pre-clinical assessment of pharmacological and molecular properties

SARS-CoV-2, the cause of the COVID-19 pandemic, exploits host cell proteins for viral entry into human lung cells. One of them, the protease TMPRSS2, is required to activate the viral spike protein (S). Even though two inhibitors, camostat and nafamostat, are known to inhibit TMPRSS2 and block cell entry of SARS-CoV-2, finding further potent therapeutic options is still an important task. In this study, we report that a late-stage drug candidate, otamixaban, inhibits SARS-CoV-2 cell entry. We show that otamixaban suppresses TMPRSS2 activity and SARS-CoV-2 infection of a human lung cell line, although with lower potency than camostat or nafamostat. In contrast, otamixaban inhibits SARS-CoV-2 infection of precision cut lung slices with the same potency as camostat. Furthermore, we report that otamixaban's potency can be significantly enhanced by (sub-) nanomolar nafamostat or camostat supplementation. Dominant molecular TMPRSS2-otamixaban interactions are assessed by extensive 109 μs of atomistic molecular dynamics simulations. Our findings suggest that combinations of otamixaban with supplemental camostat or nafamostat are a promising option for the treatment of COVID-19

Quantitative analysis of airway obstruction in lymphangio-leio-myomatosis

Lymphangioleiomyomatosis (LAM) is a rare, cystic lung disease with progressive pulmonary function loss caused by progressively proliferating LAM cells. The degree of airway obstruction has not been well investigated within the pathogenesis of LAM. Using a combination of ex vivo computed tomography (CT), microCT and histology, the site and nature of airway obstruction in LAM explant lungs was compared with matched control lungs (n=5 each). The total number of airways per generation, total airway counts, terminal bronchioles number and surface density were compared in LAM versus control. Ex vivo CT analysis demonstrated a reduced number of airways from generation 7 on (p<0.0001) in LAM compared with control, whereas whole-lung microCT analysis confirmed the three- to four-fold reduction in the number of airways. Specimen microCT analysis further demonstrated a four-fold decrease in the number of terminal bronchioles (p=0.0079) and a decreased surface density (p=0.0079). Serial microCT and histology images directly showed the loss of functional airways by collapse of airways on the cysts and filling of the airway by exudate. LAM lungs show a three- to four-fold decrease in the number of (small) airways, caused by cystic destruction which is the likely culprit for the progressive loss of pulmonary function

Omicron‐induced interferon signaling prevents influenza A H1N1 and H5N1 virus infection

Recent findings in permanent cell lines suggested that SARS‐CoV‐2 Omicron BA.1 induces a stronger interferon response than Delta. Here, we show that BA.1 and BA.5 but not Delta induce an antiviral state in air‐liquid interface cultures of primary human bronchial epithelial cells and primary human monocytes. Both Omicron subvariants caused the production of biologically active types I (α/β) and III (λ) interferons and protected cells from super‐infection with influenza A viruses. Notably, abortive Omicron infection of monocytes was sufficient to protect monocytes from influenza A virus infection. Interestingly, while influenza‐like illnesses surged during the Delta wave in England, their spread rapidly declined upon the emergence of Omicron. Mechanistically, Omicron‐induced interferon signaling was mediated via double‐stranded RNA recognition by MDA5, as MDA5 knockout prevented it. The JAK/STAT inhibitor baricitinib inhibited the Omicron‐mediated antiviral response, suggesting it is caused by MDA5‐mediated interferon production, which activates interferon receptors that then trigger JAK/STAT signaling. In conclusion, our study (1) demonstrates that only Omicron but not Delta induces a substantial interferon response in physiologically relevant models, (2) shows that Omicron infection protects cells from influenza A virus super‐infection, and (3) indicates that BA.1 and BA.5 induce comparable antiviral states

Distinct systemic cytokine networks in symptomatic and asymptomatic carotid stenosis

Inflammatory processes are crucial in atherosclerosis and atherothrombosis. This study aimed to identify a cytokine-pattern that is associated with plaque-vulnerability or symptomatic state in comprehensively investigated patients with symptomatic (sCS) and asymptomatic carotid stenosis (aCS). Twenty-two patients with sCS and twenty-four patients with aCS undergoing carotid endarterectomy (CEA) were considered. A cytokine-panel was measured in plasma-specimens prior to surgery and at a 90 day follow-up. Doppler-ultrasound detecting microembolic signals (MES) in the ipsilateral middle cerebral artery was performed. Carotid plaques were analysed regarding histopathological criteria of plaque-vulnerability and presence of chemokine receptor CXCR4. Correction for multiple comparisons and logistic regression analysis adjusting for vascular risk factors, grade of stenosis, antithrombotic and statin pretreatment were applied. In sCS-patients higher plasma-levels of Fractalkine (CX3CL1), IFN-α2, IL-1β, IL-2, IL-3, IL-7 were found compared to aCS-patients. CXCR4-expression on inflammatory cells was more evident in sCS- compared to aCS-plaques and was associated with vulnerability-criteria. In contrast, plasma-cytokine-levels were not related to CXCR4-expression or other vulnerability-criteria or MES. However, in both groups distinct inter-cytokine correlation patterns, which persisted at follow-up and were more pronounced in the sCS-group could be detected. In conclusion, we identified a distinct cytokine/chemokine-network in sCS-patients with elevated and closely correlated mediators of diverse functions

The Adult Sprague-Dawley Sugen-Hypoxia Rat Is Still "the One:" A Model of Group 1 Pulmonary Hypertension: Reply to Le Cras and Abman.

To the editor: Kojonazarov et al. recently reported severe emphysema in the SU5416/Hypoxia (SuHx) rat model of pulmonary hypertension (1). The authors found that adult male Wistar Kyoto (WKY) rats had increased air-to-tissue ratio as judged by non-gated in vivo micro-computed tomography (CT), and an increased mean linear intercept (MLI) as surrogate of emphysema (1, 2). Le Cras and Abman now responded to the Kojonazarov report by underlining the “important role of the developmental timing of disrupted VEGF signaling” (3). They cite earlier studies conducted on the ovine fetus showing that VEGF inhibition caused vascular remodeling, reduction in vascular/airway growth, and neonatal pulmonary hypertension at birth (4). Although SU5416 is known to induce emphysema in rats housed in room air at Denver altitude (1609m altitude) (5, 6), we contended in our response letter (11) that, at least in male Sprague Dawley (SD) rats, the combination of VEGFR inhibition and hypoxia does not lead to any biologically relevant emphysema or other significant parenchymal lung disease (7) but to pulmonary arterial hypertension (PAH) due to severe angioproliferative remodeling (7, 8)

A multiscale X-ray phase-contrast tomography dataset of a whole human left lung

Technological advancements in X-ray imaging using bright and coherent synchrotron sources now allows the decoupling of sample size and resolution while maintaining high sensitivity to the microstructures of soft, partially dehydrated tissues. The continuous developments in multiscale X-ray imaging resulted in hierarchical phase-contrast tomography, a comprehensive approach to address the challenge of organ-scale (up to tens of centimeters) soft tissue imaging with resolution and sensitivity down to the cellular level. Using this technique, we imaged ex vivo an entire human left lung at an isotropic voxel size of 25.08 μm along with local zooms down to 6.05–6.5 μm and 2.45–2.5 μm in voxel size. The high tissue contrast offered by the fourth-generation synchrotron source at the European Synchrotron Radiation Facility reveals the complex multiscale anatomical constitution of the human lung from the macroscopic (centimeter) down to the microscopic (micrometer) scale. The dataset provides comprehensive organ-scale 3D information of the secondary pulmonary lobules and delineates the microstructure of lung nodules with unprecedented detail

Omicron-induced interferon signalling prevents influenza A virus infection

Recent findings in permanent cell lines suggested that SARS-CoV-2 Omicron BA.1 induces a stronger interferon response than Delta. Here, we show that BA.1 and BA.5 but not Delta induce an antiviral state in air-liquid interface (ALI) cultures of primary human bronchial epithelial (HBE) cells and primary human monocytes. Both Omicron subvariants caused the production of biologically active type I (α/β) and III (λ) interferons and protected cells from super-infection with influenza A viruses. Notably, abortive Omicron infection of monocytes was sufficient to protect monocytes from influenza A virus infection. Interestingly, while influenza-like illnesses surged during the Delta wave in England, their spread rapidly declined upon the emergence of Omicron. Mechanistically, Omicron-induced interferon signalling was mediated via double-stranded RNA recognition by MDA5, as MDA5 knock-out prevented it. The JAK/ STAT inhibitor baricitinib inhibited the Omicron-mediated antiviral response, suggesting it is caused by MDA5-mediated interferon production, which activates interferon receptors that then trigger JAK/ STAT signalling. In conclusion, our study 1) demonstrates that only Omicron but not Delta induces a substantial interferon response in physiologically relevant models, 2) shows that Omicron infection protects cells from influenza A virus super-infection, and 3) indicates that BA.1 and BA.5 induce comparable antiviral states

Quantitative analysis of airway obstruction in lymphangioleiomyomatosis

Lymphangioleiomyomatosis (LAM) is a rare, cystic lung disease with progressive pulmonary function loss caused by progressively proliferating LAM cells. The degree of airway obstruction has not been well investigated within the pathogenesis of LAM.Using a combination of ex vivo computed tomography (CT), microCT and histology, the site and nature of airway obstruction in LAM explant lungs was compared with matched control lungs (n=5 each). The total number of airways per generation, total airway counts, terminal bronchioles number and surface density were compared in LAM versus control.Ex vivo CT analysis demonstrated a reduced number of airways from generation 7 on (p<0.0001) in LAM compared with control, whereas whole-lung microCT analysis confirmed the three- to four-fold reduction in the number of airways. Specimen microCT analysis further demonstrated a four-fold decrease in the number of terminal bronchioles (p=0.0079) and a decreased surface density (p=0.0079). Serial microCT and histology images directly showed the loss of functional airways by collapse of airways on the cysts and filling of the airway by exudate.LAM lungs show a three- to four-fold decrease in the number of (small) airways, caused by cystic destruction which is the likely culprit for the progressive loss of pulmonary function.status: publishe