formulations, but increasing proportions of this polymer in PPG41 and PPG21 mats caused a larger number of defects, such as beading and branching. FTIR analysis confirmed the presence of PCL and PVP in PP scaffolds, as well as the addition of gelatin in all PPG blends. Moreover, as expected, all scaffolds were hydrophilic, with water contact angles below 90°, being suitable for protein adsorption and cell adhesion. Regarding 16HBE and A549 cell viability, surprisingly, no major differences were found between the different formulations over the two-week culture period, showing that all polymer blends were equally capable of promoting cell adhesion and growth. While PP scaffolds significantly outperformed PPG electrospun mats in early timepoints, no such differences were identified at the end of the experimental period. Conclusion/Significance: These results suggested that PCL, PVP, and/or gelatin blend electrospun scaffolds are conducive to lung epithelial cell adhesion and proliferation. Nevertheless, further studies investigating epithelial cell differentiation and function should be conducted to fully assess the suitability of these biomaterials as platforms for in vitro lung tissue engineering.

PLEOMORPHIC CARCINOMA IN THE MIDST OF COVID

SESSION TITLE: Pathology Identifying Chest Infections Case Report Posters SESSION TYPE: Case Report Posters PRESENTED ON: 10/17/2022 12:15 pm - 01:15 pm INTRODUCTION: Pleomorphic carcinoma is a subtype of sarcomatoid carcinomas that represents <1 % of all primary lung neoplasms. This case highlights a recent diagnosis of a patient with pleomorphic carcinoma in the midst of COVID-19 pneumonia. CASE PRESENTATION: A 75 year old female with a 180-pack year smoking history presented to the emergency department with dyspnea and chest discomfort. Vital signs significant for oxygen saturation at 93% on room air. The patient had been admitted to the hospital 7 months prior for acute hypoxemic respiratory failure due to COVID-19 pneumonia. At that point, computed tomography (CT) of the chest showed a right lower lobe 5.5 cm juxtapleural lesion measuring fluid attenuation by Hounsfield units without intralesional enhancement. The lesion was initially thought to be secondary to the patient's COVID-19 pneumonia and was not investigated further. The patient was subsequently lost to follow up. Seven months later the patient presented with worsening shortness of breath. Chest CT revealed large right complex pleural effusion with near complete lung collapse. The patient underwent pigtail catheter placement with partial re-expansion of the lung. Pleural fluid analysis showed an exudative effusion with no malignant cells on cytology. Follow-up CT imaging showed a large mass-like area in the right mid and lower hemithorax. Video assisted thorascopic surgery (VATS) decortication and thoracotomy revealed a right lower lobe abscess and empyema. Pathology samples collected during procedure showed malignant cells of sarcamatoid features found in right lung and intraparenchymal lymph nodes. Histology and immunostaining showed a tumor composed of a component of poorly differentiated adenocarcinoma and more than 10% spindle/pleomorphic cells. Immunostaining showed the poorly differentiated adenocarcinoma component was positive for moc 31, Ber-EP4, cytokeratin AE1/AE3, CAM 5.2, lack TTF-1 and p40. The spindle/pleomorphic component was negative for cytokeratins. DISCUSSION: Pulmonary pleomorphic carcinoma (PC) is a rare, poorly differentiated non-small cell lung cancer (NSCLC) that contains at least 10% spindle and/or giant cells or a carcinoma consisting only of spindle and giant cells. PC has poor response to conventional treatments for NSCLC and subsequently poor 5 year survival. It more common in men and smokers. COVID-19 causes a variety of pulmonary radiographic manifestations, including nodules and mass-like consolidations. Superimposed bacterial infections are also common. Our case, however, highlights the importance of serial radiographic monitoring and, when indicated, tissue sampling to rule out alternative explanations for abnormal CT findings. CONCLUSIONS: Appropriate screening and careful follow up of suspicious lung lesions is vital to ensure prompt diagnosis and treatment of lung malignancy. Reference #1: WHO Classification of Tumours Editorial Board. Thoracic Tumours. In: WHO Classification of Tumours,Earke 5th ed, IARC Publications, 2021. Vol 5. Reference #2: Ito K, Oizumi S, Fukumoto S, Harada M, Ishida T, Fujita Y, Harada T, Kojima T, Yokouchi H, Nishimura M;Hokkaido Lung Cancer Clinical Study Group. Clinical characteristics of pleomorphic carcinoma of the lung. Lung Cancer. 2010 May;68(2):204-10. doi: 10.1016/j.lungcan.2009.06.002. Epub 2009 Jul 3. PMID: 19577320. Reference #3: Maneenil K, Xue Z, Liu M, Boland J, Wu F, Stoddard SM, Molina J, Yang P. Sarcomatoid Carcinoma of the Lung: The Mayo Clinic Experience in 127 Patients. Clin Lung Cancer. 2018 May;19(3):e323-e333. doi: 10.1016/j.cllc.2017.12.008. Epub 2017 Dec 21. PMID: 29454534. DISCLOSURES: No relevant relationships by Rachel Earle No relevant relationships by Samantha Gillenwater No relevant relationships by Miquel Gonzalez No relevant relationships by Sikandar Khan No relevant relationships by Christopher Lau no disclosure submitted for Jinesh Mehta;

People with cystic fibrosis do not show an increased interferonresponse transcriptomic signature in nasal epithelial cells

Objectives: People with CF (PwCF) are at increased risk of respiratory infections and chronic inflammation.We sought to determine whether the inflammatory response is different in nasal epithelium of PwCF compared to healthy volunteers (HV). Since Interferons can increase ACE2 expression, a protein required for SARS-CoV-2 entry,we focused our analysis on the the focusing on the interferon-response signature. Methods: We reanalysed nasal curettage sample bulk RNA-seq signatures of pilot and validation datasets for which the study methods and demographics of the recruited cohort have already been reported. For this analysis, we performed in-silico deconvolution of bulk RNA-seq data using publicly available single-cell RNA-seq data from nasal epitheliumas a reference to determine the abundance of the specific cell types in each sample. Results: Hierarchical clustering of the pilot and validation cohorts revealed 3 clusters. Analysis of the larger validation cohort revealed that Cluster A included HV (11 out of 11 subjects) and both homozygous (7 out of 13 subjects) and heterozygous (3 out of 10 subjects) PwCF. Subject cluster A was characterised by increased expression of genes related to secretory and ciliated epithelial cells, whereas Clusters B and C contained both homozygous and heterozygous PwCF only and were characterised by genes restricted to neutrophils and involved in immune responses. We then compared samples from cluster A that contained samples from HV (n = 11), PwCF homozygous (n = 7) and heterozygous (n = 3) for F508del. This analysis identified 379 genes upregulated in HV and 146 genes upregulated in PwCF homozygous for F508del and only 44 and 6 genes upregulated in HV and PwCF heterozygous for F508del, respectively (FDR q < 0.05). ACE2, TMPRS2 or other interferon-response genes were not deferentially expressed in either comparison of cluster A. Conclusion: PwCF do not have higher expression of interferon-response genes in nasal epithelial cells

Innate Immune Responses by Respiratory Viruses, Including Rhinovirus, During Asthma Exacerbation.

Viral infection, especially with rhinovirus (RV), is a major cause of asthma exacerbation. The production of anti-viral cytokines such as interferon (IFN)-ß and IFN-α from epithelial cells or dendritic cells is lower in patients with asthma or those with high IgE, which can contribute to viral-induced exacerbated disease in these patients. As for virus-related factors, RV species C (RV-C) induces more exacerbated disease than other RVs, including RV-B. Neutrophils activated by viral infection can induce eosinophilic airway inflammation through different mechanisms. Furthermore, virus-induced or virus-related proteins can directly activate eosinophils. For example, CXCL10, which is upregulated during viral infection, activates eosinophils in vitro. The role of innate immune responses, especially type-2 innate lymphoid cells (ILC2) and epithelial cell-related cytokines including IL-33, IL-25, and thymic stromal lymphopoietin (TSLP), in the development of viral-induced airway inflammation has recently been established. For example, RV infection induces the expression of IL-33 or IL-25, or increases the ratio of ILC2 in the asthmatic airway, which is correlated with the severity of exacerbation. A mouse model has further demonstrated that virus-induced mucous metaplasia and ILC2 expansion are suppressed by antagonizing or deleting IL-33, IL-25, or TSLP. For treatment, IFNs including IFN-ß suppress not only viral replication but also ILC2 activation in vitro. Agonists of toll-like receptor (TLR) 3 or 7 can induce IFNs, which can then suppress viral replication and ILC2 activation. Therefore, if delivered in the airway, IFNs or TLR agonists could become innovative treatments for virus-induced asthma exacerbation.