Effects of Pycnogenol® in people with post-COVID-19 condition (PYCNOVID): study protocol for a single-center, placebo controlled, quadruple-blind, randomized trial.

BACKGROUND: A significant proportion of the global population has been infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at some point since the onset of the pandemic. Although most individuals who develop coronavirus disease 2019 (COVID-19) recover without complications, about 6% have persistent symptoms, referred to as post-COVID-19 condition (PCC). Intervention studies investigating treatments that potentially alleviate PCC-related symptoms and thus aim to mitigate the global public health burden and healthcare costs linked to PCC are desperately needed. The PYCNOVID trial investigates the effects of Pycnogenol®, a French maritime pine bark extract with anti-inflammatory and antioxidative properties, versus placebo on patient-reported health status in people with PCC. METHODS: This is a single-center, placebo-controlled, quadruple blind, randomized trial. We aim to randomly assign 150 individuals with PCC (1:1 ratio) to receive either 200 mg Pycnogenol® or placebo daily for 12 weeks. Randomization is stratified for duration of PCC symptoms (≤ 6 months versus > 6 months) and presence of symptomatic chronic disease(s). The primary endpoint is perceived health status at 12 weeks (EuroQol-Visual Analogue Scale) adjusted for baseline values and stratification factors. Secondary endpoints include change in self-reported PCC symptoms, health-related quality of life, symptoms of depression and anxiety, cognitive function, functional exercise capacity, physical activity measured with accelerometry, and blood biomarkers for endothelial health, inflammation, coagulation, platelet function, and oxidative stress. Investigators, study participants, outcome assessors, and data analysts are blinded regarding the intervention assignment. Individuals with PCC were involved in the design of this study. DISCUSSION: This is the first trial to investigate the effects of Pycnogenol® versus placebo on patient-reported health status in people with PCC. Should the trial proof clinical effectiveness, Pycnogenol® may serve as a therapeutic approach to mitigate symptoms associated with PCC. TRIAL REGISTRATION: The study is registered at ClinicalTrials.gov. :NCT05890534, June 6, 2023.

C-di-AMP levels modulate Staphylococcus aureus cell wall thickness, response to oxidative stress, and antibiotic resistance and tolerance.

IMPORTANCE: Antibiotic resistance and tolerance are substantial healthcare-related problems, hampering effective treatment of bacterial infections. Mutations in the phosphodiesterase GdpP, which degrades cyclic di-3', 5'-adenosine monophosphate (c-di-AMP), have recently been associated with resistance to beta-lactam antibiotics in clinical Staphylococcus aureus isolates. In this study, we show that high c-di-AMP levels decreased the cell size and increased the cell wall thickness in S. aureus mutant strains. As a consequence, an increase in resistance to cell wall targeting antibiotics, such as oxacillin and fosfomycin as well as in tolerance to ceftaroline, a cephalosporine used to treat methicillin-resistant S. aureus infections, was observed. These findings underline the importance of investigating the role of c-di-AMP in the development of tolerance and resistance to antibiotics in order to optimize treatment in the clinical setting.

Transport of Designed Ankyrin Repeat Proteins through reconstituted human bronchial epithelia and protection against SARS-CoV-2.

Clinical studies have proven antiviral effectiveness of treatment with a Designed Ankyrin Repeat Protein (DARPin) specific against the spike protein of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2). More information on transport mechanisms and efficiency to the site of action is desirable. Transepithelial migration through air-liquid interface (ALI) cultures of reconstituted human bronchial epithelia (HBE) was assessed by Enzyme-Linked Immunosorbent Assays and Confocal Laser Scanning Microscopy for different DARPin designs in comparison to a monoclonal antibody. Antiviral efficacy against authentic SARS-CoV-2, applied apically on HBE, was investigated based on viral titers and genome equivalents, after administration of therapeutic candidates on the basal side. Transepithelial translocation of all DARPin candidates and the monoclonal antibody was efficient and dose dependent. Small DARPins and the antibody migrated more efficiently than larger molecules, indicating different transport mechanisms involved. Microscopic analyses support this, demonstrating passive paracellular transport of smaller DARPins and transcellular migration of the larger molecules. All therapeutic candidates applied to the basal side of HBE conferred effective protection against SARS-CoV-2 infection. In summary, we have shown that DARPins specific against SARS-CoV-2 translocate across intact airway epithelia and confer effective protection against infection and viral replication.

Cystic Fibrosis Lung Disease in the Aging Population.

The demographics of the population with cystic fibrosis (CF) is continuously changing, with nowadays adults outnumbering children and a median predicted survival of over 40 years. This leads to the challenge of treating an aging CF population, while previous research has largely focused on pediatric and adolescent patients. Chronic inflammation is not only a hallmark of CF lung disease, but also of the aging process. However, very little is known about the effects of an accelerated aging pathology in CF lungs. Several chronic lung disease pathologies show signs of chronic inflammation with accelerated aging, also termed "inflammaging"; the most notable being chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF). In these disease entities, accelerated aging has been implicated in the pathogenesis via interference with tissue repair mechanisms, alterations of the immune system leading to impaired defense against pulmonary infections and induction of a chronic pro-inflammatory state. In addition, CF lungs have been shown to exhibit increased expression of senescence markers. Sustained airway inflammation also leads to the degradation and increased turnover of cystic fibrosis transmembrane regulator (CFTR). This further reduces CFTR function and may prevent the novel CFTR modulator therapies from developing their full efficacy. Therefore, novel therapies targeting aging processes in CF lungs could be promising. This review summarizes the current research on CF in an aging population focusing on accelerated aging in the context of chronic airway inflammation and therapy implications.

Cigarette smoke activates the parthanatos pathway of cell death in human bronchial epithelial cells.

Tobacco smoke negatively affects human bronchial epithelial (HBE) cells and is directly implicated in the etiology of smoking related respiratory diseases. Smoke exposure causes double-stranded DNA breaks and DNA damage activates PARP-1, the key mediator of the parthanatos pathway of cell death. We hypothesize that smoke exposure activates the parthanatos pathway in HBE cells and represents a cell death mechanism that contributes to smoking related lung diseases. We exposed fully differentiated, primary HBE cells grown at the air liquid interface to cigarette smoke and evaluated them for parthanatos pathway activation. Smoke exposure induced mitochondrial to nuclear translocation of Apoptosis-Inducing Factor (AIF) and Endonuclease G (EndoG) within the first three hours characteristic of the parthanatos pathway. Exposing cells to an increasing number of cigarettes revealed that significant activation of the parthanatos pathway occurs after exposure to higher levels of smoke. Use of the specific PARP-1 inhibitor, BMN673, abrogated the effect of smoke induced activation of the parthanatos pathway. Smoke-mediated activation of the parthanatos pathway is increased in HBE cells originating from habitual smokers compared to non-smokers. This suggests that chronic smoke exposure leads to an increase in smoke-mediated activation of the parthanatos pathway and implicates its contribution in the pathogenesis of smoke-related lung diseases.

Wood combustion particles induce adverse effects to normal and diseased airway epithelia.

Residential wood burning is a major source of poorly characterized, deleterious particulate matter, whose composition and toxicity may vary with wood type, burning condition and photochemical age. The causative link between ambient wood particle constituents and observed adverse health effects is currently lacking. Here we investigate the relationship between chemical properties of primary and atmospherically aged wood combustion particles and acute toxicity in human airway epithelial cells. Emissions from a log wood burner were diluted and injected into a smog chamber for photochemical aging. After concentration-enrichment and removal of oxidizing gases, directly emitted and atmospherically aged particles were deposited on cell cultures at the air-liquid interface for 2 hours in an aerosol deposition chamber mimicking physiological conditions in lungs. Cell models were fully differentiated normal and diseased (cystic fibrosis and asthma) human bronchial epithelia (HBE) and the bronchial epithelial cell line BEAS-2B. Cell responses were assessed at 24 hours after aerosol exposure. Atmospherically relevant doses of wood combustion particles significantly increased cell death in all but the asthma cell model. Expression of oxidative stress markers increased in HBE from all donors. Increased cell death and inflammatory responses could not be assigned to a single chemical fraction of the particles. Exposure to primary and aged wood combustion particles caused adverse effects to airway epithelia, apparently induced by several interacting components.

Acute toxicity of silver and carbon nanoaerosols to normal and cystic fibrosis human bronchial epithelial cells.

Inhalation of engineered nanoparticles (NP) poses a still unknown risk. Individuals with chronic lung diseases are expected to be more vulnerable to adverse effects of NP than normal subjects, due to altered respiratory structures and functions. Realistic and dose-controlled aerosol exposures were performed using the deposition chamber NACIVT. Well-differentiated normal and cystic fibrosis (CF) human bronchial epithelia (HBE) with established air-liquid interface and the human bronchial epithelial cell line BEAS-2B were exposed to spark-generated silver and carbon nanoaerosols (20 nm diameter) at three different doses. Necrotic and apoptotic cell death, pro-inflammatory response, epithelial function and morphology were assessed within 24 h after aerosol exposure. NP exposure resulted in significantly higher necrosis in CF than normal HBE and BEAS-2B cells. Before and after NP treatment, CF HBE had higher caspase-3 activity and secreted more IL-6 and MCP-1 than normal HBE. Differentiated HBE had higher baseline secretion of IL-8 and less caspase-3 activity and MCP-1 secretion compared to BEAS-2B cells. These biomarkers increased moderately in response to NP exposure, except for MCP-1, which was reduced in HBE after AgNP treatment. No functional and structural alterations of the epithelia were observed in response to NP exposure. Significant differences between cell models suggest that more than one and fully differentiated HBE should be used in future toxicity studies of NP in vitro. Our findings support epidemiologic evidence that subjects with chronic airway diseases are more vulnerable to adverse effects of particulate air pollution. Thus, this sub-population needs to be included in nano-toxicity studies.

Toxicity of aged gasoline exhaust particles to normal and diseased airway epithelia.

Particulate matter (PM) pollution is a leading cause of premature death, particularly in those with pre-existing lung disease. A causative link between particle properties and adverse health effects remains unestablished mainly due to complex and variable physico-chemical PM parameters. Controlled laboratory experiments are required. Generating atmospherically realistic aerosols and performing cell-exposure studies at relevant particle-doses are challenging. Here we examine gasoline-exhaust particle toxicity from a Euro-5 passenger car in a uniquely realistic exposure scenario, combining a smog chamber simulating atmospheric ageing, an aerosol enrichment system varying particle number concentration independent of particle chemistry, and an aerosol deposition chamber physiologically delivering particles on air-liquid interface (ALI) cultures reproducing normal and susceptible health status. Gasoline-exhaust is an important PM source with largely unknown health effects. We investigated acute responses of fully-differentiated normal, distressed (antibiotics-treated) normal, and cystic fibrosis human bronchial epithelia (HBE), and a proliferating, single-cell type bronchial epithelial cell-line (BEAS-2B). We show that a single, short-term exposure to realistic doses of atmospherically-aged gasoline-exhaust particles impairs epithelial key-defence mechanisms, rendering it more vulnerable to subsequent hazards. We establish dose-response curves at realistic particle-concentration levels. Significant differences between cell models suggest the use of fully-differentiated HBE is most appropriate in future toxicity studies.

In situ fiber-optical monitoring of cytosolic calcium in tissue explant cultures.

We present a fluorescence-lifetime based method for monitoring cell and tissue activity in situ, during cell culturing and in the presence of a strong autofluorescence background. The miniature fiber-optic probes are easily incorporated in the tight space of a cell culture chamber or in an endoscope. As a first application we monitored the cytosolic calcium levels in porcine tracheal explant cultures using the Calcium Green-5N (CG5N) indicator. Despite the simplicity of the optical setup we are able to detect changes of calcium concentration as small as 2.5 nM, with a monitoring time resolution of less than 1 s.

Nanoparticle uptake by airway phagocytes after fungal spore challenge in murine allergic asthma and chronic bronchitis.

BACKGROUND: In healthy lungs, deposited micrometer-sized particles are efficiently phagocytosed by macrophages present on airway surfaces; however, uptake of nanoparticles (NP) by macrophages appears less effective and is largely unstudied in lung disease. Using mouse models of allergic asthma and chronic obstructive pulmonary disease (COPD), we investigated NP uptake after challenge with common biogenic ambient air microparticles. METHODS: Bronchoalveolar lavage (BAL) cells from diseased mice (allergic asthma: ovalbumin [OVA] sensitized and COPD: Scnn1b-transgenic [Tg]) and their respective healthy controls were exposed ex vivo first to 3-µm fungal spores of Calvatia excipuliformis and then to 20-nm gold (Au) NP. Electron microscopic imaging was performed and NP uptake was assessed by quantitative morphometry. RESULTS: Macrophages from diseased mice were significantly larger compared to controls in OVA-allergic versus sham controls and in Scnn1b-Tg versus wild type (WT) mice. The percentage of macrophages containing AuNP tended to be lower in Scnn1b-Tg than in WT mice. In all animal groups, fungal spores were localized in macrophage phagosomes, the membrane tightly surrounding the spore, whilst AuNP were found in vesicles largely exceeding NP size, co-localized in spore phagosomes and occasionally, in the cytoplasm. AuNP in vesicles were located close to the membrane. In BAL from OVA-allergic mice, 13.9 ± 8.3% of all eosinophils contained AuNP in vesicles exceeding NP size and close to the membrane. CONCLUSIONS: Overall, AuNP uptake by BAL macrophages occurred mainly by co-uptake together with other material, including micrometer-sized ambient air particles like fungal spores. The lower percentage of NP containing macrophages in BAL from Scnn1b-Tg mice points to a change in the macrophage population from a highly to a less phagocytic phenotype. This likely contributes to inefficient macrophage clearance of NP in lung disease. Finally, the AuNP containing eosinophils in OVA-allergic mice show that other inflammatory cells present on airway surfaces may substantially contribute to NP uptake.

A compact and portable deposition chamber to study nanoparticles in air-exposed tissue.

BACKGROUND: Epidemiological studies show that elevated levels of particulate matter in ambient air are highly correlated with respiratory and cardiovascular diseases. Atmospheric particles originate from a large number of sources and have a highly complex and variable composition. An assessment of their potential health risks and the identification of the most toxic particle sources would require a large number of investigations. Due to ethical and economic reasons, it is desirable to reduce the number of in vivo studies and to develop suitable in vitro systems for the investigation of cell-particle interactions. METHODS: We present the design of a new particle deposition chamber in which aerosol particles are deposited onto cell cultures out of a continuous air flow. The chamber allows for a simultaneous exposure of 12 cell cultures. RESULTS: Physiological conditions within the deposition chamber can be sustained constantly at 36-37°C and 90-95% relative humidity. Particle deposition within the chamber and especially on the cell cultures was determined in detail, showing that during a deposition time of 2 hr 8.4% (24% relative standard deviation) of particles with a mean diameter of 50 nm [mass median diameter of 100 nm (geometric standard deviation 1.7)] are deposited on the cell cultures, which is equal to 24-34% of all charged particles. The average well-to-well variability of particles deposited simultaneously in the 12 cell cultures during an experiment is 15.6% (24.7% relative standard deviation). CONCLUSIONS: This particle deposition chamber is a new in vitro system to investigate realistic cell-particle interactions at physiological conditions, minimizing stress on the cell cultures other than from deposited particles. A detailed knowledge of particle deposition characteristics on the cell cultures allows evaluating reliable dose-response relationships. The compact and portable design of the deposition chamber allows for measurements at any particle sources of interest.