Nanoparticles and Airway Epithelial Cells: Exploring the Impacts and Methodologies in Toxicity Assessment.

The production of nanoparticles has recently surged due to their varied applications in the biomedical, pharmaceutical, textile, and electronic sectors. However, this rapid increase in nanoparticle manufacturing has raised concerns about environmental pollution, particularly its potential adverse effects on human health. Among the various concerns, inhalation exposure to nanoparticles poses significant risks, especially affecting the respiratory system. Airway epithelial cells play a crucial role as the primary defense against inhaled particulate matter and pathogens. Studies have shown that nanoparticles can disrupt the airway epithelial barrier, triggering inflammatory responses, generating reactive oxygen species, and compromising cell viability. However, our understanding of how different types of nanoparticles specifically impact the airway epithelial barrier remains limited. Both in vitro cell culture and in vivo murine models are commonly utilized to investigate nanoparticle-induced cellular responses and barrier dysfunction. This review discusses the methodologies frequently employed to assess nanoparticle toxicity and barrier disruption. Furthermore, we analyze and compare the distinct effects of various nanoparticle types on the airway epithelial barrier. By elucidating the diverse responses elicited by different nanoparticles, we aim to provide insights that can guide future research endeavors in assessing and mitigating the potential risks associated with nanoparticle exposure.

8-Bromo-cAMP attenuates human airway epithelial barrier disruption caused by titanium dioxide fine and nanoparticles.

Titanium dioxide fine particles (TiO2-FPs) and nanoparticles (TiO2-NPs) are the most widely used whitening pigments worldwide. Inhalation of TiO2-FPs and TiO2-NPs can be harmful as it triggers toxicity in the airway epithelial cells. The airway epithelium serves as the respiratory system's first line of defense in which airway epithelial cells are significant targets of inhaled pathogens and environmental particles. Our group previously found that TiO2-NPs lead to a disrupted barrier in the polarized airway epithelial cells. However, the effect of TiO2-FPs on the respiratory epithelial barrier has not been examined closely. In this study, we aimed to compare the effects of TiO2-FPs and TiO2-NPs on the structure and function of the airway epithelial barrier. Additionally, we hypothesized that 8-Bromo-cAMP, a cyclic adenosine monophosphate (cAMP) derivative, would alleviate the disruptive effects of both TiO2-FPs and TiO2-NPs. We observed increased epithelial membrane permeability in both TiO2-FPs and TiO2-NPs after exposure to 16HBE cells. Immunofluorescent labeling showed that both particle sizes disrupted the structural integrity of airway epithelial tight junctions and adherens junctions. TiO2-FPs had a slightly more, but insignificant impact on the epithelial barrier disruption than TiO2-NPs. Treatment with 8-Bromo-cAMP significantly attenuated the barrier-disrupting impact of both TiO2-FPs and TiO2-NPs on cell monolayers. Our study demonstrates that both TiO2-FPs and TiO2-NPs cause comparable barrier disruption and suggests a protective role for cAMP signaling. The observed effects of TiO2-FPs and TiO2-NPs provide a necessary understanding for characterizing the pathways involved in the defensive role of the cAMP pathway on TiO2-induced airway barrier disruption.

Electronic cigarette exposure disrupts airway epithelial barrier function and exacerbates viral infection.

The use of electronic cigarettes (e-cigs), especially among teenagers, has reached alarming and epidemic levels, posing a significant threat to public health. However, the short- and long-term effects of vaping on the airway epithelial barrier are unclear. Airway epithelial cells are the forefront protectors from viruses and pathogens. They contain apical junctional complexes (AJCs), which include tight junctions (TJs) and adherens junctions (AJs) formed between adjacent cells. Previously, we reported respiratory syncytial virus (RSV) infection, the leading cause of acute lower respiratory infection-related hospitalization in children and high-risk adults, induces a "leaky airway" by disrupting the epithelial AJC structure and function. We hypothesized chemical components of e-cigs disrupt airway epithelial barrier and exacerbate RSV-induced airway barrier dysfunction. Using confluent human bronchial epithelial (16HBE) cells and well-differentiated normal human bronchial epithelial (NHBE) cells, we found that exposure to extract and aerosol e-cig nicotine caused a significant decrease in transepithelial electrical resistance (TEER) and the structure of the AJC even at noncytotoxic concentrations. Western blot analysis of 16HBE cells exposed to e-cig nicotine extract did not reveal significant changes in AJC proteins. Exposure to aerosolized e-cig cinnamon or menthol flavors also induced barrier disruption and aggravated nicotine-induced airway barrier dysfunction. Moreover, preexposure to nicotine aerosol increased RSV infection and the severity of RSV-induced airway barrier disruption. Our findings demonstrate that e-cig exposure disrupts the airway epithelial barrier and exacerbates RSV-induced damage. Knowledge gained from this study will provide awareness of adverse e-cig respiratory effects and positively impact the mitigation of e-cig epidemic.NEW & NOTEWORTHY Electronic cigarette (e-cig) use, especially in teens, is alarming and at epidemic proportions, threatening public health. Our study shows that e-cig nicotine exposure disrupts airway epithelial tight junctions and increases RSV-induced barrier dysfunction. Furthermore, exposure to aerosolized flavors exaggerates e-cig nicotine-induced airway barrier dysfunction. Our study confirms that individual and combined components of e-cigs deleteriously impact the airway barrier and that e-cig exposure increases susceptibility to viral infection.

Vitamin D3 protects against respiratory syncytial virus-induced barrier dysfunction in airway epithelial cells via PKA signaling pathway.

Respiratory syncytial virus (RSV) is the leading cause of acute lower respiratory tract infection in infants and young children globally and is responsible for hospitalization and mortality in the elderly population. Virus-induced airway epithelial barrier damage is a critical step during RSV infection, and emerging studies suggest that RSV disrupts the tight junctions (TJs) and adherens junctions (AJs) between epithelial cells, increasing the permeability of the airway epithelial barrier. The lack of commercially available vaccines and effective antiviral drugs for RSV emphasizes the need for new management strategies. Vitamin D3 is a promising intervention for viral infection due to its critical role in modulating innate immune responses. However, there is limited evidence on the effect of vitamin D3 on RSV pathogenies. Here, we investigated the impact of vitamin D3 on RSV-induced epithelial barrier dysfunction and the underlying mechanisms. We found that pre-incubation with 1,25(OH)2D3, the active form of vitamin D3, alleviated RSV-induced epithelial barrier disruption in a dose-dependent manner without affecting viability in 16HBE cells. 1,25(OH)2D3 induced minor changes in the protein expression level of TJ/AJ proteins in RSV-infected cells. We observed increased CREB phosphorylation at Ser133 during 1,25(OH)2D3 exposure, indicating that vitamin D3 triggered protein kinase A (PKA) activity in 16HBE. PKA inhibitors modified the restoration of barrier function by 1,25(OH)2D3 in RSV-infected cells, implying that PKA signaling is responsible for the protective effects of vitamin D3 against RSV-induced barrier dysfunction in airway epithelial cells. Our findings suggest vitamin D3 as a prophylactic intervention to protect the respiratory epithelium during RSV infections.

Airway Epithelial Cell Junctions as Targets for Pathogens and Antimicrobial Therapy.

Intercellular contacts between epithelial cells are established and maintained by the apical junctional complexes (AJCs). AJCs conserve cell polarity and build epithelial barriers to pathogens, inhaled allergens, and environmental particles in the respiratory tract. AJCs consist of tight junctions (TJs) and adherens junctions (AJs), which play a key role in maintaining the integrity of the airway barrier. Emerging evidence has shown that different microorganisms cause airway barrier dysfunction by targeting TJ and AJ proteins. This review discusses the pathophysiologic mechanisms by which several microorganisms (bacteria and viruses) lead to the disruption of AJCs in airway epithelial cells. We present recent progress in understanding signaling pathways involved in the formation and regulation of cell junctions. We also summarize the potential chemical inhibitors and pharmacological approaches to restore the integrity of the airway epithelial barrier. Understanding the AJCs-pathogen interactions and mechanisms by which microorganisms target the AJC and impair barrier function may further help design therapeutic innovations to treat these infections.

Respiratory syncytial virus disrupts the airway epithelial barrier by decreasing cortactin and destabilizing F-actin.

Respiratory syncytial virus (RSV) infection is the leading cause of acute lower respiratory tract infection in young children worldwide. Our group recently revealed that RSV infection disrupts the airway epithelial barrier in vitro and in vivo. However, the underlying molecular pathways were still elusive. Here, we report the critical roles of the filamentous actin (F-actin) network and actin-binding protein cortactin in RSV infection. We found that RSV infection causes F-actin depolymerization in 16HBE cells, and that stabilizing the F-actin network in infected cells reverses the epithelial barrier disruption. RSV infection also leads to significantly decreased cortactin in vitro and in vivo. Cortactin-knockout 16HBE cells presented barrier dysfunction, whereas overexpression of cortactin protected the epithelial barrier against RSV. The activity of Rap1 (which has Rap1A and Rap1B forms), one downstream target of cortactin, declined after RSV infection as well as in cortactin-knockout cells. Moreover, activating Rap1 attenuated RSV-induced epithelial barrier disruption. Our study proposes a key mechanism in which RSV disrupts the airway epithelial barrier via attenuating cortactin expression and destabilizing the F-actin network. The identified pathways will provide new targets for therapeutic intervention toward RSV-related disease. This article has an associated First Person interview with the first author of the paper.

Effects of Vertical Transmission of Respiratory Viruses to the Offspring.

Overt and subclinical maternal infections in pregnancy can have multiple and significant pathological consequences for the developing fetus, leading to acute perinatal complications and/or chronic disease throughout postnatal life. In this context, the current concept of pregnancy as a state of systemic immunosuppression seems oversimplified and outdated. Undoubtedly, in pregnancy the maternal immune system undergoes complex changes to establish and maintain tolerance to the fetus while still protecting from pathogens. In addition to downregulated maternal immunity, hormonal changes, and mechanical adaptation (e.g., restricted lung expansion) make the pregnant woman more susceptible to respiratory pathogens, such as influenza virus, respiratory syncytial virus (RSV), and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Depending on the infectious agent and timing of the infection during gestation, fetal pathology can range from mild to severe, and even fatal. Influenza is associated with a higher risk of morbidity and mortality in pregnant women than in the general population, and, especially during the third trimester of pregnancy, mothers are at increased risk of hospitalization for acute cardiopulmonary illness, while their babies show higher risk of complications such as prematurity, respiratory and neurological illness, congenital anomalies, and admission to neonatal intensive care. RSV exposure in utero is associated with selective immune deficit, remodeling of cholinergic innervation in the developing respiratory tract, and abnormal airway smooth muscle contractility, which may predispose to postnatal airway inflammation and hyperreactivity, as well as development of chronic airway dysfunction in childhood. Although there is still limited evidence supporting the occurrence of vertical transmission of SARS-CoV-2, the high prevalence of prematurity among pregnant women infected by SARS-CoV-2 suggests this virus may alter immune responses at the maternal-fetal interface, affecting both the mother and her fetus. This review aims at summarizing the current evidence about the short- and long-term consequences of intrauterine exposure to influenza, RSV, and SARS-CoV-2 in terms of neonatal and pediatric outcomes.

RSV-induced changes in a 3-dimensional organoid model of human fetal lungs.

We have shown that respiratory syncytial virus (RSV) can spread hematogenously from infected airways of a pregnant woman to the developing fetal lungs in utero. This study sought to measure RSV replication, cytopathic effects, and protein expression in human lung organoids (HLOs) reproducing architecture and transcriptional profiles of human fetal lungs during the 1st trimester of gestation. HLOs derived from human pluripotent stem cells were microinjected after 50 or 100 days in culture with medium or recombinant RSV-A2 expressing the red fluorescent protein gene (rrRSV). Infection was monitored by fluorescent microscopy and PCR. Immunohistochemistry and proteomic analysis were performed. RSV infected HLOs in a dose- and time-dependent manner. RSV-infected HLOs increased expression of CC10 (Club cells), but had sparse FOXJ1 (ciliated cells). Disruption of F-actin cytoskeleton was consistent with proteomic data showing a significant increase in Rho GTPases proteins. RSV upregulated the transient receptor potential vanilloid 1 (TRPV1) channel and, while ß2 adrenergic receptor (ß2AR) expression was decreased overall, its phosphorylated form increased. Our data suggest that prenatal RSV infection produces profound changes in fetal lungs' architecture and expression profiles and maybe an essential precursor of chronic airway dysfunction. expression profiles, and possibly be an important precursor of chronic airway dysfunction.

Role of air pollutants in airway epithelial barrier dysfunction in asthma and COPD.

Chronic exposure to environmental pollutants is a major contributor to the development and progression of obstructive airway diseases, including asthma and COPD. Understanding the mechanisms underlying the development of obstructive lung diseases upon exposure to inhaled pollutants will lead to novel insights into the pathogenesis, prevention and treatment of these diseases. The respiratory epithelial lining forms a robust physicochemical barrier protecting the body from inhaled toxic particles and pathogens. Inhalation of airborne particles and gases may impair airway epithelial barrier function and subsequently lead to exaggerated inflammatory responses and airway remodelling, which are key features of asthma and COPD. In addition, air pollutant-induced airway epithelial barrier dysfunction may increase susceptibility to respiratory infections, thereby increasing the risk of exacerbations and thus triggering further inflammation. In this review, we discuss the molecular and immunological mechanisms involved in physical barrier disruption induced by major airborne pollutants and outline their implications in the pathogenesis of asthma and COPD. We further discuss the link between these pollutants and changes in the lung microbiome as a potential factor for aggravating airway diseases. Understanding these mechanisms may lead to identification of novel targets for therapeutic intervention to restore airway epithelial integrity in asthma and COPD.

Pediatric pneumothorax: Case studies and review of current literature.

Pneumothorax is an abnormal collection of air between the lung and chest wall. Pneumothorax management guidelines put forth by the American College of Chest Physicians, European Respiratory Society, and British Thoracic Society are specific to adult patients. These guidelines' utility has not been addressed in pediatric populations, which causes significant management variation in younger patients. Additionally, pneumothorax management ranges from conservative to surgical treatment, but these approaches, timelines, and effectiveness have not been validated in significant numbers of pediatric patients. Here, we present three cases of pediatric pneumothorax with variable clinical courses-one with persistent air leak despite chest tube presence who underwent VATS and surgical resection of apical blebs. In contrast, the other two cases were managed more conservatively. We reviewed the current literature for diagnostic and management of pediatric patients with pneumothorax, which underscores the need for guidelines specific to this population.

Respiratory syncytial virus induces ß2-adrenergic receptor dysfunction in human airway smooth muscle cells.

Pharmacologic agonism of the ß2-adrenergic receptor (ß2AR) induces bronchodilation by activating the enzyme adenylyl cyclase to generate cyclic adenosine monophosphate (cAMP). ß2AR agonists are generally the most effective strategy to relieve acute airway obstruction in asthmatic patients, but they are much less effective when airway obstruction in young patients is triggered by infection with respiratory syncytial virus (RSV). Here, we investigated the effects of RSV infection on the abundance and function of ß2AR in primary human airway smooth muscle cells (HASMCs) derived from pediatric lung tissue. We showed that RSV infection of HASMCs resulted in proteolytic cleavage of ß2AR mediated by the proteasome. RSV infection also resulted in ß2AR ligand-independent activation of adenylyl cyclase, leading to reduced cAMP synthesis compared to that in uninfected control cells. Last, RSV infection caused stronger airway smooth muscle cell contraction in vitro due to increased cytosolic Ca2+ concentrations. Thus, our results suggest that RSV infection simultaneously induces loss of functional ß2ARs and activation of multiple pathways favoring airway obstruction in young patients, with the net effect of counteracting ß2AR agonist-induced bronchodilation. These findings not only provide a potential mechanism for the reported lack of clinical efficacy of ß2AR agonists for treating virus-induced wheezing but also open the path to developing more precise therapeutic strategies.

RSV attenuates epithelial cell restitution by inhibiting actin cytoskeleton-dependent cell migration.

The airway epithelium's ability to repair itself after injury, known as epithelial restitution, is an essential mechanism enabling the respiratory tract's normal functions. Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections worldwide. We sought to determine whether RSV delays the airway epithelium wound repair process both in vitro and in vivo. We found that RSV infection attenuated epithelial cell migration, a step in wound repair, promoted stress fiber formation, and mediated assembly of large focal adhesions. Inhibition of Rho-associated kinase, a master regulator of actin function, reversed these effects. There was increased RhoA and phospho-myosin light chain 2 following RSV infection. In vivo, mice were intraperitoneally inoculated with naphthalene to induce lung injury, followed by RSV infection. RSV infection delayed reepithelialization. There were increased concentrations of phospho-myosin light chain 2 in day 7 naphthalene + RSV animals, which normalized by day 14. This study suggests a key mechanism by which RSV infection delays wound healing.

RSV infection potentiates TRPV1-mediated calcium transport in bronchial epithelium of asthmatic children.

The transient receptor potential vanilloid 1 (TRPV1) channel is expressed in human bronchial epithelium (HBE), where it transduces Ca2+ in response to airborne irritants. TRPV1 activation results in bronchoconstriction, cough, and mucus production, and may therefore contribute to the pathophysiology of obstructive airway disease. Since children with asthma face the greatest risk of developing virus-induced airway obstruction, we hypothesized that changes in TRPV1 expression, localization, and function in the airway epithelium may play a role in bronchiolitis and asthma in childhood. We sought to measure TRPV1 protein expression, localization, and function in HBE cells from children with versus without asthma, both at baseline and after RSV infection. We determined changes in TRPV1 protein expression, subcellular localization, and function both at baseline and after RSV infection in primary HBE cells from normal children and children with asthma. Basal TRPV1 protein expression was higher in HBE from children with versus without asthma and primarily localized to plasma membranes (PMs). During RSV infection, TRPV1 protein increased more in the PM of asthmatic HBE as compared with nonasthmatic cells. TRPV1-mediated increase in intracellular Ca2+ was greater in RSV-infected asthmatic cells, but this increase was attenuated when extracellular Ca2+ was removed. Nerve growth factor (NGF) recapitulated the effect of RSV on TRPV1 activation in HBE cells. Our data suggest that children with asthma have intrinsically hyperreactive airways due in part to higher TRPV1-mediated Ca2+ influx across epithelial membranes, and this abnormality is further exacerbated by NGF overexpression during RSV infection driving additional Ca2+ from intracellular stores.

Airway tight junctions as targets of viral infections.

The apical junctional complexes (AJCs) of airway epithelial cells are a key component of the innate immune system by creating barriers to pathogens, inhaled allergens, and environmental particles. AJCs form between adjacent cells and consist of tight junctions (TJs) and adherens junctions (AJs). Respiratory viruses have been shown to target various components of the AJCs, leading to airway epithelial barrier dysfunction by different mechanisms. Virus-induced epithelial permeability may allow for allergens and bacterial pathogens to subsequently invade. In this review, we discuss the pathophysiologic mechanisms leading to disruption of AJCs and the potential ensuing ramifications. We focus on the following viruses that affect the pulmonary system: respiratory syncytial virus, rhinovirus, influenza viruses, immunodeficiency virus, and other viruses such as coxsackievirus, adenovirus, coronaviruses, measles, parainfluenza virus, bocavirus, and vaccinia virus. Understanding the mechanisms by which viruses target the AJC and impair barrier function may help design therapeutic innovations to treat these infections.

Respiratory syncytial virus seropositivity at birth is associated with adverse neonatal respiratory outcomes.

BACKGROUND: More than 60 years since the discovery of the respiratory syncytial virus (RSV), the effects of prenatal exposure to this virus remain largely unknown. In this investigation, we sought to find evidence of RSV seroconversion in cord blood and explore its clinical implications for the newborn. METHODS: Offspring from 22 pregnant women with a history of viral respiratory infection during the third trimester of pregnancy (respiratory viral illness [RVI] group) and 40 controls were enrolled in this study between 1 September 2016 and 31 March 2019. Cord blood sera were tested for anti-RSV antibodies by indirect fluorescent antibody assay. RSV seropositivity was defined as the presence of anti-RSV immunoglobulin M (IgM) or immunoglobulin A (IgA), in addition to IgG in cord blood serum at ≥1:20 dilution. RESULTS: Anti-RSV IgG was present in all cord blood serum samples from infants born to RVI mothers (95% confidence interval [CI] = 82%-100%), with 16 samples also having elevated titers for either anti-RSV IgA or IgM (73%; 95% CI = 52%-87%). No controls had evidence of anti-RSV antibodies. Eight (50%) seropositive newborns developed at least one respiratory tract finding, including respiratory distress syndrome (N = 8), respiratory failure (N = 3), and pneumonia (N = 1). RSV seropositive newborns also required more days on oxygen, had leukocytosis and elevated C-reactive protein (P = .025, P = .047, and P < .001, respectively). CONCLUSION: This study provides evidence of acute seropositivity against RSV in cord blood of newborns delivered from mothers with a history of upper respiratory tract illness in the third trimester. Cord blood seropositivity for anti-RSV IgA or IgM was associated with adverse clinical and laboratory outcomes in newborns.

Titanium dioxide nanoparticles exaggerate respiratory syncytial virus-induced airway epithelial barrier dysfunction.

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in children worldwide. While most develop a mild, self-limiting illness, some develop severe acute lower respiratory infection and persistent airway disease. Exposure to ambient particulate matter has been linked to asthma, bronchitis, and viral infection in multiple epidemiological studies. We hypothesized that coexposure to nanoparticles worsens RSV-induced airway epithelial barrier dysfunction. Bronchial epithelial cells were incubated with titanium dioxide nanoparticles (TiO2-NP) or a combination of TiO2-NP and RSV. Structure and function of epithelial cell barrier were analyzed. Viral titer and the role of reactive oxygen species (ROS) generation were evaluated. In vivo, mice were intranasally incubated with TiO2-NP, RSV, or a combination. Lungs and bronchoalveolar lavage (BAL) fluid were harvested for analysis of airway inflammation and apical junctional complex (AJC) disruption. RSV-induced AJC disruption was amplified by TiO2-NP. Nanoparticle exposure increased viral infection in epithelial cells. TiO2-NP induced generation of ROS, and pretreatment with antioxidant, N-acetylcysteine, reversed said barrier dysfunction. In vivo, RSV-induced injury and AJC disruption were augmented in the lungs of mice given TiO2-NP. Airway inflammation was exacerbated, as evidenced by increased white blood cell infiltration into the BAL, along with exaggeration of peribronchial inflammation and AJC disruption. These data demonstrate that TiO2-NP exposure exacerbates RSV-induced AJC dysfunction and increases inflammation by mechanisms involving generation of ROS. Further studies are required to determine whether NP exposure plays a role in the health disparities of asthma and other lung diseases, and why some children experience more severe airway disease with RSV infection.

The importance of anti-vaping vigilance-EVALI in seven adolescent pediatric patients in Northeast Ohio.

As of 18 February 2020, the e-cigarette or vaping product use-associated lung injury (EVALI) epidemic has claimed the lives of 68 patients in the USA with the total number of reported cases standing at 2807 to date. We present the clinical and radiologic findings, course of illness, and treatment of EVALI in seven adolescent patients in Northeast Ohio. Five of our patients required supplemental oxygen with four requiring intensive care unit care for respiratory support during admission. Three patients were treated with systemic steroids while inpatient. Bilateral opacities were seen on radiographic imaging of all seven of our patients. All patients were discharged alive on room air. However, impaired diffusing capacity of the lungs for carbon monoxide (DLCO) with nonobstructive spirometry was seen in patients that were tested postdischarge. This suggests that although recovery from the acute illness process of EVALI is achieved, there may be long-term impact on lung function in these patients. We recommend close follow-up with a pediatric pulmonologist where spirometry and DLCO can be performed.

Respiratory syncytial virus exhibits differential tropism for distinct human placental cell types with Hofbauer cells acting as a permissive reservoir for infection.

BACKGROUND: Respiratory syncytial virus (RSV) is capable of transient viremia and extrapulmonary dissemination. Recently, this virus has been identified in fetal cord blood, suggesting the possibility of in utero acquisition in humans. Here, we assess permissivity and kinetics of RSV replication in primary human placental cells, examine their potential to transfer this infection to neighboring cells, and measure the inflammatory response evoked by the virus. METHODS AND FINDINGS: Human placental villus tissue was collected immediately upon delivery and processed for isolation of placental cytotrophoblast, fibroblast, and macrophage (Hofbauer) cells. Isolated cells were infected with a recombinant RSV-A2 strain (rrRSV) expressing red fluorescent protein (RFP) and analyzed by fluorescence microscopy, Western blot, and quantitative PCR (qPCR). Based on RFP expression, rrRSV exhibited differential tropism for the three major placental cell types. Placental fibroblasts and Hofbauer cells were permissive and supported productive rrRSV replication. While infected cytotrophoblast cells expressed viral glycoprotein (G protein), only limited RSV replication was detected. Importantly, qPCR and fluorescence-focused unit assay revealed that the viral progeny remains trapped within infected Hofbauer cells for up to 30 days, with no release into surrounding media. Yet, Hofbauer cells passed the infection onto overlaid naïve 16HBE cells, suggesting contact-dependent trans-infection. Lastly, a significant increase in proinflammatory cytokines, particularly IL-6, TNF-alpha, and IFN-gamma was measured in the supernatant of infected Hofbauer cells by multiplex cytokine assay and conventional ELISA. CONCLUSIONS: This study demonstrates that RSV can replicate in human placenta, exhibits differential tropism for distinct placental cell types, can be stored and transferred to neighboring cells by Hofbauer cells, and elicits an inflammatory response. It also supports the hypothesis that this respiratory virus can be vertically transferred to the fetus and potentially affect its development and the outcome of pregnancies.

Serum neurotrophins at birth correlate with respiratory and neurodevelopmental outcomes of premature infants.

OBJECTIVE: Preterm birth is a significant cause of infant morbidity and mortality, which are primarily the result of respiratory and neurodevelopmental complications. However, no objective biomarker is currently available to predict at birth the risk and severity of such complications. Thus, we sought to determine whether serum neurotrophins concentrations measured at birth correlate with risk for later development of bronchopulmonary dysplasia (BPD) and long-term neurodevelopmental outcomes. METHODS: This study prospectively included 223 newborns admitted to neonatal intensive care units (NICU) and divided into three groups: (i) preterm infants who developed BPD; (ii) preterm infants who did not develop BPD; (iii) term infants. An exploratory cohort was enrolled in West Virginia, followed by a validation cohort recruited in four NICUs in Ohio. Specimens for serum and tracheal neurotrophins concentrations were collected within 48 h of admission. Infants requiring a fraction of inspired oxygen >0.21 for at least 28 days were diagnosed with BPD. Neurodevelopmental outcomes were extrapolated from Bayley Scales of Infant Development-Third Edition (BSID-III) administered at the 24-month follow-up visit. RESULTS: Serum brain-derived neurotrophic factor (BDNF) concentration at birth had significant negative correlation with later diagnosis of BPD (P = 0.011) and with duration of invasive ventilation and oxygen supplementation (P = 0.009 and 0.015, respectively). Serum nerve growth factor (NGF) concentration at birth had significant positive correlation with BSID-III cognitive and language composite scores at 24 months (P < 0.001 and 0.010, respectively). CONCLUSIONS: These data suggest that serum neurotrophins concentrations measured at birth provide prognostic information on subsequent respiratory and neurodevelopmental outcomes.

Disruption of the airway epithelial barrier in a murine model of respiratory syncytial virus infection.

Respiratory syncytial virus (RSV) is a major cause of hospitalization for infants and young children worldwide. RSV is known to infect epithelial cells and increase the permeability of model airway epithelial monolayers in vitro. We hypothesized that RSV infection also induces airway barrier dysfunction in vivo. C57BL/6 mice were intranasally inoculated with RSV, and on day 4 post-inoculation were examined for viral replication, lung inflammation, and barrier integrity as well as the structure and molecular composition of epithelial junctions. In parallel, primary mouse tracheal epithelial cells (mTEC) were cultured for in vitro studies. RSV-infected mice lost weight and showed significant peribronchial inflammation compared with noninfected controls and UV-inactivated RSV-inoculated animals. RSV infection increased the permeability of the airway epithelial barrier and altered the molecular composition of epithelial tight junctions. The observed RSV-induced barrier disruption was accompanied by decreased expression of several tight-junction proteins and accumulation of cleaved extracellular fragments of E-cadherin in bronchoalveolar lavage and mTEC supernatants. Similarly, in vitro RSV infection of mTEC monolayers resulted in enhanced permeability and disruption of tight-junction structure. Furthermore, incubation of mTEC monolayers with a recombinant fragment of E-cadherin caused tight-junction disassembly. Taken together, these data indicate that RSV infection leads to airway barrier dysfunction in vivo, mediated by either decreased expression or cleavage of junctional proteins. Our observations provide further insights into the pathophysiology of RSV infection and provide a rationale for development of barrier-protecting agents to alleviate the pathogenic effects of RSV infection.