RSV-infected Airway Epithelial Cells Research Articles

Respiratory syncytial virus infection changes the piwi-interacting RNA content of airway epithelial cells.

Piwi-interacting RNAs (piRNAs) are small non-coding RNAs (sncRNAs) of about 26–32 nucleotides in length and represent the largest class of sncRNA molecules expressed in animal cells. piRNAs have been shown to play a crucial role to safeguard the genome, maintaining genome complexity and integrity, as they suppress the insertional mutations caused by transposable elements. However, there is growing evidence for the role of piRNAs in controlling gene expression in somatic cells as well. Little is known about changes in piRNA expression and possible function occurring in response to viral infections. In this study, we investigated the piRNA expression profile, using a human piRNA microarray, in human small airway epithelial (SAE) cells infected with respiratory syncytial virus (RSV), a leading cause of acute respiratory tract infections in children. We found a time-dependent increase in piRNAs differentially expressed in RSV-infected SAE cells. We validated the top piRNAs upregulated and downregulated at 24 h post-infection by RT-qPCR and identified potential targets. We then used Gene Ontology (GO) tool to predict the biological processes of the predicted targets of the most represented piRNAs in infected cells over the time course of RSV infection. We found that the most significant groups of targets of regulated piRNAs are related to cytoskeletal or Golgi organization and nucleic acid/nucleotide binding at 15 and 24 h p.i. To identify common patterns of time-dependent responses to infection, we clustered the significantly regulated expression profiles. Each of the clusters of temporal profiles have a distinct set of potential targets of the piRNAs in the cluster Understanding changes in piRNA expression in RSV-infected airway epithelial cells will increase our knowledge of the piRNA role in viral infection and might identify novel therapeutic targets for viral lung-mediated diseases.

MLKL Regulates Rapid Cell Death-independent HMGB1 Release in RSV Infected Airway Epithelial Cells.

Respiratory syncytial virus (RSV)-induced bronchiolitis is a significant contributor to infant morbidity and mortality. Previously, we identified that necroptosis, a pro-inflammatory form of cell death mediated by receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and RIPK3, and mixed lineage kinase domain like protein (MLKL), occurs in RSV-infected human airway epithelial cells (hAECs), mediating the release of the alarmin high mobility group box 1 (HMGB1). Here, we show that RSV infection of hAECs induces the biphasic release of HMGB1 at 6 (“early”) and 24 (“late”) hours post infection (hpi). The early phase of HMGB1 release at 6 hpi is cell death-independent, however, this release is nonetheless attenuated by inhibition of MLKL (primarily associated with necroptosis). The early release of HMGB1 promotes the late phase of HMGB1 release via the activation of RAGE (receptor for advanced glycation endproducts) and occurs with cell death. Treatment of hAECS with exogenous HMGB1 combined with a pan-caspase inhibitor induces hAEC necroptosis, and is attenuated by the RAGE antagonist, FPS-ZM1. Together, these findings demonstrate that RSV infection of hAECs leads to the early release of HMGB1, followed by a paracrine feed-forward amplification loop that further increases HMGB1 levels and promotes cell death. As the inhibition of MLKL or targeting of HMGB1/RAGE pathway attenuates the release of pro-inflammatory HMGB1 and decreases viral load, this suggests that the pharmacological targeting of these pathways may be of benefit for the treatment of severe RSV bronchiolitis.

ITGB4 deficiency induces mucus hypersecretion by upregulating MUC5AC in RSV-infected airway epithelial cells.

Respiratory syncytial virus (RSV) infection is the main cause of bronchiolitis in children. Excessive mucus secretion is one of the primary symbols in RSV related lower respiratory tract infections (RSV-related LRTI), which is closely associated with the occurrence and development of asthma in later life. Integrin β4 (ITGB4) is down-regulated in the airway epithelial cells (AECs) of asthma patients which plays a critical role in the pathogenesis of asthma. However, whether ITGB4 is involved in the pathological processes of RSV infection remains unclear. In this study, we found that decreased expression of ITGB4 was negatively correlated with the level of MUC5AC in childhood AECs following RSV infection. Moreover, ITGB4 deficiency led to mucus hypersecretion and MUC5AC overexpression in the small airway of RSV-infected mice. MUC5AC expression was upregulated by ITGB4 in HBE cells through EGFR, ERK and c-Jun pathways. EGFR inhibitors treatment inhibited mucus hypersecretion and MUC5AC overexpression in ITGB4-deficient mice after RSV infection. Together, these results demonstrated that epithelial ITGB4 deficiency induces mucus hypersecretion by upregulating the expression of MUC5AC through EGFR/ERK/c-Jun pathway, which further associated with RSV-related LRTI.

MiRNA-34b/c regulates mucus secretion in RSV-infected airway epithelial cells by targeting FGFR1.

Respiratory syncytial virus (RSV) infection in airway epithelial cells is the main cause of bronchiolitis in children. Excessive mucus secretion is one of the primary symbols in RSV related lower respiratory tract infections (RSV‐related LRTI). However, the pathological processes of mucus hypersecretion in RSV‐infected airway epithelial cells remains unclear. The current study explores the involvement of miR‐34b/miR‐34c in mucus hypersecretion in RSV‐infected airway epithelial cells by targeting FGFR1. First, miR‐34b/miR‐34c and FGFR1 mRNA were quantified by qPCR in throat swab samples and cell lines, respectively. Then, the luciferase reporters’ assay was designed to verify the direct binding between FGFR1 and miR‐34b/miR‐34c. Finally, the involvement of AP‐1 signalling was assessed by western blot. This study identified that miR‐34b/miR‐34c was involved in c‐Jun‐regulated MUC5AC production by targeting FGFR1 in RSV‐infected airway epithelial cells. These results provide some useful insights into the molecular mechanisms of mucus hypersecretion which may also bring new potential strategies to improve mucus hypersecretion in RSV disease.

Endogenous hydrogen sulfide is directly correlated with age and inversely correlated with intensive care length of stay in children with respiratory syncytial virus (RSV) bronchiolitis

Hydrogen sulfide (H2S) is an endogenous gasotransmitter that is involved in the regulation of important physiological functions in the respiratory tract. We have recently described previously unknown antiviral properties of H2S in RSV-infected airway epithelial cells and mice, but its role in naturally-acquired RSV infections remains to be determined.

Respiratory syncytial virus infection-induced mucus secretion by down-regulation of miR-34b/c-5p expression in airway epithelial cells.

Severe RSV infection is the main cause of hospitalization to children under the age of five. The regulation of miRNAs on the severity of RSV infection is unclear. The aim of the study was to identify the critical differential expression miRNAs (DE miRNAs) that can regulate the pathological response in RSV‐infected airway epithelial cells. In this study, miRNA and mRNA chips of RSV‐infected airway epithelia from Gene Expression Omnibus (GEO) were screened and analysed, separately. DE miRNAs‐targeted genes were performed for further pathway and process enrichment analysis. DE miRNA‐targeted gene functional network was constructed on the basis of miRNA‐mRNA interaction. The screened critical miRNA was also investigated by bioinformatics analysis. Then, RSV‐infected human bronchial epithelial cells (HBECs) were constructed to verify the expression of the DE miRNAs. Finally, specific synthetic DE miRNAs mimics were used to confirm the effect of DE miRNAs on the RSV‐infected HBECs. 45 DE miRNAs were identified from GEO62306 dataset. Our results showed that hsa‐mir‐34b‐5p and hsa‐mir‐34c‐5p decreased significantly in HBECs after RSV infection. Consistent with the biometric analysis, hsa‐mir‐34b/c‐5p is involved in the regulation of mucin expression gene MUC5AC. In RSV‐infected HBECs, the inducement of MUC5AC production by decreased hsa‐mir‐34b/c‐5p was partly mediated through activation of c‐Jun. These findings provide new insights into the mechanism of mucus obstruction after RSV infection and represent valuable targets for RSV infection and airway obstruction treatment.

Respiratory Syncytial Virus Infection Induced Mucus Secretion by Down Regulation of miR-34b/c-5p Expression in Airway Epithelial Cells

Background: Severe RSV infection is the main cause of hospitalization to children under age of five. The regulation of miRNAs on the severity of RSV infection are unclear. The aim of the study is to identify the critical differential expression miRNAs (DE miRNAs) that can regulate the pathological response in RSV-infected airway epithelial cells. Methods: miRNA and mRNA chips of RSV-infected airway epithelia from Gene Expression Omnibus (GEO) was screened and analyzed. DE miRNAs from different groups were identified and performed for further pathway and process enrichment analysis. DE miRNAs-targeted gene functional network was constructed based on miRNA-mRNA interaction. The screened critical miRNA was also investigated by bioinformatics analysis. Then, RSV-infected human bronchial epithelial cells (HBECs) were constructed to verify the expression of the DE miRNAs. Finally, specific synthetic DE miRNAs mimics was used to confirm effect of the DE miRNAs on the of RSV-infected HBECs. Results: 14 DE miRNAs were identified from GEO62306 dataset. Hsa-mir-34b/c-5p decreased significantly in HBECs after RSV infection. Consistent with the biometric analysis, hsa-mir-34b/c-5p is involved in the regulation of mucin expression gene MUC5AC. In RSV-infected HBECs, the inducement of MUC5AC production by decreased hsa-mir-34b/c-5p was partly mediated through activation of c-Jun. Conclusions: We conclude that the decreased expression of hsa-mir-34b/c-5p in HBECs after RSV infection play a critical role in MUC5AC expression through activation of c-Jun. These findings provide new insights into the mechanism of mucus obstruction after RSV infection and represent a useful biomarker of RSV infection and valuable targets of airway obstruction treatment. Funding Statement: This work was funded by grants #81970033，#31900424，#81570026, #81670002 and #3167188 from the NSFC; grants #2017JJ2402, #2019JJ50760 from the Hunan Natural Science Foundation; grant #16K097 from open Foundation of Hunan College Innovation Program and grant #2018zzts813, #2018zzts812, #2019zzts327, #2019zzts1008 from the Fundamental Research Funds for the Central Universities of Central South University. Declaration of Interests: The authors declare that they have no competing interests. Ethics Approval Statement: The authors stated: Not applicable.

Histone deacetylase inhibitors suppress RSV infection and alleviate virus-induced airway inflammation.

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in infants and young children. However, the majority of RSV-infected patients only show mild symptoms. Different severities of infection and responses among the RSV-infected population indicate that epigenetic regulation as well as personal genetic background may affect RSV infectivity. Histone deacetylase (HDAC) is an important epigenetic regulator in lung diseases. The present study aimed to explore the possible connection between HDAC expression and RSV-induced lung inflammation. To address this question, RSV-infected airway epithelial cells (BEAS-2B) were prepared and a mouse model of RSV infection was established, and then treated with various concentrations of HDAC inhibitors (HDACis), namely trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA). Viral replication and markers of virus-induced airway inflammation or oxidative stress were assessed. The activation of the nuclear factor-κB (NF-κB), cyclo-oxygenase-2 (COX-2), mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription 3 (STAT3) signaling pathways was evaluated by western blot analysis. Our results showed that RSV infection in airway epithelial cells (AECs) significantly decreased histone acetylation levels by altering HDAC2 expression. The treatment of RSV-infected AECs with HDACis significantly restricted RSV replication by upregulating the interferon-α (IFN-α) related signaling pathways. The treatment of RSV-infected AECs with HDACis also significantly inhibited RSV-induced pro-inflammatory cytokine release [interleukin (IL)-6 and IL-8] and oxidative stress-related molecule production [malondialdehyde (MDA), and nitrogen monoxide (NO)]. The activation of NF-κB, COX-2, MAPK and Stat3, which orchestrate pro-inflammatory gene expression and oxidative stress injury, was also significantly inhibited. Our in vivo study using a mouse model of RSV infection validated these results. Treatment with HDACis alleviated airway inflammation and reduced in vivo RSV replication. Our data demonstrated that RSV reduced histone acetylation by enhancing HDAC2 expression. Treatment with HDACis (TSA/SAHA) significantly inhibited RSV replication and decreased RSV-induced airway inflammation and oxidative stress. Therefore, the inhibition of HDACs represents a novel therapeutic approach in modulating RSV-induced lung disease.

Respiratory Syncytial Virus Induced Type I IFN Production by pDC Is Regulated by RSV-Infected Airway Epithelial Cells, RSV-Exposed Monocytes and Virus Specific Antibodies

Innate immune responses elicited upon virus exposure are crucial for the effective eradication of viruses, the onset of adaptive immune responses and for establishing proper immune memory. Respiratory syncytial virus (RSV) is responsible for a high disease burden in neonates and immune compromised individuals, causing severe lower respiratory tract infections. During primary infections exuberant innate immune responses may contribute to disease severity. Furthermore, immune memory is often insufficient to protect during RSV re-exposure, which results in frequent symptomatic reinfections. Therefore, identifying the cell types and pattern recognition receptors (PRRs) involved in RSV-specific innate immune responses is necessary to understand incomplete immunity against RSV. We investigated the innate cellular response triggered upon infection of epithelial cells and peripheral blood mononuclear cells. We show that CD14+ myeloid cells and epithelial cells are the major source of IL-8 and inflammatory cytokines, IL-6 and TNF-α, when exposed to live RSV Three routes of RSV-induced IFN-α production can be distinguished that depend on the cross-talk of different cell types and the presence or absence of virus specific antibodies, whereby pDC are the ultimate source of IFN-α. RSV-specific antibodies facilitate direct TLR7 access into endosomal compartments, while in the absence of antibodies, infection of monocytes or epithelial cells is necessary to provide an early source of type I interferons, required to engage the IFN-α,β receptor (IFNAR)-mediated pathway of IFN-α production by pDC. However, at high pDC density infection with RSV causes IFN-α production without the need for a second party cell. Our study shows that cellular context and immune status are factors affecting innate immune responses to RSV. These issues should therefore be addressed during the process of vaccine development and other interventions for RSV disease.

Human metapneumovirus infection induces expression of thymic stromal lymphopoetin and IL-33 in airway epithelial cells in vitro and lung infiltration of OX40L+ DCs in vivo (P6200)

Abstract Human metapneumovirus (hMPV) is a common virus known to induce acute respiratory tract infection. The specific mechanism by which hMPV modulates the immune response in infected airway epithelial cells (AECs) leading to tissue damage, still remains unknown. Thymic stromal lymphopoetin (TSLP) and IL-33, epithelial cell derived cytokines, induce high expression levels of OX40L on DCs, leading to Th2 responses and allergic inflammation. Recent reports have shown that Th2 inflammatory responses in RSV infections are mediated by TSLP-dependent OX40L expression in DCs. We hypothesize that the TSLP, IL-33 and OX40L pathway mediates lung inflammation in hMPV-infected individuals. We tested the expression of TSLP and IL-33 in human AECs after hMPV infection and compared them with RSV-infected AECs by qRT-PCR. We also evaluated lung infiltration of GR1+CD11b+ and CD11c+CD11b+OX40L+ cells in hMPV-infected mice by flow cytometry. We found an induction of TSLP mRNA levels in hMPV-infected AECs in a MOI dependent-manner. We also proved that hMPV induces higher TSLP mRNA levels than RSV in AECs, and hMPV but not RSV up-regulates IL-33 levels in AECs. Furthermore, we showed that hMPV infection in mice induces an increased lung infiltration of neutrophiles and OX40L+ DCs than the mock control group. These results suggest that hMPV infection in lungs may be inducing an augmented Th2 inflammatory response, through TSLP and IL-33 produced by hMPV-infected AECs and activated DCs expressing OX40L.

TSLP from RSV‐stimulated rat airway epithelial cells activates myeloid dendritic cells

The respiratory syncytial virus (RSV) is a primary cause of lower respiratory tract infections in children, the elderly and in people who are immune suppressed, and is also the cause for the development of asthma primarily in infants. However, the immunological mechanisms by which RSV enhances allergic sensitization and asthma remain unclear. The aim of this study was to examine the influence of RSV-infected airway epithelial cells on the activation and functions of rat myeloid dendritic cells (mDCs).We found that the exposure of primary rat airway epithelial cells (PRAECs) to RSV induced a rapid (6 h), high (12 h) and persistent (18 h) increase in thymic stromal lymphopoietin (TSLP) mRNA compared with untreated PRAECs. TSLP protein expression was also enhanced by RSV infection. Functional maturation of mDCs was induced by RSV-treated PRAECs, as shown by their enhanced levels of OX40L and thymus- and activation-regulated chemokine (TARC) mRNAs, which increased the expressions of major histocompatibility complex II (MHCII) and CD86 costimulatory molecules and promoted enhanced T-cell proliferation in mixed lymphocyte reactions. These activities were inhibited in cocultures with RSV-infected RTECs (rat tracheal epithelial cells, an immortalized cell strain) that had been pretreated with TSLP-targeted small interfering RNA. These results suggest that RSV can induce epithelial cells to produce TSLP, which in turn promotes the maturation of mDCs that might support Th2 cell polarization.

Fosfomycin Suppresses Chemokine Induction in Airway Epithelial Cells Infected with Respiratory Syncytial Virus

Respiratory syncytial virus (RSV) infects airway epithelial cells, causing bronchiolitis and pneumonia. Inflammation is mediated by various cytokines secreted from RSV-infected airway epithelial cells, and it promotes the pathogenesis of RSV-related diseases. Fosfomycin (FOF) is approved as a treatment for various bacterial infectious diseases, including respiratory infectious diseases, in Japan. FOF is suggested to exhibit immunomodulatory effects on lipopolysaccharide-stimulated monocytes and T lymphocytes, in addition to its antimicrobial activity. We investigated the effect of FOF on the cytokine production of an airway epithelial cell line, A549, infected with RSV. RSV-induced cytokines, such as regulated on activation, normal T-cell expressed and secreted (RANTES), interleukin-8 (IL-8), and IL-6, in infected A549 cells. We found that FOF decreased the levels of RSV-induced RANTES and IL-8 but not the level of RSV-induced IL-6. The RANTES promoter was activated by RSV infection. Site-directed mutagenesis analysis of the RANTES promoter showed that NF-kappaB-binding motifs had a critical role in RSV-induced RANTES promoter activity. A luciferase reporter gene assay and a DNA-binding assay indicated that FOF suppressed the NF-kappaB activity induced by RSV infection. These results demonstrate that FOF treatment suppresses the RSV-induced transcription of the chemokines RANTES and IL-8 in airway epithelial cells.

Respiratory Syncytial Virus Inhibits Lung Epithelial Na+ Channels by Up-regulating Inducible Nitric-oxide Synthase

Respiratory syncytial virus (RSV) infection has been shown to reduce Na+-driven alveolar fluid clearance in BALB/c mice in vivo. To investigate the cellular mechanisms by which RSV inhibits amiloride-sensitive epithelial Na+ channels (ENaC), the main pathways through which Na+ ions enter lung epithelial cells, we infected human Clara-like lung (H441) cells with RSV that expresses green fluorescent protein (rRA2). 3-6 days later patch clamp recordings showed that infected cells (i.e. cells expressing green fluorescence; GFP+) had significantly lower whole-cell amiloride-sensitive currents and single channel activity (NPo) as compared with non-infected (GFP-), non-inoculated, or cells infected with UV-inactivated RSV. Both alpha and beta ENaC mRNA levels were significantly reduced in GFP+ cells as measured by real-time reverse transcription-PCR. Infection with RSV increased expression of the inducible nitric-oxide synthase (iNOS) and nitrite concentration in the culture medium; nuclear translocation of NF-kappaB p65 subunit and NF-kappaB activation were also up-regulated. iNOS up-regulation in GFP+ cells was prevented by knocking down IkappaB kinase gamma before infection. Furthermore, pretreatment of H441 cells with the specific iNOS inhibitor 1400W (1 microM) resulted in a doubling of the amiloride-sensitive Na+ current in GFP+ cells. Additionally, preincubation of H441 cells with A77-1726 (20 microM), a de novo UTP synthesis inhibitor, and 1400W completely reversed the RSV inhibition of amiloride-sensitive currents in GFP+ cells. Thus, both UTP- and iNOS-generated reactive species contribute to ENaC down-regulation in RSV-infected airway epithelial cells.

Respiratory syncytial virus-induced chemokine production: linking viral replication to chemokine production in vitro and in vivo.

Respiratory syncytial virus (RSV) is a negative-sense, single-strand RNA virus that can initiate severe bronchiolitis in infants, as well as in elderly adults. Although RSV preferentially infects and replicates in the airway epithelium, studies have shown that RSV has the ability to infect and, to a limited extent, replicate in alveolar macrophages. In the present study, we sought to characterize the RSV-induced chemokine production in vitro and in vivo, because chemokines have been shown to contribute to both the inflammation and pathophysiology of disease. Our results show that RSV-infected airway epithelial cells and alveolar macrophages display differential profiles of chemokine production: airway epithelial cells produce CCL2/monocyte chemoattractant protein-1, CCL5/RANTES, CXCL10/gamma interferon inducible protein-10, and kerotinocyte cytokine (KC); and alveolar macrophages up-regulate CCL5 and macrophage inflammatory protein (MIP)-2 after RSV infection. In vivo, we observed the induction of CCL2, CCL3/MIP-1 alpha, CCL5, CXCL10, and KC after RSV infection. In the present study, we also addressed the necessity for viral infection and/or replication in chemokine induction by use of ultraviolet (UV)-inactivated RSV, as well as RSV inhibitors of binding/infection and replication, that is, NMSO3, a sulfated sialyl lipid compound, and ribavirin, respectively. Our results suggest that viral replication is necessary for optimal chemokine production.

Specific Inhibition of Type I Interferon Signal Transduction by Respiratory Syncytial Virus

Respiratory viruses often express mechanisms to resist host antiviral systems, but the biochemical basis for evasion of interferon effects by respiratory syncytial virus (RSV) is poorly defined. In this study, we identified RSV effects on interferon (IFN)-dependent signal transduction and gene expression in human airway epithelial cells. Initial experiments demonstrated inhibition of antiviral gene expression induced by IFN-alpha and IFN-beta, but not IFN-gamma, in epithelial cells infected with RSV. Selective viral effects on type I IFN-dependent signaling were confirmed when we observed impaired type I, but not type II, IFN-induced activation of the transcription factor Stat1 in RSV-infected cells. RSV infection of airway epithelial cells resulted in decreased Stat2 expression and function with preservation of upstream signaling events, providing a molecular mechanism for viral inhibition of the type I IFN JAK-STAT pathway. Furthermore, nonspecific pharmacologic inhibition of proteasome function in RSV-infected cells restored Stat2 levels and IFN-dependent activation of Stat1. The results indicate that RSV acts on epithelial cells in the airway to directly modulate the type I IFN JAK-STAT pathway, and this effect is likely mediated though proteasome-dependent degradation of Stat2. Decreased antiviral gene expression in RSV-infected airway epithelial cells may allow RSV replication and establishment of a productive viral infection through subversion of IFN-dependent immunity.

EXPOSURE TO URBAN AIR PARTICULATES ALTERS THE MACROPHAGE-MEDIATED INFLAMMATORY RESPONSE TO RESPIRATORY VIRAL INFECTION

Epidemiology studies associate increased pulmonary morbidity with episodes of high particulate air pollution (size range 0.1-10 mum diameter, PM10). Pneumonia, often viral in origin, is increased following episodes of high PM10 pollution. Therefore, this study was undertaken to investigate how PM10 alters airway inflammatory responses to respiratory syncytial virus (RSV), a frequent cause of viral pneumonia in infants and the elderly. Supernatants of unexposed and PM10-exposed alveolar macrophage (AM) cultured with uninfected or RSV-infected airway epithelial cells were assessed for a number of chemokines responsible for inflammatory responses in the lung. AM exposure to PM10 in the absence of infection resulted in a significant increase in interleukin (IL)-8 and macrophage inflammatory protein (MIP)-1 apha production but not in MIP-1 beta or monocyte chemotactic protein (MCP)-1. AM responded to RSV infection by the production of IL-8, MIP-1 alpha, MIP-1 beta, and MCP-1, while RANTES was derived solely from the RSV-infected bronchial epithelial cell line BEAS-2B. In the presence of PM10, the AM response to RSV was blunted. RSV-induced MCP-1 was significantly decreased, and the levels of MIP-1 and IL-8 were lower than expected from a combined response to PM10 and RSV. Furthermore, AM analyzed for uptake of virus showed a 50% decrease in viral antigen when exposed to PM10. RSV-induced production of RANTES by epithelial cells was decreased in the presence of AM but not affected by PM10 exposure. Taken together, these results suggest that AM-regulated inflammatory responses to viral infection are altered by exposure to PM10 in a manner that may result in increased spread of infection and thus may increase viral pneumonia-related hospital admissions.