Search Results (348)

The evolution of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its impact on public health continue to demand attention as the virus continues to evolve, demonstrating a remarkable ability to adapt to diverse selective pressures including immune responses, therapeutic treatments, and prophylactic interventions. The SARS-CoV-2 variant landscape remains dynamic, with new subvariants continuously emerging, many harboring spike protein mutations linked to immune evasion. In this study, we characterized a panel of live SARS-CoV-2 strains, including those key subvariants implicated in recent waves of infection. Our findings revealed a significant variability in mutation patterns in the spike protein across the strains analyzed. Commercial antibodies and human convalescent plasma (HCoP) samples from unvaccinated donors were ineffective in neutralizing the most recent Omicron subvariants, particularly after the emergence of JN.1 subvariant. Using human airway epithelial cells derived from healthy bronchiolar tissue (hBAEC), we established both monoinfections and coinfections involving SARS-CoV-2, Influenza A virus H1N1 (IFAV_H1N1) and Respiratory Syncytial Virus (RSV). Assessments were conducted to compare viral infectivity and the production and release of immune mediators in the apical and basolateral compartments. Notably, Omicron KP.3.1.1 subvariant induced a more pronounced cytopathic effect in hBAEC compared to its parental strain JN.1 and even surpassed the impact observed with the ancestral wild-type virus (WA1/2020, Washington strain). Furthermore, the coinfection of KP.3.1.1 subvariant with IFAV_H1N1 or RSV did not attenuate SARS-CoV-2 infectivity; instead, it significantly exacerbated the pathogenic synergy in the lung epithelium. Our study demonstrated that pro-inflammatory cytokines IL-6, IFN-β, and IL-10 were upregulated in hBAEC following SARS-CoV-2 monoinfection with recent Omicron subvariants as well as during coinfection with IFAV_H1N1 and RSV. Taken together, our findings offer new insights into the immune evasion strategies and pathogenic potential of evolving SARS-CoV-2 Omicron subvariants, as well as their interactions with other respiratory viruses, carrying important implications for therapeutic development and public health preparedness. Full article

Saliva was used as non-invasive alternative to blood for diagnosing pathophysiological conditions. This study aimed to assess changes in protein profile in people with obesity after bariatric surgery and to assess the impact of exercise on these changes. The saliva proteome was determined from two-dimensional gels of twenty adults (ten people with normal weight and ten people with obesity). The effects of bariatric surgery and exercise were assessed. A decrease in body weight, body mass index, and waist-to-height ratio was observed after bariatric surgery. Low levels of carbonic anhydrase VI (CA-VI), short palate, lung, and nasal epithelium clone 2 (SPLUNC2), and haptoglobin were observed. One month after bariatric surgery, spots of haptoglobin and SPLUNC2 increased, although one CA-VI spot decreased. Zn-alpha-2 glycoprotein, immunoglobulin chains, and actin-related protein-3, which are high in people with obesity, decreased 1 month after bariatric surgery. Five months after bariatric surgery, the most significant change was the amylase decrease. The exercise-induced changes in salivary proteins increased SPLUNC, CA-VI, type S cystatins, actin cytoplasmic 1, and zinc alpha-2 glycoprotein levels and decrease Ig kappa chain C region and Rab GDP dissociation inhibitor beta. It can be concluded that the salivary proteins change between people with normal weight vs. patients with obesity, as well as after bariatric surgery and exercise programmes. Salivary proteins may be useful biomarkers in non-invasive samples for monitoring and assessing the impact of interventions on people with obesity. Full article

Background: Pulmonary fibrosis is a major disease that leads to the progressive loss of lung function. The disease manifests early, resulting in type 2 respiratory failure. This is likely due to the bronchocentric fibrosis around the major airways, which causes airflow limitation. It affects approximately three million patients worldwide and has a poor prognosis. Skin fibroblasts isolated from patients offer valuable insights into understanding the disease mechanisms, identifying the genetic causes, and developing personalized therapies. However, the use of skin fibroblasts to study a disease that exclusively impacts the lungs is often questioned, particularly since lung fibrosis primarily affects the alveolar epithelium. Method: We report the reprogramming of skin fibroblasts from patients with an atypical early-onset form of lung fibrosis into induced pluripotent stem cells (iPSCs) and subsequently into alveolar epithelial cells. This was achieved using a Sendai virus approach. Results: We show that the reprogrammed cells carry mutations in the calcium-binding protein genes S100A3 and S100A13, leading to diminished protein expression, thus mimicking the patients’ cells. Additionally, we demonstrate that the generated patient iPSCs exhibit aberrant calcium and mitochondrial functions. Conclusions: Due to the lack of a suitable animal model that accurately resembles the human disease, generating patient lung cells from these iPSCs can provide a valuable “disease in a dish” model for studying the atypical form of inherited lung fibrosis. This condition is associated with mutations in the calcium-binding protein genes S100A3 (NM_002960) and S100A13 (NM_001024210), aiding in the understanding of its pathogenesis. Full article

Current animal and in vitro cell culture models do not fully recapitulate the physiological and pathophysiological characteristics of the human lung. As a result, the translation of these models to clinical practice is very limited, and clinical trials initiated on the extrapolation of such data fail. Although current models are beneficial in fundamental research, there is a need to constantly improve models to more accurately predict outcomes in clinical trials and personalized medicine. Here, we report important strategies to develop a 3D lung model with human primary lung cells. Starting from the well-established air-liquid interface (ALI) culture system, we describe a gradual increase in the complexity of the system by co-culturing different primary cell types, by testing different coatings, and by adding a three-dimensional matrix. As a result, we have established a reproducible 3D in vitro model of the airway consisting of human primary cells representing a differentiated mucociliary airway epithelium, an underlying submucosa with fibroblasts, and an endothelial interface. Full article

Background and Objective: Primary ciliary dyskinesia (PCD) is a rare genetic disorder that affects the mucociliary system, leading to progressive lung damage. This deterioration can result in bronchiectasis, atelectasis, and respiratory failure, necessitating lung transplantation in severe cases. This study aims to assess ciliary motility and ultrastructure in the bronchial epithelium of transplanted lungs in patients with PCD to determine whether mucociliary function is preserved post-transplantation. The findings seek to enhance scientific understanding and provide prognostic insights for these patients. Materials and Methods: A prospective observational study was conducted on two patients with PCD and advanced lung disease who underwent bilateral lung transplantation. Nasal and bronchial cilia samples were analyzed using high-speed videomicroscopy and transmission electron microscopy. Follow-up assessments included ciliary function analysis, lung rejection monitoring, and quality-of-life evaluations, with follow-up extending up to 30 months post-transplant. Results: Post-transplant evaluations demonstrated normal ciliary motility and ultrastructure in the transplanted lungs throughout the study period (up to 30 months), indicating the long-term preservation of mucociliary function. Conclusions: Transplanted lungs in patients with PCD maintain normal bronchial ciliary motility and structure in the long term, suggesting a favorable prognosis for both the graft and the recipient. These findings support the feasibility and long-term effectiveness of lung transplantation in patients with PCD. Full article

Congenital diaphragmatic hernia (CDH) is a complex disorder whereby improper formation of the diaphragm allows herniation of the internal organs into the thoracic cavity, resulting in pulmonary hypoplasia among other complications. Although epithelial dysfunction is central to CDH pathology, relatively little attention has been paid to the underlying mechanisms orchestrating epithelial malfunction. Proinflammatory signaling downstream of impaired mechanotransduction due to in utero lung compression has been elucidated to drive epithelial cell phenotypes. This has been illustrated by a reduction in nuclear YAP and the upregulation of NF-kB in CDH models. In this review, we draw from recent findings using emerging technologies to examine epithelial cell mechanisms in CDH and discuss the role of compression as a central and, crucially, sufficient driver of CDH phenotypes. In recognition of the limitations of using genetic knockout models to recapitulate such a heterogenic and etiologically complicated disease, we discuss alternative models such as the established nitrofen rat model, air–liquid interface (ALI) cultures, organoids and ex vivo lung explants. Throughout, we acknowledge the importance of involving mechanical compression in the modeling of CDH in order to faithfully recapitulate the disease. Finally, we explore novel therapeutic strategies from stem cell and regenerative therapies to precision medicine and the importance of defining CDH endotypes in order to guide treatments. Full article

Pemphigus-associated interstitial lung disease (P-ILD) is a severe complication observed in pemphigus patients that is characterized by pulmonary interstitial inflammation and fibrosis. This study investigated the role of anti-desmoglein (Dsg) 1/3 antibodies in P-ILD pathogenesis and evaluated the therapeutic potential of molecular hydrogen (H₂). Using a BALB/cJGpt mouse model, we demonstrated that anti-Dsg 1 antibodies, but not anti-Dsg 3 antibodies, induced interstitial inflammation and fibrosis. Immunofluorescence staining confirmed IgG deposition in the alveolar epithelium, suggesting immune complex formation and epithelial damage. Gene expression analysis revealed elevated pro-inflammatory cytokines (IL-1β, IL-13) and upregulated pro-fibrotic markers (α-SMA, S100A4, TGF-β, and collagen genes) in P-ILD progression. Elevated oxidative stress and impaired ROS metabolism further implied the role of oxidative damage in disease pathogenesis. To assess H₂’s therapeutic potential, hydrogen-rich water was administered to P-ILD mice. H₂ treatment significantly reduced oxidative stress, attenuated interstitial inflammation, and prevented pulmonary fibrosis. These protective effects were attributed to H₂’s antioxidant properties, which restored the pro-oxidant–antioxidant balance. Our findings underscore the critical role of anti-Dsg 1 antibodies and oxidative stress in P-ILD and highlight H₂ as a promising therapeutic agent for mitigating anti-Dsg 1 antibody-induced lung injury. Full article

Hypercapnia, the elevation of CO₂ in blood and tissue, is a risk factor for mortality in patients with severe lung disease and pulmonary infections. We previously showed that hypercapnia increases viral replication and mortality in mice infected with influenza A virus (IAV). Elevated CO₂ also augmented cholesterol content and pseudo-SARS-CoV-2 entry in bronchial epithelial cells. Interestingly, cellular cholesterol facilitates IAV uptake, replication, assembly, and egress from cells. Here, we report that hypercapnia increases viral protein expression in airway epithelium of mice infected with IAV. Elevated CO₂ also enhanced IAV adhesion and internalization, viral protein expression, and viral replication in bronchial epithelial cells. Hypercapnia increased the expression and activation of the transcription factor sterol-regulatory element binding protein 2 (SREBP2), resulting in elevated expression of cholesterol synthesis enzymes, decreased expression of a cholesterol efflux transporter, and augmented cellular cholesterol. Moreover, reducing cellular cholesterol with an SREBP2 inhibitor or statins blocked hypercapnia-induced increases in viral adhesion and internalization, viral protein expression, and IAV replication. Inhibitors of mTOR and Akt also blocked the effect of hypercapnia on viral growth. Our findings suggest that targeting cholesterol synthesis and/or mTOR/Akt signaling may hold promise for reducing susceptibility to influenza infection in patients with advanced lung disease and hypercapnia. Full article

The COVID-19 pandemic highlighted the importance of mathematical modeling for understanding viral infection dynamics and accelerated its application into immunological research. Collaborative efforts among international research groups yielded a wealth of experimental data, which facilitated model development and validation. This study focuses on developing a modular mathematical model of the immune response, capturing the interactions between innate and adaptive immunity, with an application to SARS-CoV-2 infection. The model was validated using experimental data from middle-aged individuals with moderate COVID-19 progression, including measurements of viral load in the upper and lower airways, serum antibodies, CD4+ and CD8+ T cells, and interleukin-6 levels. Parameter optimization and sensitivity analysis were performed to improve the model accuracy. Additionally, identifiability analysis was conducted to assess whether the data were sufficient for reliable parameter estimation. The verified model simulates the dynamics of moderate, severe, and critical COVID-19 progressions using measured data on lung epithelium damage, viral load, and IL-6 levels as key indicators of disease severity. We also performed a series of validation scenarios to assess whether the model correctly reproduces biologically relevant behaviors under various conditions, such as immunity hyperactivation, co-infection with HIV, and interferon administration as a therapeutic strategy. The model was developed as a component of the Digital Twin project and represents a general immune module that integrates both innate and adaptive immunity. It can be utilized for further COVID-19 research or serve as a foundation for studying other infectious diseases, provided sufficient data are available. Full article

Interstitial lung diseases (ILD) represent a diverse group of disorders that primarily affect the pulmonary interstitium and, less commonly, involve the alveolar and vascular epithelium. Overlapping clinical, radiological and histopathological features make proper classification difficult, requiring multiple complementary methodologies, including flow cytometry of bronchoalveolar lavages (BAL). This retrospective study analyzed BAL flow cytometry data from 1074 real-life patients, quantifying alveolar macrophages, CD4/CD8 lymphocytes, neutrophils, eosinophils, and CD1a+ Langerhans cells, with the aim of evaluating its diagnostic utility in ILD and pulmonary infection. Clustering and logistic regression analyses identified seven distinct leukocyte profiles: lymphocytic (associated with hypersensitivity pneumonitis, cryptogenic organizing pneumonia, and lymphocytic interstitial pneumonia), sarcoidosis, macrophagic (including nonspecific interstitial pneumonia, desquamative interstitial pneumonitis, pneumoconiosis, and unclassifiable ILD), neutrophilic (including usual interstitial pneumonia, respiratory bronchiolitis ILD, and acute interstitial pneumonia), infectious diseases, eosinophilic ILD, and Langerhans cell histiocytosis. The estimated leukocyte profiles were associated with different overall survival (OS) outcomes. Neutrophilic profiles, both infectious and non-infectious, correlated with poorer OS, particularly in patients without pulmonary fibrosis. Furthermore, corticosteroids and other immunosuppressive therapies did not show significant OS differences across leukocyte profiles. Although the gold standard in BAL cytology continues to be cytopathology, these results support BAL flow cytometry as a rapid and reliable complementary tool to aid in the classification of interstitial lung diseases based on immune cell profiles, providing valuable predictive information and contributing to personalized therapeutic approaches. Full article

Parainfluenza viruses are a common cause of respiratory illness in many species. In this study, experimental, alphavirus-derived RNA particle vaccines either with or without adjuvant were evaluated against porcine parainfluenza virus 1 (PPIV1) challenge and compared to live virus exposure. Groups of ten, three-week-old pigs were vaccinated intramuscularly with an adjuvanted RNA particle (RPAdj/C) or non-adjuvanted RP (RP/C) or administered an intranasal live exposure (LE/C) dose of PPIV1 at 0- and 21-days post vaccination (DPV) followed by challenge with PPIV1 at 40 DPV. In addition, two groups were included as non-vaccinated, non-challenged (NV/NC) and non-vaccinated, challenged (NV/C) controls. Intranasal virus exposure and RP vaccination, regardless of adjuvant, reduced PPIV1 shedding in nasal swabs by 5 days post inoculation (DPI). All vaccinated or exposed pigs seroconverted as shown by enzyme-linked immunosorbent assay and serum virus neutralization. The antibody isotype detected in bronchoalveolar lavage fluid (BALF) LE/C was predominantly IgA while RP vaccination induced an IgG response. Reduced PPIV1 antigen was observed in the LE/C, RP/C and RPAdj/C groups in lung, trachea, or nasal turbinate epithelium. Additionally, the RPAdj vaccine significantly reduced nasal shedding compared to NV/C pigs although not as much as LE/C pigs. These results suggest vaccination could mitigate PPIV1 infection in commercial systems. Full article

Alveolar active sodium transport is essential for clearing edema from airspaces, in a process known as alveolar fluid clearance (AFC). Although it has been reported that atrial natriuretic peptide (ANP) attenuates AFC, little is known about the underlying molecular effects of natriuretic peptides (NPs). Therefore, we examined the contribution of NPs to AFC and their effects as mediators of active sodium transport. By using the isolated liquid-filled lungs model, we investigated the effects of NPs on AFC. The expression of NPs, Na⁺, K⁺-ATPase, and Na⁺ channels was assessed in alveolar epithelial cells. Congestive heart failure (CHF) was induced by using the aortocaval fistula model. ANP and brain NP (BNP) significantly reduced AFC rate from 0.49 ± 0.02 mL/h in sham rats to 0.26 ± 0.013 and 0.19 ± 0.005 in ANP and BNP-treated groups, respectively. These effects were mediated by downregulating the active Na⁺ transport components in the alveolar epithelium while enhancing the ubiquitination and degradation of αENaC in the lungs, as reflected by increased levels of Nedd4-2. In addition, AFC was reduced in compensated CHF rats treated with ANP, while in decompensated CHF, ANP partially restored AFC. In conclusion, NPs regulate AFC in health and CHF. This research could help optimize pharmacological treatments for severe CHF. Full article

The protease–antiprotease balance is involved in many biological processes, including blood coagulation, tissue remodeling, inflammation and immune responses. The aim of this study is to determine the balance between SERPINs and some related proteases in the lungs of stable COPD patients. In this cross-sectional study, the expression and localization of human SERPINs (anti-proteases) and some related proteases were measured in the lung parenchyma of mild-moderate COPD (MCOPD, n = 13) patients, control smokers (CS, n = 14) and control nonsmokers (CNS, n = 12) using transcriptome analysis, immunohistochemistry, and ELISA tests. Peripheral lung transcriptomic data showed increased mRNA levels of tissue plasminogen activator (tPA), cathepsin-L and caspase-1 as well as increased SERPINs A6, B3, B5, B11, B13 in the COPD group compared to the CNS group. At the protein level, IHC analysis showed that tPA and cathepsin-L increased in the bronchiolar epithelium and alveolar septa of the CS and COPD groups compared to the CNS group, as well as SERPINB5 and B13 in the alveolar macrophages and alveolar septa of the CS and COPD groups compared to the CNS group. SERPINA6 was shown to be decreased in the bronchiolar epithelium, bronchiolar lamina propria, and alveolar septa of the CS and COPD groups compared to the CNS group and was positively correlated with lung function. SERPINB3 was decreased in the alveolar septa of the CS group compared to the CNS group. The ELISA tests showed that in the total lung extracts, decreased levels of SERPINA6 and increased caspase-1 were shown in the COPD group compared to the CNS or both control groups, respectively. These data show an imbalance, at the protein level, of SERPINs and some related proteases in the lungs of the CS and stable COPD groups. These alterations may play a role in damaging the lung parenchyma of susceptible COPD patients. Full article

ARDS is a challenging syndrome in which the hallmark is alveolar epithelium damage, with the consequent extravasation of fluids into the interstitium and alveolar space. Patients with severe ARDS almost always require mechanical ventilation and aggressive fluid resuscitation, at least in the initial phases. The increased intrathoracic pressure during positive pressure ventilation reduces cardiac output, worsening the circulatory status of these patients even more. In this pathological context, fluid therapies serve as a means to restore intravascular volume but can simultaneously play a detrimental role, increasing the amount of liquid in the lungs and worsening gas exchange and lung mechanics. Indeed, clinical research suggests that fluid overload leads to worsening outcomes, mostly in terms of gas exchange, days of mechanical ventilation, and ICU stay duration. For these reasons, this review aims to provide basic information about ARDS pathophysiology and heart–lung interactions, the understanding of which is essential to guide fluid therapy, together with the close monitoring of hemodynamics and fluid responsiveness. Full article

The mpox virus (MPXV) Clade IIb outbreak in 2022 was the biggest one ever to occur outside Africa, causing different types of clinical symptoms and levels of disease severity. There is no currently approved treatment for mpox, but Tecovirimat has proven effective against known orthopoxviruses in several animal models and Vero cell cultures. Since serious complications, including lung involvement, have been reported, especially in immunocompromised people, we investigated the effects of MPXV infection on the in vitro model of lung airway epithelium (Calu-3 cell line) and examined MPXV replication kinetic and related ultrastructural changes, also performing dose–response studies to measure Tecovirimat antiviral activity. Our results highlighted an active replication of MPXV in Calu-3 cells linked to mitochondrial structural modifications with perinuclear relocation and the formation of cytoplasmic vacuoles. Treatment with Tecovirimat consistently reduced viral replication both in supernatants (81%) and inside cells (77%) and ultimately stopped viral infectivity (92% of cytopathic effect reduction) after 48 h of infection. Drug administration inhibited the final wrapping of mature viral particles, causing extensive cytoplasmic vacuolation. Our results demonstrated Tecovirimat’s in vitro effectiveness against MPXV at the nanomolar concentration on Calu-3 cells. This suggests a potential rationale for using this drug for patients with mpox severe disease and lung involvement. Full article