RESUMO
Cytotoxic T lymphocytes (CTLs) mediate host defense against viral and intracellular bacterial infections and tumors. However, the magnitude of CTL response and their function needed to confer heterosubtypic immunity against influenza virus infection are unknown. We addressed the role of CD8+ T cells in the absence of any cross-reactive antibody responses to influenza viral proteins using an adenoviral vector expressing a 9mer amino acid sequence recognized by CD8+ T cells. Our results indicate that both CD8+ T cell frequency and function are crucial for heterosubtypic immunity. Low morbidity, lower viral lung titers, low to minimal lung pathology, and better survival upon heterosubtypic virus challenge correlated with the increased frequency of NP-specific CTLs. NP-CD8+ T cells induced by differential infection doses displayed distinct RNA transcriptome profiles and functional properties. CD8+ T cells induced by a high dose of influenza virus secreted significantly higher levels of IFN-γ and exhibited higher levels of cytotoxic function. The mice that received NP-CD8+ T cells from the high-dose virus recipients through adoptive transfer had lower viral titers following viral challenge than those induced by the low dose of virus, suggesting differential cellular programming by antigen dose. Enhanced NP-CD8+ T-cell functions induced by a higher dose of influenza virus strongly correlated with the increased expression of cellular and metabolic genes, indicating a shift to a more glycolytic metabolic phenotype. These findings have implications for developing effective T cell vaccines against infectious diseases and cancer. IMPORTANCE: Cytotoxic T lymphocytes (CTLs) are an important component of the adaptive immune system that clears virus-infected cells or tumor cells. Hence, developing next-generation vaccines that induce or recall CTL responses against cancer and infectious diseases is crucial. However, it is not clear if the frequency, function, or both are essential in conferring protection, as in the case of influenza. In this study, we demonstrate that both CTL frequency and function are crucial for providing heterosubtypic immunity to influenza by utilizing an Ad-viral vector expressing a CD8 epitope only to rule out the role of antibodies, single-cell RNA-seq analysis, as well as adoptive transfer experiments. Our findings have implications for developing T cell vaccines against infectious diseases and cancer.
Assuntos
Linfócitos T CD8-Positivos , Infecções por Orthomyxoviridae , Linfócitos T Citotóxicos , Animais , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/virologia , Camundongos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T Citotóxicos/imunologia , Camundongos Endogâmicos C57BL , Feminino , Transferência Adotiva , Interferon gama/imunologia , Interferon gama/metabolismo , Proteínas do Nucleocapsídeo/imunologia , Pulmão/imunologia , Pulmão/virologia , Proteínas de Ligação a RNA/imunologia , Proteínas de Ligação a RNA/genética , Nucleoproteínas/imunologia , Nucleoproteínas/genética , Proteínas do Core Viral/imunologia , Proteínas do Core Viral/genéticaRESUMO
Preventing and controlling influenza virus infection remains a global public health challenge, as it causes seasonal epidemics to unexpected pandemics. These infections are responsible for high morbidity, mortality, and substantial economic impact. Vaccines are the prophylaxis mainstay in the fight against influenza. However, vaccination fails to confer complete protection due to inadequate vaccination coverages, vaccine shortages, and mismatches with circulating strains. Antivirals represent an important prophylactic and therapeutic measure to reduce influenza-associated morbidity and mortality, particularly in high-risk populations. Here, we review current FDA-approved influenza antivirals with their mechanisms of action, and different viral- and host-directed influenza antiviral approaches, including immunomodulatory interventions in clinical development. Furthermore, we also illustrate the potential utility of machine learning in developing next-generation antivirals against influenza.
Assuntos
Vacinas contra Influenza , Influenza Humana , Infecções por Orthomyxoviridae , Orthomyxoviridae , Humanos , Influenza Humana/tratamento farmacológico , Influenza Humana/prevenção & controle , Antivirais/farmacologia , Antivirais/uso terapêutico , Infecções por Orthomyxoviridae/tratamento farmacológico , Vacinas contra Influenza/uso terapêuticoRESUMO
Influenza is a highly contagious respiratory virus that causes mild to severe respiratory illness, as well as death, and remains a serious threat to human health. Annual vaccination is the most cost-effective way to control influenza; however, the vaccine does not provide protection against emerging strains with epidemic and pandemic potential. Several antivirals have been developed to treat influenza but there is a rapid emergence of antiviral resistant strains. Therefore, there is an urgent need to understand the virus and its interactions with the host immune system so that novel strategies can be developed for prophylactic and therapeutic interventions. Innate lymphoid cells (ILCs), a family of immune cells present in the peripheral circulation and in mucosal tissues, play an important role in regulation of tissue homeostasis, inflammation, and immunity. This review examines the current understanding and therapeutic potential of ILCs during influenza virus infection in humans.
Assuntos
Vacinas contra Influenza , Influenza Humana , Humanos , Imunidade Inata , Vacinas contra Influenza/uso terapêutico , Influenza Humana/epidemiologia , Influenza Humana/prevenção & controle , Linfócitos , VacinaçãoRESUMO
Neonatal hypoxic-ischemic encephalopathy (HIE) is one of the most important reasons for morbidity and mortality in term-born infants. HIE impacts early somatic, neurological, and motor development including social. To illustrate the damages in the sensorimotor system, an adapted and validated model of neonatal anoxia is used. This study evaluated the sex differences in Wistar rats, neurological reflex, and motor development at the suckling period. Short- and long-term impairments associated with sex differences were observed. In general, anoxic males were more affected in comparison to their control group and to anoxic females. Long-lasting effects of the injury in adolescent rats predominately affected males. Similar to previous studies, we also found a decrease in the number of the substantia nigra cells in both sexes, compared to their control. So far, the results indicate that HIE caused neurobehavioral alterations and asymmetrical motor behavior with brain damage, possibly related to cognitive impairments previously observed at adolescence. These alterations may represent a useful endpoint for studying the efficacy of potential strategies that may improve the developmental consequences of a perinatal asphyxia insult in humans.
Assuntos
Hipóxia-Isquemia Encefálica , Humanos , Lactente , Gravidez , Animais , Ratos , Feminino , Masculino , Ratos Wistar , Animais Recém-Nascidos , Modelos Animais de Doenças , HipóxiaRESUMO
Ferrets represent an invaluable animal model to study influenza virus pathogenesis and transmission. To further characterize this model, we developed a differentiated primary ferret nasal epithelial cell (FNEC) culture model for investigation of influenza A virus infection and virus-host interactions. This well-differentiated culture consists of various cell types, a mucociliary clearance system, and tight junctions, representing the nasal ciliated pseudostratified respiratory epithelium. Both α2,6-linked and α2,3-linked sialic acid (SA) receptors, which preferentially bind the hemagglutinin (HA) of human and avian influenza viruses, respectively, were detected on the apical surface of the culture with different cellular tropisms. In accordance with the distribution of SA receptors, we observed that a pre-2009 seasonal A(H1N1) virus infected both ciliated and nonciliated cells, whereas a highly pathogenic avian influenza (HPAI) A(H5N1) virus primarily infected nonciliated cells. Transmission electron microscopy revealed that virions were released from or associated with the apical membranes of ciliated, nonciliated, and mucin-secretory goblet cells. Upon infection, the HPAI A(H5N1) virus replicated to titers higher than those of the human A(H1N1) virus at 37°C; however, replication of the A(H5N1) virus was significantly attenuated at 33°C. Furthermore, we found that infection with the A(H5N1) virus induced higher expression levels of immune mediator genes and resulted in more cell damage/loss than with the human A(H1N1) virus. This primary differentiated FNEC culture model, recapitulating the structure of the nasal epithelium, provides a useful model to bridge in vivo and in vitro studies of cellular tropism, infectivity, and pathogenesis of influenza viruses during the initial stages of infection.IMPORTANCE Although ferrets serve as an important model of influenza virus infection, much remains unknown about virus-host interactions in this species at the cellular level. The development of differentiated primary cultures of ferret nasal epithelial cells is an important step toward understanding cellular tropism and the mechanisms of influenza virus infection and replication in the airway milieu of this model. Using lectin staining and microscopy techniques, we characterized the sialic acid receptor distribution and the cellular composition of the culture model. We then evaluated the replication of and immune response to human and avian influenza viruses at relevant physiological temperatures. Our findings offer significant insight into this first line of defense against influenza virus infection and provide a model for the evaluation of emerging influenza viruses in a well-controlled in vitro environmental setting.
Assuntos
Vírus da Influenza A Subtipo H1N1/genética , Virus da Influenza A Subtipo H5N1/genética , Tropismo Viral/genética , Animais , Brônquios/virologia , Técnicas de Cultura de Células/métodos , Cílios/virologia , Modelos Animais de Doenças , Células Epiteliais/virologia , Furões/virologia , Células Caliciformes/metabolismo , Células Caliciformes/virologia , Humanos , Vírus da Influenza A Subtipo H1N1/metabolismo , Vírus da Influenza A Subtipo H1N1/patogenicidade , Virus da Influenza A Subtipo H5N1/patogenicidade , Vírus da Influenza A/fisiologia , Influenza Humana/virologia , Mucosa Nasal/metabolismo , Mucosa Nasal/virologia , Cultura Primária de Células , Receptores de Superfície Celular/metabolismo , Receptores Virais/metabolismo , Mucosa Respiratória/virologia , Traqueia/virologia , Viroses/genéticaRESUMO
This article presents the development of a stretchable sensor network with high signal-to-noise ratio and measurement accuracy for real-time distributed sensing and remote monitoring. The described sensor network was designed as an island-and-serpentine type network comprising a grid of sensor "islands" connected by interconnecting "serpentines." A novel high-yield manufacturing process was developed to fabricate networks on recyclable 4-inch wafers at a low cost. The resulting stretched sensor network has 17 distributed and functionalized sensing nodes with low tolerance and high resolution. The sensor network includes Piezoelectric (PZT), Strain Gauge (SG), and Resistive Temperature Detector (RTD) sensors. The design and development of a flexible frame with signal conditioning, data acquisition, and wireless data transmission electronics for the stretchable sensor network are also presented. The primary purpose of the frame subsystem is to convert sensor signals into meaningful data, which are displayed in real-time for an end-user to view and analyze. The challenges and demonstrated successes in developing this new system are demonstrated, including (a) developing separate signal conditioning circuitry and components for all three sensor types (b) enabling simultaneous sampling for PZT sensors for impact detection and (c) configuration of firmware/software for correct system operation. The network was expanded with an in-house developed automated stretch machine to expand it to cover the desired area. The released and stretched network was laminated into an aerospace composite wing with edge-mount electronics for signal conditioning, processing, power, and wireless communication.
RESUMO
Although influenza viruses typically cause respiratory tract disease, some viruses, particularly those with an H7 hemagglutinin, have been isolated from the eyes of conjunctivitis cases. Previous work has shown that isolates of multiple subtypes from both ocular and respiratory infections are capable of replication in human ex vivo ocular tissues and corneal or conjunctival cell monolayers, leaving the determinants of ocular tropism unclear. Here, we evaluated the effect of several variables on tropism for ocular cells cultured in vitro and examined the potential effect of the tear film on viral infectivity. All viruses tested were able to replicate in primary human corneal epithelial cell monolayers subjected to aerosol inoculation. The temperature at which cells were cultured postinoculation minimally affected infectivity. Replication efficiency, in contrast, was reduced at 33°C relative to that at 37°C, and this effect was slightly greater for the conjunctivitis isolates than for the respiratory ones. With the exception of a seasonal H3N2 virus, the subset of viruses studied in multilayer corneal tissue constructs also replicated productively after either aerosol or liquid inoculation. Human tears significantly inhibited the hemagglutination of both ocular and nonocular isolates, but the effect on viral infectivity was more variable, with tears reducing the infectivity of nonocular isolates more than ocular isolates. These data suggest that most influenza viruses may be capable of establishing infection if they reach the surface of ocular cells but that this is more likely for ocular-tropic viruses, as they are better able to maintain their infectivity during passage through the tear film.IMPORTANCE The potential spread of zoonotic influenza viruses to humans represents an important threat to public health. Unfortunately, despite the importance of cellular and tissue tropism to pathogenesis, determinants of influenza virus tropism have yet to be fully elucidated. Here, we sought to identify factors that limit the ability of most influenza viruses to cause ocular infection. Although ocular symptoms in humans caused by avian influenza viruses tend to be relatively mild, these infections are concerning due to the potential of the ocular surface to serve as a portal of entry for viruses that go on to establish respiratory infections. Furthermore, a better understanding of the factors that influence infection and replication in this noncanonical site may point toward novel determinants of tropism in the respiratory tract.
Assuntos
Conjuntivite Viral/metabolismo , Córnea/virologia , Células Epiteliais/virologia , Vírus da Influenza A Subtipo H3N2/fisiologia , Influenza Humana/metabolismo , Tropismo Viral/fisiologia , Replicação Viral/fisiologia , Animais , Conjuntivite Viral/patologia , Conjuntivite Viral/virologia , Córnea/metabolismo , Córnea/patologia , Cães , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Humanos , Influenza Humana/patologia , Células Madin Darby de Rim CaninoRESUMO
Monkeypox virus (MPXV) is a human pathogen that is a member of the Orthopoxvirus genus, which includes Vaccinia virus and Variola virus (the causative agent of smallpox). Human monkeypox is considered an emerging zoonotic infectious disease. To identify host factors required for MPXV infection, we performed a genome-wide insertional mutagenesis screen in human haploid cells. The screen revealed several candidate genes, including those involved in Golgi trafficking, glycosaminoglycan biosynthesis, and glycosylphosphatidylinositol (GPI)-anchor biosynthesis. We validated the role of a set of vacuolar protein sorting (VPS) genes during infection, VPS51 to VPS54 (VPS51-54), which comprise the Golgi-associated retrograde protein (GARP) complex. The GARP complex is a tethering complex involved in retrograde transport of endosomes to the trans-Golgi apparatus. Our data demonstrate that VPS52 and VPS54 were dispensable for mature virion (MV) production but were required for extracellular virus (EV) formation. For comparison, a known antiviral compound, ST-246, was used in our experiments, demonstrating that EV titers in VPS52 and VPS54 knockout (KO) cells were comparable to levels exhibited by ST-246-treated wild-type cells. Confocal microscopy was used to examine actin tail formation, one of the viral egress mechanisms for cell-to-cell dissemination, and revealed an absence of actin tails in VPS52KO- or VPS54KO-infected cells. Further evaluation of these cells by electron microscopy demonstrated a decrease in levels of wrapped viruses (WVs) compared to those seen with the wild-type control. Collectively, our data demonstrate the role of GARP complex genes in double-membrane wrapping of MVs necessary for EV formation, implicating the host endosomal trafficking pathway in orthopoxvirus infection.IMPORTANCE Human monkeypox is an emerging zoonotic infectious disease caused by Monkeypox virus (MPXV). Of the two MPXV clades, the Congo Basin strain is associated with severe disease, increased mortality, and increased human-to-human transmission relative to the West African strain. Monkeypox is endemic in regions of western and central Africa but was introduced into the United States in 2003 from the importation of infected animals. The threat of MPXV and other orthopoxviruses is increasing due to the absence of routine smallpox vaccination leading to a higher proportion of naive populations. In this study, we have identified and validated candidate genes that are required for MPXV infection, specifically, those associated with the Golgi-associated retrograde protein (GARP) complex. Identifying host targets required for infection that prevents extracellular virus formation such as the GARP complex or the retrograde pathway can provide a potential target for antiviral therapy.
Assuntos
Endossomos/metabolismo , Interações Hospedeiro-Patógeno , Proteínas de Membrana/genética , Monkeypox virus/fisiologia , Proteínas de Transporte Vesicular/metabolismo , Actinas/efeitos dos fármacos , Actinas/metabolismo , Animais , Benzamidas/farmacologia , Transporte Biológico , Linhagem Celular , Genoma Humano , Glicosaminoglicanos/biossíntese , Glicosaminoglicanos/genética , Glicosilfosfatidilinositóis/biossíntese , Complexo de Golgi/genética , Complexo de Golgi/metabolismo , Haploidia , Humanos , Isoindóis/farmacologia , Proteínas de Membrana/metabolismo , Mpox/virologia , Mutagênese Insercional , Proteínas de Transporte Vesicular/genética , Carga Viral , Replicação ViralRESUMO
A role for pulmonary endothelial cells in the orchestration of cytokine production and leukocyte recruitment during influenza virus infection, leading to severe lung damage, has been recently identified. As the mechanistic pathway for this ability is not fully known, we extended previous studies on influenza virus tropism in cultured human pulmonary endothelial cells. We found that a subset of avian influenza viruses, including potentially pandemic H5N1, H7N9, and H9N2 viruses, could infect human pulmonary endothelial cells (HULEC) with high efficiency compared to human H1N1 or H3N2 viruses. In HULEC, human influenza viruses were capable of binding to host cellular receptors, becoming internalized and initiating hemifusion but failing to uncoat the viral nucleocapsid and to replicate in host nuclei. Unlike numerous cell types, including epithelial cells, we found that pulmonary endothelial cells constitutively express a high level of the restriction protein IFITM3 in endosomal compartments. IFITM3 knockdown by small interfering RNA (siRNA) could partially rescue H1N1 virus infection in HULEC, suggesting IFITM3 proteins were involved in blocking human influenza virus infection in endothelial cells. In contrast, selected avian influenza viruses were able to escape IFITM3 restriction in endothelial cells, possibly by fusing in early endosomes at higher pH or by other, unknown mechanisms. Collectively, our study demonstrates that the human pulmonary endothelium possesses intrinsic immunity to human influenza viruses, in part due to the constitutive expression of IFITM3 proteins. Notably, certain avian influenza viruses have evolved to escape this restriction, possibly contributing to virus-induced pneumonia and severe lung disease in humans. IMPORTANCE: Avian influenza viruses, including H5N1 and H7N9, have been associated with severe respiratory disease and fatal outcomes in humans. Although acute respiratory distress syndrome (ARDS) and progressive pulmonary endothelial damage are known to be present during severe human infections, the role of pulmonary endothelial cells in the pathogenesis of avian influenza virus infections is largely unknown. By comparing human seasonal influenza strains to avian influenza viruses, we provide greater insight into the interaction of influenza virus with human pulmonary endothelial cells. We show that human influenza virus infection is blocked during the early stages of virus entry, which is likely due to the relatively high expression of the host antiviral factors IFITMs (interferon-induced transmembrane proteins) located in membrane-bound compartments inside cells. Overall, this study provides a mechanism by which human endothelial cells limit replication of human influenza virus strains, whereas avian influenza viruses overcome these restriction factors in this cell type.
Assuntos
Células Endoteliais/imunologia , Interações Hospedeiro-Patógeno , Células Endoteliais da Veia Umbilical Humana/imunologia , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A Subtipo H3N2/imunologia , Proteínas de Membrana/imunologia , Proteínas de Ligação a RNA/imunologia , Animais , Aves , Linhagem Celular , Endossomos/química , Endossomos/imunologia , Endossomos/virologia , Células Endoteliais/virologia , Células Epiteliais/imunologia , Células Epiteliais/virologia , Regulação da Expressão Gênica , Células Endoteliais da Veia Umbilical Humana/virologia , Humanos , Concentração de Íons de Hidrogênio , Vírus da Influenza A Subtipo H1N1/crescimento & desenvolvimento , Vírus da Influenza A Subtipo H3N2/crescimento & desenvolvimento , Virus da Influenza A Subtipo H5N1/crescimento & desenvolvimento , Virus da Influenza A Subtipo H5N1/imunologia , Subtipo H7N9 do Vírus da Influenza A/crescimento & desenvolvimento , Subtipo H7N9 do Vírus da Influenza A/imunologia , Vírus da Influenza A Subtipo H9N2/crescimento & desenvolvimento , Vírus da Influenza A Subtipo H9N2/imunologia , Pulmão , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Especificidade de Órgãos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/genética , Transdução de Sinais , Especificidade da Espécie , Internalização do Vírus , Replicação Viral/imunologiaRESUMO
The NLR protein, NLRC5 is an important regulator of MHC class I gene expression, however, the role of NLRC5 in other innate immune responses is less well defined. In the present study, we report that NLRC5 binds RIG-I and that this interaction is critical for robust antiviral responses against influenza virus. Overexpression of NLRC5 in the human lung epithelial cell line, A549, and normal human bronchial epithelial cells resulted in impaired replication of influenza virus A/Puerto Rico/8/34 virus (PR8) and enhanced IFN-ß expression. Influenza virus leads to induction of IFN-ß that drives RIG-I and NLRC5 expression in host cells. Our results suggest that NLRC5 extends and stabilizes influenza virus induced RIG-I expression and delays expression of the viral inhibitor protein NS1. We show that NS1 binds to NLRC5 to suppress its function. Interaction domain mapping revealed that NLRC5 interacts with RIG-I via its N-terminal death domain and that NLRC5 enhanced antiviral activity in an leucine-rich repeat domain independent manner. Taken together, our findings identify a novel role for NLRC5 in RIG-I-mediated antiviral host responses against influenza virus infection, distinguished from the role of NLRC5 in MHC class I gene regulation.
Assuntos
RNA Helicases DEAD-box/imunologia , Regulação da Expressão Gênica/imunologia , Vírus da Influenza A/imunologia , Influenza Humana/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Mucosa Respiratória/imunologia , Proteína DEAD-box 58 , Células Epiteliais/imunologia , Células Epiteliais/patologia , Células Epiteliais/virologia , Células HEK293 , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Influenza Humana/patologia , Estrutura Terciária de Proteína , Receptores Imunológicos , Mucosa Respiratória/patologia , Mucosa Respiratória/virologiaRESUMO
UNLABELLED: Influenza A viruses (IAVs) express the PB1-F2 protein from an alternate reading frame within the PB1 gene segment. The roles of PB1-F2 are not well understood but appear to involve modulation of host cell responses. As shown in previous studies, we find that PB1-F2 proteins of mammalian IAVs frequently have premature stop codons that are expected to cause truncations of the protein, whereas avian IAVs usually express a full-length 90-amino-acid PB1-F2. However, in contrast to other avian IAVs, recent isolates of highly pathogenic H5N1 influenza viruses had a high proportion of PB1-F2 truncations (15% since 2010; 61% of isolates in 2013) due to several independent mutations that have persisted and expanded in circulating viruses. One natural H5N1 IAV containing a mutated PB1-F2 start codon (i.e., lacking ATG) was 1,000-fold more virulent for BALB/c mice than a closely related H5N1 containing intact PB1-F2. In vitro, we detected expression of an in-frame protein (C-terminal PB1-F2) from downstream ATGs in PB1-F2 plasmids lacking the well-conserved ATG start codon. Transient expression of full-length PB1-F2, truncated (24-amino-acid) PB1-F2, and PB1-F2 lacking the initiating ATG in mammalian and avian cells had no effect on cell apoptosis or interferon expression in human lung epithelial cells. Full-length and C-terminal PB1-F2 mutants colocalized with mitochondria in A549 cells. Close monitoring of alterations of PB1-F2 and their frequency in contemporary avian H5N1 viruses should continue, as such changes may be markers for mammalian virulence. IMPORTANCE: Although most avian influenza viruses are harmless for humans, some (such as highly pathogenic H5N1 avian influenza viruses) are capable of infecting humans and causing severe disease with a high mortality rate. A number of risk factors potentially associated with adaptation to mammalian infection have been noted. Here we demonstrate that the protein PB1-F2 is frequently truncated in recent isolates of highly pathogenic H5N1 viruses. Truncation of PB1-F2 has been proposed to act as an adaptation to mammalian infection. We show that some forms of truncation of PB1-F2 may be associated with increased virulence in mammals. Our data support the assessment of PB1-F2 truncations for genomic surveillance of influenza viruses.
Assuntos
Virus da Influenza A Subtipo H5N1/isolamento & purificação , Virus da Influenza A Subtipo H5N1/fisiologia , Infecções por Orthomyxoviridae/virologia , Proteínas Virais/genética , Proteínas Virais/metabolismo , Fatores de Virulência/genética , Fatores de Virulência/metabolismo , Animais , Apoptose , Linhagem Celular , Códon sem Sentido , Modelos Animais de Doenças , Células Epiteliais/fisiologia , Células Epiteliais/virologia , Feminino , Humanos , Virus da Influenza A Subtipo H5N1/genética , Interferons/biossíntese , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae/patologia , VirulênciaRESUMO
To enhance the immunogenicity of the Influenza H5N1 vaccine, we developed an oil-in-water nanoemulsion (NE) adjuvant. NE displayed good temperature stability and maintained particle size. More importantly, it significantly enhanced IL-6 and MCP-1 production to recruit innate cells, including neutrophils, monocytes/macrophages and dendritic cells to the local environment. Furthermore, NE enhanced dendritic cell function to induce robust antigen-specific T and B cell immune responses. NE-adjuvanted H5N1 vaccine not only elicited significantly higher and long-lasting antibody responses, but also conferred enhanced protection against homologous clade 1 as well as heterologous clade 2 H5N1 virus challenge in young as well as in aged mice. The pre-existing immunity to seasonal influenza did not affect the immunogenicity of NE-adjuvanted H5N1 vaccine.
Assuntos
Virus da Influenza A Subtipo H5N1 , Vacinas contra Influenza/administração & dosagem , Nanopartículas/química , Adjuvantes Imunológicos , Animais , Anticorpos Antivirais , Emulsões , Humanos , Influenza Humana/prevenção & controle , CamundongosRESUMO
We compared the innate immune response to a newly emerged swine-origin influenza A(H3N2) variant containing the M gene from 2009 pandemic influenza A(H1N1), termed "A(H3N2)vpM," to the immune responses to the 2010 swine-origin influenza A(H3N2) variant and seasonal influenza A(H3N2). Our results demonstrated that A(H3N2)vpM-induced myeloid dendritic cells secreted significantly lower levels of type I interferon (IFN) but produced significantly higher levels of proinflammatory cytokines and induced potent inflammasome activation. The reduction in antiviral immunity with increased inflammatory responses upon A(H3N2)vpM infection suggest that these viruses have the potential for increased disease severity in susceptible hosts.
Assuntos
Inflamassomos/metabolismo , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A Subtipo H3N2/imunologia , Leucócitos Mononucleares/imunologia , Animais , Linhagem Celular , Citocinas/metabolismo , Células Dendríticas/imunologia , Humanos , Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Vírus da Influenza A Subtipo H3N2/isolamento & purificação , Infecções por Orthomyxoviridae/virologia , Suínos , Doenças dos Suínos/virologiaRESUMO
Redundancies in both the ubiquitin and epithelial sodium transport pathways allude to their importance of proteolytic degradation and ion transport in maintaining normal cell function. The classical pathway implicated in ubiquitination of the epithelial sodium channel (ENaC) involves Nedd4-2 regulation of sodium channel subunit expression and has been studied extensively studied. However, less attention has been given to the role of the ubiquitin-like protein Nedd8. Here we show that Nedd8 plays an important role in the ubiquitination of ENaC in alveolar epithelial cells. We report that the Nedd8 pathway is redox-sensitive and that under oxidizing conditions Nedd8 conjugation to Cullin-1 is attenuated, resulting in greater surface expression of α-ENaC. This observation was confirmed in our electrophysiology studies in which we inhibited Nedd8-activating enzyme using MLN4924 (a specific Nedd8-activating enzyme inhibitor) and observed a marked increase in ENaC activity (measured as the product of the number of channels (N) and the open probability (Po) of a channel). These results suggest that ubiquitination of lung ENaC is redox-sensitive and may have significant implications for our understanding of the role of ENaC in pulmonary conditions where oxidative stress occurs, such as pulmonary edema and acute lung injury.
Assuntos
Canais Epiteliais de Sódio/metabolismo , Peróxido de Hidrogênio/farmacologia , Ubiquitinas/metabolismo , Células Epiteliais Alveolares/efeitos dos fármacos , Células Epiteliais Alveolares/metabolismo , Animais , Células Cultivadas , Proteínas Culina/metabolismo , Ciclopentanos/farmacologia , Canais Epiteliais de Sódio/genética , Feminino , Expressão Gênica , Potenciais da Membrana , Camundongos , Camundongos Endogâmicos C57BL , Proteína NEDD8 , Oxirredução , Técnicas de Patch-Clamp , Pirimidinas/farmacologia , Ratos , Enzimas Ativadoras de Ubiquitina/antagonistas & inibidores , Enzimas Ativadoras de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Regulação para CimaRESUMO
The mechanisms by which enteric commensal microbiota influence maturation and repair of the epithelial barrier are relatively unknown. Epithelial restitution requires active cell migration, a process dependent on dynamic turnover of focal cell-matrix adhesions (FAs). Here, we demonstrate that natural, commensal bacteria stimulate generation of reactive oxygen species (ROS) in intestinal epithelia. Bacteria-mediated ROS generation induces oxidation of target cysteines in the redox-sensitive tyrosine phosphatases, LMW-PTP and SHP-2, which in turn results in increased phosphorylation of focal adhesion kinase (FAK), a key protein regulating the turnover of FAs. Accordingly, phosphorylation of FAK substrate proteins, focal adhesion formation, and cell migration are all significantly enhanced by bacterial contact in both in vitro and in vivo models of wound closure. These results suggest that commensal bacteria regulate cell migration via induced generation of ROS in epithelial cells.
Assuntos
Enterobacteriaceae/metabolismo , Células Epiteliais/metabolismo , Quinase 1 de Adesão Focal/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Movimento Celular , Adesões Focais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Monoéster Fosfórico Hidrolases , CicatrizaçãoRESUMO
BACKGROUND: Medical Imaging and radiotherapy (MIRT) are at the forefront of artificial intelligence applications. The exponential increase of these applications has made governance frameworks necessary to uphold safe and effective clinical adoption. There is little information about how healthcare practitioners in MIRT in the UK use AI tools, their governance and associated challenges, opportunities and priorities for the future. METHODS: This cross-sectional survey was open from November to December 2022 to MIRT professionals who had knowledge or made use of AI tools, as an attempt to map out current policy and practice and to identify future needs. The survey was electronically distributed to the participants. Statistical analysis included descriptive statistics and inferential statistics on the SPSS statistical software. Content analysis was employed for the open-ended questions. RESULTS: Among the 245 responses, the following were emphasised as central to AI adoption: governance frameworks, practitioner training, leadership, and teamwork within the AI ecosystem. Prior training was strongly correlated with increased knowledge about AI tools and frameworks. However, knowledge of related frameworks remained low, with different professionals showing different affinity to certain frameworks related to their respective roles. Common challenges and opportunities of AI adoption were also highlighted, with recommendations for future practice.
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Inteligência Artificial , Humanos , Estudos Transversais , Diagnóstico por Imagem , Reino UnidoRESUMO
BACKGROUND: Artificial intelligence (AI)-enabled applications are increasingly being used in providing healthcare services, such as medical imaging support. Sufficient and appropriate education for medical imaging professionals is required for successful AI adoption. Although, currently, there are AI training programmes for radiologists, formal AI education for radiographers is lacking. Therefore, this study aimed to evaluate and discuss a postgraduate-level module on AI developed in the UK for radiographers. METHODOLOGY: A participatory action research methodology was applied, with participants recruited from the first cohort of students enrolled in this module and faculty members. Data were collected using online, semi-structured, individual interviews and focus group discussions. Textual data were processed using data-driven thematic analysis. RESULTS: Seven students and six faculty members participated in this evaluation. Results can be summarised in the following four themes: a. participants' professional and educational backgrounds influenced their experiences, b. participants found the learning experience meaningful concerning module design, organisation, and pedagogical approaches, c. some module design and delivery aspects were identified as barriers to learning, and d. participants suggested how the ideal AI course could look like based on their experiences. CONCLUSIONS: The findings of our work show that an AI module can assist educators/academics in developing similar AI education provisions for radiographers and other medical imaging and radiation sciences professionals. A blended learning delivery format, combined with customisable and contextualised content, using an interprofessional faculty approach is recommended for future similar courses.
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Technological advancements in computer science have started to bring artificial intelligence (AI) from the bench closer to the bedside. While there is still lots to do and improve, AI models in medical imaging and radiotherapy are rapidly being developed and increasingly deployed in clinical practice. At the same time, AI governance frameworks are still under development. Clinical practitioners involved with procuring, deploying, and adopting AI tools in the UK should be well-informed about these AI governance frameworks. This scoping review aimed to map out available literature on AI governance in the UK, focusing on medical imaging and radiotherapy. Searches were performed on Google Scholar, Pubmed, and the Cochrane Library, between June and July 2022. Of 4225 initially identified sources, 35 were finally included in this review. A comprehensive conceptual AI governance framework was proposed, guided by the need for rigorous AI validation and evaluation procedures, the accreditation rules and standards, and the fundamental ethical principles of AI. Fairness, transparency, trustworthiness, and explainability should be drivers of all AI models deployed in clinical practice. Appropriate staff education is also mandatory to ensure AI's safe and responsible use. Multidisciplinary teams under robust leadership will facilitate AI adoption, and it is crucial to involve patients, the public, and practitioners in decision-making. Collaborative research should be encouraged to enhance and promote innovation, while caution should be paid to the ongoing auditing of AI tools to ensure safety and clinical effectiveness.
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Inteligência Artificial , Radioterapia (Especialidade) , Humanos , Diagnóstico por Imagem , Radiografia , Reino UnidoRESUMO
Artificial intelligence (AI) has transitioned from the lab to the bedside, and it is increasingly being used in healthcare. Radiology and Radiography are on the frontline of AI implementation, because of the use of big data for medical imaging and diagnosis for different patient groups. Safe and effective AI implementation requires that responsible and ethical practices are upheld by all key stakeholders, that there is harmonious collaboration between different professional groups, and customised educational provisions for all involved. This paper outlines key principles of ethical and responsible AI, highlights recent educational initiatives for clinical practitioners and discusses the synergies between all medical imaging professionals as they prepare for the digital future in Europe. Responsible and ethical AI is vital to enhance a culture of safety and trust for healthcare professionals and patients alike. Educational and training provisions for medical imaging professionals on AI is central to the understanding of basic AI principles and applications and there are many offerings currently in Europe. Education can facilitate the transparency of AI tools, but more formalised, university-led training is needed to ensure the academic scrutiny, appropriate pedagogy, multidisciplinarity and customisation to the learners' unique needs are being adhered to. As radiographers and radiologists work together and with other professionals to understand and harness the benefits of AI in medical imaging, it becomes clear that they are faced with the same challenges and that they have the same needs. The digital future belongs to multidisciplinary teams that work seamlessly together, learn together, manage risk collectively and collaborate for the benefit of the patients they serve.
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To define roles for reactive oxygen species (ROS) and epithelial sodium channel (ENaC) in maintaining lung fluid balance in vivo, we used two novel whole animal imaging approaches. Live X-ray fluoroscopy enabled quantification of air space fluid content of C57BL/6J mouse lungs challenged by intratracheal (IT) instillation of saline; results were confirmed by using conventional lung wet-to-dry weight ratios and Evans blue as measures of pulmonary edema. Visualization and quantification of ROS produced in lungs was performed in mice that had been administered a redox-sensitive dye, hydro-Cy7, by IT instillation. We found that inhibition of NADPH oxidase with a Rac-1 inhibitor, NSC23766, resulted in alveolar flooding, which correlated with a decrease in lung ROS production in vivo. Consistent with a role for Nox2 in alveolar fluid balance, Nox2(-/-) mice showed increased retention of air space fluid compared with wild-type controls. Interestingly, fluoroscopic analysis of C57BL/6J lungs IT instilled with LPS showed an acute stimulation of lung fluid clearance and ROS production in vivo that was abrogated by the ROS scavenger tetramethylpiperidine-N-oxyl (TEMPO). Acute application of LPS increased the activity of 20 pS nonselective ENaC channels in rat type 1 cells; the average number of channel and single-channel open probability (NPo) increased from 0.14 ± 0.04 to 0.62 ± 0.23. Application of TEMPO to the same cell-attached recording caused an immediate significant decrease in ENaC NPo to 0.04 ± 0.03. These data demonstrate that, in vivo, ROS has the capacity to stimulate lung fluid clearance by increasing ENaC activity.