RESUMO
BACKGROUND: The purpose of this study was to examine the health-related quality of life of sexual minority survivors in comparison with heterosexual survivors. METHODS: Four hundred eighty eligible survivors participated in a telephone survey that measured survivors' outcomes, which consisted of physical and mental quality of life and self-rated fair or poor health. These survivors were diagnosed with stage I, II, or III colorectal cancer an average of 3 years before the survey and were recruited from 4 cancer registries. Using forward selection with generalized linear models or logistic regression models, the authors considered 4 domains-personal factors, environmental factors, health condition characteristics, and body function and structure-as correlates for each survivorship outcome. RESULTS: The authors found that unadjusted physical quality of life and self-rated fair/poor health were similar for all survivors. Sexual minority survivors had poorer unadjusted mental quality of life in comparison with heterosexual survivors. After adjustments for covariates, this difference was no longer statistically significant. Three domains (personal factors, health condition characteristics, and body function and structure) explained colorectal cancer survivors' fair/poor health and 46% of the variance in physical quality of life, whereas 56% of the variance in mental quality of life was explained by personal factors, body function and structure, and environmental factors. CONCLUSIONS: This study has identified modifiable factors that can be used to improve cancer survivors' quality of life and are, therefore, relevant to ongoing efforts to improve the survivorship experience.
Assuntos
Sobreviventes de Câncer , Neoplasias Colorretais , Feminino , Humanos , Masculino , Qualidade de Vida , Comportamento Sexual , SobreviventesRESUMO
Seasonal influenza carrying key hemagglutinin (HA) head region glycosylation sites can be removed from the lung by pulmonary surfactant protein D (SP-D). Little is known about HA head glycosylation of low-pathogenicity avian influenza virus (LPAIV) subtypes. These can pose a pandemic threat through reassortment and emergence in human populations. Since the presence of head region high-mannose glycosites dictates SP-D activity, the ability to predict these glycosite glycan subtypes may be of value. Here, we investigate the activities of two recombinant human SP-D forms against representative LPAIV strains, including H2N1, H5N1, H6N1, H11N9, an avian H3N8, and a human seasonal H3N2 subtype. Using mass spectrometry, we determined the glycan subclasses and heterogeneities at each head glycosylation site. Sequence alignment and molecular structure analysis of the HAs were performed for LPAIV strains in comparison to seasonal H3N2 and avian H3N8. Intramolecular contacts were determined between the protein backbone and glycosite glycan based on available three-dimensional structure data. We found that glycosite "N165" (H3 numbering) is occupied by high-mannose glycans in H3 HA but by complex glycans in all LPAIV HAs. SP-D was not active on LPAIV but was on H3 HAs. Since SP-D affinity for influenza HA depends on the presence of high-mannose glycan on the head region, our data demonstrate that SP-D may not protect against virus containing these HA subtypes. Our results also demonstrate that glycan subtype can be predicted at some glycosites based on sequence comparisons and three-dimensional structural analysis.IMPORTANCE Low-pathogenicity avian influenza virus (LPAIV) subtypes can reassort with circulating human strains and pandemic viruses can emerge in human populations, as was seen in the 1957 pandemic, in which an H2 virus reassorted with the circulating H1N1 to create a novel H2N2 genotype. Lung surfactant protein D (SP-D), a key factor in first-line innate immunity defense, removes influenza type A virus (IAV) through interaction with hemagglutinin (HA) head region high-mannose glycan(s). While it is known that both H1 and H3 HAs have one or more key high-mannose glycosites in the head region, little is known about similar glycosylation of LPAIV strains H2N1, H5N1, H6N1, or H11N9, which may pose future health risks. Here, we demonstrate that the hemagglutinins of LPAIV strains do not have the required high-mannose glycans and do not interact with SP-D, and that sequence analysis can predict glycan subtype, thus predicting the presence or absence of this virulence marker.
Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Interações Hospedeiro-Patógeno/fisiologia , Vírus da Influenza A/metabolismo , Polissacarídeos/metabolismo , Proteína D Associada a Surfactante Pulmonar/metabolismo , Sequência de Aminoácidos , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Humanos , Vírus da Influenza A Subtipo H1N1 , Vírus da Influenza A Subtipo H3N2 , Vírus da Influenza A Subtipo H3N8 , Virus da Influenza A Subtipo H5N1 , Modelos Moleculares , Polissacarídeos/química , Conformação Proteica , Análise de Sequência de Proteína , VirulênciaRESUMO
Development of pneumonia is the most lethal consequence of influenza, increasing mortality more than 50-fold compared with uncomplicated infection. The spread of viral infection from conducting airways to the alveolar epithelium is therefore a pivotal event in influenza pathogenesis. We found that mitogenic stimulation with keratinocyte growth factor (KGF) markedly accelerated mortality after infectious challenge with influenza A virus (IAV). Coadministration of KGF with IAV markedly accelerated the spread of viral infection from the airways to alveoli compared with challenge with IAV alone, based on spatial and temporal analyses of viral nucleoprotein staining of lung tissue sections and dissociated lung cells. To better define the temporal relationship between KGF administration and susceptibility to IAV infection in vivo, we administered KGF 120, 48, 24, and 0 h before intrapulmonary IAV challenge and assessed the percentages of proliferating and IAV-infected, alveolar type II (AECII) cells in dispersed lung cell populations. Peak AECII infectivity coincided with the timing of KGF administration that also induced peak AECII proliferation. AECII from mice that were given intrapulmonary KGF before isolation and then infected with IAV ex vivo exhibited the same temporal pattern of proliferation and infectious susceptibility. KGF-induced increases in mortality, AECII proliferation, and enhanced IAV susceptibility were all reversed by pretreatment of the animals with the mTOR inhibitor rapamycin before mitogenic stimulation. Taken together, these data suggest mTOR signaling-dependent, mitogenic conditioning of AECII is a determinant of host susceptibility to infection with IAV.
Assuntos
Células Epiteliais Alveolares/metabolismo , Proliferação de Células/efeitos dos fármacos , Fator 7 de Crescimento de Fibroblastos/farmacologia , Vírus da Influenza A/metabolismo , Mitógenos/farmacologia , Infecções por Orthomyxoviridae/metabolismo , Células Epiteliais Alveolares/patologia , Animais , Suscetibilidade a Doenças/induzido quimicamente , Feminino , Camundongos , Camundongos Endogâmicos DBA , Infecções por Orthomyxoviridae/patologiaRESUMO
Innate immunity is critical in the early containment of influenza A virus (IAV) infection, and surfactant protein D (SP-D) plays a crucial role in the pulmonary defense against IAV. In pigs, which are important intermediate hosts during the generation of pandemic IAVs, SP-D uses its unique carbohydrate recognition domain (CRD) to interact with IAV. An N-linked CRD glycosylation provides interactions with the sialic acid-binding site of IAV, and a tripeptide loop at the lectin-binding site facilitates enhanced interactions with IAV glycans. Here, to investigate both mechanisms of IAV neutralization in greater detail, we produced an N-glycosylated neck-CRD fragment of porcine SP-D (RpNCRD) in HEK293 cells. X-ray crystallography disclosed that the N-glycan did not alter the CRD backbone structure, including the lectin site conformation, but revealed a potential second nonlectin-binding site for glycans. IAV hemagglutination inhibition, IAV aggregation, and neutralization of IAV infection studies showed that RpNCRD, unlike the human analogue RhNCRD, exhibits potent neutralizing activity against pandemic A/Aichi/68 (H3N2), enabled by both porcine-specific structural features of its CRD. MS analysis revealed an N-glycan site-occupancy of >98% at Asn-303 of RpNCRD with complex-type, heterogeneously branched and predominantly α(2,3)-sialylated oligosaccharides. Glycan-binding array data characterized both RpNCRD and RhNCRD as mannose-type lectins. RpNCRD also bound LewisY structures, whereas RhNCRD bound polylactosamine-containing glycans. The presence of the N-glycan in the CRD increases the glycan-binding specificity of RpNCRD. These insights increase our understanding of porcine-specific innate defense against pandemic IAV and may inform the design of recombinant SP-D-based antiviral drugs.
Assuntos
Imunidade Inata/imunologia , Vírus da Influenza A/imunologia , Lectinas/metabolismo , Infecções por Orthomyxoviridae/prevenção & controle , Polissacarídeos/metabolismo , Proteína D Associada a Surfactante Pulmonar/metabolismo , Ácidos Siálicos/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Configuração de Carboidratos , Glicosilação , Testes de Inibição da Hemaglutinação , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/virologia , Polissacarídeos/química , Proteína D Associada a Surfactante Pulmonar/química , Proteína D Associada a Surfactante Pulmonar/genética , Homologia de Sequência , SuínosRESUMO
This commentary highlights the article by Pociask et al that provides new insights into the lingering effects of influenza infection.
Assuntos
Influenza Humana/patologia , Pulmão/patologia , Pulmão/virologia , Animais , Modelos Animais de Doenças , Humanos , Influenza Humana/genética , Influenza Humana/virologia , Superinfecção/microbiologia , Superinfecção/virologia , Transcriptoma/genéticaRESUMO
Despite sustained biomedical research effort, influenza A virus remains an imminent threat to the world population and a major healthcare burden. The challenge in developing vaccines against influenza is the ability of the virus to mutate rapidly in response to selective immune pressure. Hemagglutinin is the predominant surface glycoprotein and the primary determinant of antigenicity, virulence and zoonotic potential. Mutations leading to changes in the number of HA glycosylation sites are often reported. Such genetic sequencing studies predict at best the disruption or creation of sequons for N-linked glycosylation; they do not reflect actual phenotypic changes in HA structure. Therefore, combined analysis of glycan micro and macro-heterogeneity and bioassays will better define the relationships among glycosylation, viral bioactivity and evolution. We present a study that integrates proteomics, glycomics and glycoproteomics of HA before and after adaptation to innate immune system pressure. We combined this information with glycan array and immune lectin binding data to correlate the phenotypic changes with biological activity. Underprocessed glycoforms predominated at the glycosylation sites found to be involved in viral evolution in response to selection pressures and interactions with innate immune-lectins. To understand the structural basis for site-specific glycan microheterogeneity at these sites, we performed structural modeling and molecular dynamics simulations. We observed that the presence of immature, high-mannose type glycans at a particular site correlated with reduced accessibility to glycan remodeling enzymes. Further, the high mannose glycans at sites implicated in immune lectin recognition were predicted to be capable of forming trimeric interactions with the immune-lectin surfactant protein-D.
Assuntos
Glicômica/métodos , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Vírus da Influenza A/metabolismo , Polissacarídeos/análise , Proteômica/métodos , Biologia Computacional/métodos , Cristalografia por Raios X , Glicosilação , Humanos , Imunidade Inata , Vírus da Influenza A/química , Manose/metabolismo , Modelos Moleculares , Simulação de Dinâmica Molecular , Polissacarídeos/químicaRESUMO
Pancreatic cancer is more common in older adults, who are underrepresented in clinical trials and frequently under treated. Chronological age alone should not deter clinicians from offering treatment to geriatric patients, as they are a heterogeneous population. Geriatric assessment, frailty assessment tools, and toxicity risk scores help clinicians select appropriate patients for therapy. For resectable disease, surgery can be safe but should be done at a high-volume center. Adjuvant therapy is important; though there remains controversy on the role of radiation, chemotherapy is well studied and efficacious. In locally advanced unresectable disease, chemoradiation or chemotherapy alone is an option. Neoadjuvant therapy improves the chances of resectability in borderline resectable disease. Chemotherapy extends survival in metastatic disease, but treatment goals and risk-benefit ratios have to be clarified. Adequate symptom management and supportive care are important. There are now many new treatment strategies and novel therapies for this disease.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Quimiorradioterapia , Avaliação Geriátrica/métodos , Terapia Neoadjuvante , Neoplasias Pancreáticas/terapia , Idoso , Terapia Combinada , Humanos , Neoplasias Pancreáticas/mortalidade , Seleção de Pacientes , Guias de Prática Clínica como Assunto , Prognóstico , Medição de Risco , Taxa de SobrevidaRESUMO
We recently reported that a trimeric neck and carbohydrate recognition domain (NCRD) fragment of human surfactant protein D (SP-D), a host defense lectin, with combinatorial substitutions at the 325 and 343 positions (D325A+R343V) exhibits markedly increased antiviral activity for seasonal strains of influenza A virus (IAV). The NCRD binds to glycan-rich viral envelope proteins including hemagglutinin (HA). We now show that replacement of D325 with serine to create D325S+R343V provided equal or increased neutralizing activity compared with D325A+R343V. The activity of the double mutants was significantly greater than that of either single mutant (D325A/S or R343V). D325A+R343V and D325S+R343V also strongly inhibited HA activity, and markedly aggregated, the 1968 pandemic H3N2 strain, Aichi68. D325S+R343V significantly reduced viral loads and mortality of mice infected with Aichi68, whereas wild-type SP-D NCRD did not. The pandemic H1N1 strains of 1918 and 2009 have only one N-linked glycan side on the head region of the HA and are fully resistant to inhibition by native SP-D. Importantly, we now show that D325A+R343V and D325S+R343V inhibited Cal09 H1N1 and related strains, and reduced uptake of Cal09 by epithelial cells. Inhibition of Cal09 was mediated by the lectin activity of the NCRDs. All known human pandemic strains have at least one glycan attachment on the top or side of the HA head, and our results indicate that they may be susceptible to inhibition by modified host defense lectins.
Assuntos
Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A Subtipo H3N2/imunologia , Influenza Humana/virologia , Proteína D Associada a Surfactante Pulmonar/genética , Animais , Sítios de Ligação , Células CHO , Cricetinae , Cricetulus , Resistência à Doença , Cães , Feminino , Interações Hospedeiro-Patógeno , Humanos , Influenza Humana/imunologia , Células Madin Darby de Rim Canino , Camundongos , Camundongos Endogâmicos DBA , Mutação de Sentido Incorreto , Pandemias , Ligação Proteica , Proteína D Associada a Surfactante Pulmonar/química , Proteína D Associada a Surfactante Pulmonar/metabolismo , Carga ViralRESUMO
θ-Defensins are cyclic octadecapeptides found in nonhuman primates whose broad antiviral spectrum includes HIV-1, HSV-1, severe acute respiratory syndrome coronavirus, and influenza A virus (IAV). We previously reported that synthetic θ-defensins called retrocyclins can neutralize and aggregate various strains of IAV and increase IAV uptake by neutrophils. This study describes two families of peptides, hapivirins and diprovirins, whose design was inspired by retrocyclins. The goal was to develop smaller partially cyclic peptides that retain the antiviral activity of retrocyclins, while being easier to synthesize. The novel peptides also allowed for systemic substitution of key residues to evaluate the role of charge or hydrophobicity on antiviral activity. Seventy-two hapivirin or diprovirin peptides are described in this work, including several whose anti-IAV activity equals or exceeds that of normal α- or θ-defensins. Some of these also had strong antibacterial and antifungal activity. These new peptides were active against H3N2 and H1N1 strains of IAV. Structural features imparting strong antiviral activity were identified through iterative cycles of synthesis and testing. Our findings show the importance of hydrophobic residues for antiviral activity and show that pegylation, which often increases a peptide's serum t(1/2) in vivo, can increase the antiviral activity of DpVs. The new peptides acted at an early phase of viral infection, and, when combined with pulmonary surfactant protein D, their antiviral effects were additive. The peptides strongly increased neutrophil and macrophage uptake of IAV, while inhibiting monocyte cytokine generation. Development of modified θ-defensin analogs provides an approach for creating novel antiviral agents for IAV infections.
Assuntos
Antivirais/síntese química , Antivirais/farmacologia , Defensinas/imunologia , Defensinas/farmacologia , Vírus da Influenza A/imunologia , Sequência de Aminoácidos , Animais , Antivirais/imunologia , Linhagem Celular , Técnicas de Química Sintética , Cromatografia Líquida de Alta Pressão , Defensinas/síntese química , Cães , Humanos , Microscopia Eletrônica de Transmissão , Dados de Sequência Molecular , Monócitos/virologia , Neutrófilos/virologia , Peptídeos , Relação Estrutura-Atividade , Fator de Necrose Tumoral alfa/biossínteseRESUMO
The collectins have been shown to have a role in host defense against influenza A virus (IAV) and other significant viral pathogens (e.g., HIV). The ficolins are a related group of innate immune proteins that are present at relatively high concentrations in serum, but also in respiratory secretions; however, there has been little study of the role of ficolins in viral infection. In this study, we demonstrate that purified recombinant human H-ficolin and H-ficolin in human serum and bronchoalveolar lavage fluid bind to IAV and inhibit viral infectivity and hemagglutination activity in vitro. Removal of ficolins from human serum or bronchoalveolar lavage fluid reduces their antiviral activity. Inhibition of IAV did not involve the calcium-dependent lectin activity of H-ficolin. We demonstrate that H-ficolin is sialylated and that removal of sialic acid abrogates IAV inhibition, while addition of the neuraminidase inhibitor oseltamivir potentiates neutralization, hemagglutinin inhibition, and viral aggregation caused by H-ficolin. Pandemic and mouse-adapted strains of IAV are generally not inhibited by the collectins surfactant protein D or mannose binding lectin because of a paucity of glycan attachments on the hemagglutinin of these strains. In contrast, H-ficolin inhibited both the mouse-adapted PR-8 H1N1 strain and a pandemic H1N1 strain from 2009. H-ficolin also fixed complement to a surface coated with IAV. These findings suggest that H-ficolin contributes to host defense against IAV.
Assuntos
Antivirais/farmacologia , Glicoproteínas/fisiologia , Vírus da Influenza A Subtipo H3N2/imunologia , Lectinas/fisiologia , Pandemias , Estações do Ano , Replicação Viral/imunologia , Animais , Linhagem Celular Tumoral , Células Cultivadas , Testes de Fixação de Complemento , Cães , Humanos , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A Subtipo H1N1/patogenicidade , Vírus da Influenza A Subtipo H3N2/patogenicidade , Influenza Humana/imunologia , Influenza Humana/prevenção & controle , Camundongos , Testes de Neutralização , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Proteínas da Matriz Viral/antagonistas & inibidoresRESUMO
Surfactant protein D (SP-D), a mammalian C-type lectin, is the primary innate inhibitor of influenza A virus (IAV) in the lung. Interactions of SP-D with highly branched viral N-linked glycans on hemagglutinin (HA), an abundant IAV envelope protein and critical virulence factor, promote viral aggregation and neutralization through as yet unknown molecular mechanisms. Two truncated human SP-D forms, wild-type (WT) and double mutant D325A+R343V, representing neck and carbohydrate recognition domains are compared in this study. Whereas both WT and D325A+R343V bind to isolated glycosylated HA, WT does not inhibit IAV in neutralization assays; in contrast, D325A+R343V neutralization compares well with that of full-length native SP-D. To elucidate the mechanism for these biochemical observations, we have determined crystal structures of D325A+R343V in the presence and absence of a viral nonamannoside (Man9). On the basis of the D325A+R343V-Man9 structure and other crystallographic data, models of complexes between HA and WT or D325A+R343V were produced and subjected to molecular dynamics. Simulations reveal that whereas WT and D325A+R343V both block the sialic acid receptor site of HA, the D325A+R343V complex is more stable, with stronger binding caused by additional hydrogen bonds and hydrophobic interactions with HA residues. Furthermore, the blocking mechanism of HA differs for WT and D325A+R343V because of alternate glycan binding modes. The combined results suggest a mechanism through which the mode of SP-D-HA interaction could significantly influence viral aggregation and neutralization. These studies provide the first atomic-level molecular view of an innate host defense lectin inhibiting its viral glycoprotein target.
Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Modelos Moleculares , Proteína D Associada a Surfactante Pulmonar/química , Adesividade , Substituição de Aminoácidos , Sítios de Ligação , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Humanos , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Vírus da Influenza A Subtipo H3N2/química , Vírus da Influenza A Subtipo H3N2/crescimento & desenvolvimento , Vírus da Influenza A Subtipo H3N2/metabolismo , Viabilidade Microbiana , Simulação de Dinâmica Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Polissacarídeos/química , Polissacarídeos/metabolismo , Conformação Proteica , Proteína D Associada a Surfactante Pulmonar/genética , Proteína D Associada a Surfactante Pulmonar/metabolismo , Receptores de Superfície Celular/antagonistas & inibidores , Receptores de Superfície Celular/química , Receptores de Superfície Celular/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
Pigs can act as intermediate hosts by which reassorted influenza A virus (IAV) strains can be transmitted to humans and cause pandemic influenza outbreaks. The innate host defense component surfactant protein D (SP-D) interacts with glycans on the hemagglutinin of IAV and contributes to protection against IAV infection in mammals. This study shows that a recombinant trimeric neck lectin fragment derived from porcine SP-D (pSP-D) exhibits profound inhibitory activity against IAV, in contrast to comparable fragments derived from human SP-D. Crystallographic analysis of the pSP-D fragment complexed with a viral sugar component shows that a unique tripeptide loop alters the lectin site conformation of pSP-D. Molecular dynamics simulations highlight the role of this flexible loop, which adopts a more stable conformation upon sugar binding and may facilitate binding to viral glycans through contact with distal portions of the branched mannoside. The combined data demonstrate that porcine-specific structural features of SP-D contribute significantly to its distinct anti-IAV activity. These findings could help explain why pigs serve as important reservoirs for newly emerging pathogenic IAV strains.
Assuntos
Antivirais/farmacologia , Metabolismo dos Carboidratos , Vírus da Influenza A/efeitos dos fármacos , Proteína D Associada a Surfactante Pulmonar/farmacologia , Animais , Antivirais/química , Sequência de Bases , Sítios de Ligação , Células Cultivadas , Cristalização , Primers do DNA , Cães , Testes de Sensibilidade Microbiana , Modelos Moleculares , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Proteína D Associada a Surfactante Pulmonar/química , Proteína D Associada a Surfactante Pulmonar/genética , SuínosRESUMO
The early inflammatory response to influenza A virus infection contributes to severe lung disease and continues to pose a serious threat to human health. The mechanisms by which inflammatory cells invade the respiratory tract remain unclear. Uncontrolled inflammation and oxidative stress cause lung damage in response to influenza A infection. We have previously shown that the fatty acid binding protein 5 (FABP5) has anti-inflammatory properties. We speculate that, as a transporter of fatty acids, FABP5 plays an important protective role against oxidative damage to lipids during infection as well. Using FABP5-/- and wild-type (WT) mice infected with influenza A virus, we showed that FABP5-/- mice had increased cell infiltration of macrophages and neutrophils compared with WT mice. FABP5-/- mice presented lower viral burden but lost as much weight as WT mice. The adaptive immune response was also increased in FABP5-/- mice as illustrated by the accumulation of T and B cells in the lung tissues and increased levels of H1N1-specific IgG antibodies. FABP5 deficiency greatly enhanced oxidative damage and lipid peroxidation following influenza A infection and presented with sustained tissue inflammation. Interestingly, FABP5 expression decreased following influenza A infection in WT lung tissues that corresponded to a decrease in the anti-inflammatory molecule PPAR-γ activity. In conclusion, our results demonstrate a previously unknown contribution of FABP5 to influenza A virus pathogenesis by controlling excessive oxidative damage and inflammation. This property could be exploited for therapeutic purposes.
Assuntos
Proteínas de Ligação a Ácido Graxo/fisiologia , Vírus da Influenza A Subtipo H1N1/patogenicidade , Influenza Humana/imunologia , Proteínas de Neoplasias/fisiologia , Estresse Oxidativo , Pneumonia/etiologia , Imunidade Adaptativa , Animais , Western Blotting , Células Cultivadas , Suscetibilidade a Doenças , Ensaio de Imunoadsorção Enzimática , Imunofluorescência , Humanos , Técnicas Imunoenzimáticas , Imunoprecipitação , Influenza Humana/complicações , Influenza Humana/virologia , Peroxidação de Lipídeos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , PPAR gama/metabolismo , Pneumonia/metabolismo , Pneumonia/patologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
LL-37, the only human cathelicidin, is a cationic antimicrobial peptide with antibacterial and antifungal activity. LL-37 is released from neutrophil granules and produced by epithelial cells. It has been implicated in host defence against influenza A virus (IAV) in recent studies. We now demonstrate dose-related neutralizing activity of LL-37 against several seasonal and mouse-adapted IAV strains. The ability of LL-37 to inhibit these IAV strains resulted mainly from direct effects on the virus, since pre-incubation of virus with LL-37 was needed for optimal inhibition. LL-37 bound high-density lipoprotein (HDL), and pre-incubation of LL-37 with human serum or HDL reduced its antiviral activity. LL-37 did not inhibit viral association with epithelial cells as assessed by quantitative RT-PCR or confocal microscopy. This finding contrasted with results obtained with surfactant protein D (SP-D). Unlike collectins or human neutrophil defensins (HNPs), LL-37 did not induce viral aggregation under electron microscopy. In the electron microscopy studies, LL-37 appeared to cause disruption of viral membranes. LL-37 had additive antiviral activity when combined with other innate inhibitors like SP-D, surfactant protein A and HNPs. Unlike HNPs, LL-37 did not bind SP-D significantly. These findings indicate that LL-37 contributes to host defence against IAV through a mechanism distinct from that of SP-D and HNPs.
Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Defensinas/metabolismo , Vírus da Influenza A Subtipo H3N2/efeitos dos fármacos , Vírus da Influenza A Subtipo H3N2/metabolismo , Vírus da Influenza A/metabolismo , Proteína D Associada a Surfactante Pulmonar/metabolismo , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/metabolismo , Peptídeos Catiônicos Antimicrobianos/imunologia , Células CHO , Colectinas/imunologia , Colectinas/metabolismo , Cricetinae , Cricetulus , Defensinas/imunologia , Cães , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Células Epiteliais/virologia , Humanos , Vírus da Influenza A Subtipo H3N2/imunologia , Vírus da Influenza A/imunologia , Lipoproteínas HDL/imunologia , Lipoproteínas HDL/metabolismo , Células Madin Darby de Rim Canino , Camundongos , Neutrófilos/imunologia , Neutrófilos/metabolismo , Neutrófilos/virologia , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/metabolismo , Infecções por Orthomyxoviridae/virologia , Proteína A Associada a Surfactante Pulmonar/imunologia , Proteína A Associada a Surfactante Pulmonar/metabolismo , Proteína D Associada a Surfactante Pulmonar/imunologia , CatelicidinasRESUMO
We provide a comprehensive review of current approved systemic treatment strategies for advanced hepatocellular carcinoma (HCC), starting with the phase III clinical trial of sorafenib which was the first to definitively show a survival benefit. After this trial, there was an initial period of little progress. However, in recent years, an explosion of new agents and combinations of agents has resulted in a markedly improved outlook for patients. We then provide the authors' current approach to therapy, i.e., "How We Treat HCC". Promising future directions and important gaps in therapy that persist are finally reviewed. HCC is a highly prevalent cancer worldwide and the incidence is growing due not only to alcoholism, hepatitis B and C, but also to steatohepatitis. HCC, like renal cell carcinoma and melanoma, is a cancer largely resistant to chemotherapy but the advent of anti-angiogenic, targeted and immune therapies have improved survival for all of these cancers. We hope this review will heighten interest in the field of HCC therapies, provide a clear outline of the current data and strategy for treatment, and sensitize readers to new developments that are likely to emerge in the near future.
RESUMO
Porcine surfactant protein D (pSP-D) displays distinctively strong, broad-range inhibitory activity against influenza A virus (IAV). N-Linked glycosylation of the carbohydrate recognition domain (CRD) of pSP-D contributes to the high affinity of this collectin for IAV. To investigate the role of the N-linked glycan further, HEK293E protein expression was used to produce recombinant pSP-D (RpSP-D) that has similar structural and antiviral properties as NpSP-D. We introduced an additional N-linked glycan in the CRD of RpSP-D but this modification did not alter the antiviral activity. Human SP-D is unglycosylated in its CRD and less active against IAV compared with pSP-D. In an attempt to modify its antiviral properties, several recombinant human SP-D (RhSP-D) mutants were constructed with N-linked glycans introduced at various locations within its CRD. To retain lectin activity, necessary for the primary interactions between SP-D and IAV, N-linked glycosylation of RhSP-D was shown to be restricted to the corresponding position in the CRD of either pSP-D or surfactant protein A (SP-A). These N-glycosylated RhSP-D mutants, however, did not show increased neutralization activity against IAV. By developing RhSP-D mutants that also have the pSP-D-specific Ser-Gly-Ala loop inserted in the CRD, we could demonstrate that the N-linked glycan-mediated interactions between pSP-D and IAV involves additional structural prerequisites of the pSP-D CRD. Ultimately, these studies will help to develop highly effective SP-D-based therapeutic and prophylactic drugs against IAV.
Assuntos
Vírus da Influenza A/metabolismo , Lectinas , Polissacarídeos/metabolismo , Proteína D Associada a Surfactante Pulmonar/metabolismo , Animais , Cães , Glicosilação , Células HEK293 , Humanos , Vírus da Influenza A/química , Vírus da Influenza A/genética , Influenza Humana/genética , Influenza Humana/terapia , Mutação , Polissacarídeos/química , Polissacarídeos/genética , Proteína A Associada a Surfactante Pulmonar/química , Proteína A Associada a Surfactante Pulmonar/genética , Proteína A Associada a Surfactante Pulmonar/metabolismo , Proteína D Associada a Surfactante Pulmonar/química , Proteína D Associada a Surfactante Pulmonar/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , SuínosRESUMO
The recognition of influenza A virus (IAV) by surfactant protein D (SP-D) is mediated by interactions between the SP-D carbohydrate recognition domains (CRD) and glycans displayed on envelope glycoproteins. Although native human SP-D shows potent antiviral and aggregating activity, trimeric recombinant neck+CRDs (NCRDs) show little or no capacity to influence IAV infection. A mutant trimeric NCRD, D325A/R343V, showed marked hemagglutination inhibition and viral neutralization, with viral aggregation and aggregation-dependent viral uptake by neutrophils. D325A/R343V exhibited glucose-sensitive binding to Phil82 hemagglutinin trimer (HA) by surface plasmon resonance. By contrast, there was very low binding to the HA trimer from another virus (PR8) that lacks glycans on the HA head. Mass spectrometry demonstrated the presence of high mannose glycans on the Phil82 HA at positions known to contribute to IAV binding. Molecular modeling predicted an enhanced capacity for bridging interactions between HA glycans and D325A/R343V. Finally, the trimeric D325A/R343V NCRD decreased morbidity and increased viral clearance in a murine model of IAV infection using a reassortant A/WSN/33 virus with a more heavily glycosylated HA. The combined data support a model in which altered binding by a truncated mutant SP-D to IAV HA glycans facilitates viral aggregation, leading to significant viral neutralization in vitro and in vivo. These studies demonstrate the potential utility of homology modeling and protein structure analysis for engineering effective collectin antivirals as in vivo therapeutics.
Assuntos
Resistência à Doença/genética , Evolução Molecular , Vírus da Influenza A Subtipo H1N1/fisiologia , Proteína D Associada a Surfactante Pulmonar/química , Proteína D Associada a Surfactante Pulmonar/genética , Ressonância de Plasmônio de Superfície/métodos , Animais , Antivirais/metabolismo , Antivirais/farmacologia , Cristalografia por Raios X , Feminino , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Humanos , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Vírus da Influenza A Subtipo H1N1/metabolismo , Espectrometria de Massas , Camundongos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Infecções por Orthomyxoviridae/virologia , Multimerização Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Proteína D Associada a Surfactante Pulmonar/metabolismo , Especificidade da EspécieRESUMO
BACKGROUND: Influenza A virus (IAV) infection primarily targets respiratory epithelial cells and produces clinical outcomes ranging from mild upper respiratory infection to severe pneumonia. Recent studies have shown the importance of lung antioxidant defense systems against injury by IAV. Nuclear factor-erythroid 2 related factor 2 (Nrf2) activates the majority of antioxidant genes. METHODS: Alveolar type II (ATII) cells and alveolar macrophages (AM) were isolated from human lungs not suitable for transplantation and donated for medical research. In some studies ATII cells were transdifferentiated to alveolar type I-like (ATI-like) cells. Alveolar epithelial cells were infected with A/PR/8/34 (PR8) virus. We analyzed PR8 virus production, influenza A nucleoprotein levels, ROS generation and expression of antiviral genes. Immunocytofluorescence was used to determine Nrf2 translocation and western blotting to detect Nrf2, HO-1 and caspase 1 and 3 cleavage. We also analyzed ingestion of PR8 virus infected apoptotic ATII cells by AM, cytokine levels by ELISA, glutathione levels, necrosis and apoptosis by TUNEL assay. Moreover, we determined the critical importance of Nrf2 using adenovirus Nrf2 (AdNrf2) or Nrf2 siRNA to overexpress or knockdown Nrf2, respectively. RESULTS: We found that IAV induced oxidative stress, cytotoxicity and apoptosis in ATI-like and ATII cells. We also found that AM can ingest PR8 virus-induced apoptotic ATII cells (efferocytosis) but not viable cells, whereas ATII cells did not ingest these apoptotic cells. PR8 virus increased ROS production, Nrf2, HO-1, Mx1 and OAS1 expression and Nrf2 translocation to the nucleus. Nrf2 knockdown with siRNA sensitized ATI-like cells and ATII cells to injury induced by IAV and overexpression of Nrf2 with AdNrf2 protected these cells. Furthermore, Nrf2 overexpression followed by infection with PR8 virus decreased virus replication, influenza A nucleoprotein expression, antiviral response and oxidative stress. However, AdNrf2 did not increase IFN-λ1 (IL-29) levels. CONCLUSIONS: Our results indicate that IAV induces alveolar epithelial injury and that Nrf2 protects these cells from the cytopathic effects of IAV likely by increasing the expression of antioxidant genes. Identifying the pathways involved in protecting cells from injury during influenza infection may be particularly important for developing new therapeutic strategies.
Assuntos
Células Epiteliais/metabolismo , Células Epiteliais/virologia , Vírus da Influenza A/fisiologia , Alvéolos Pulmonares/metabolismo , Alvéolos Pulmonares/virologia , Adolescente , Sobrevivência Celular , Células Cultivadas , Criança , Pré-Escolar , Células Epiteliais/patologia , Feminino , Humanos , Masculino , Alvéolos Pulmonares/patologiaRESUMO
BACKGROUND: Excessive inflammatory host response increases morbidity and mortality associated with seasonal respiratory influenza, and highly pathogenic virus strains are characterized by massive infiltration of monocytes and/or macrophages that produce a storm of injurious cytokines. METHODS: Here, we examined the role in respiratory influenza of serpinB1, an endogenous inhibitor of the serine proteases elastase, cathepsin G, and proteinase-3, increasingly recognized as regulators of inflammation. RESULTS: After challenge with high-dose surfactant protein-D (SP-D)-sensitive influenza A/Philadelphia/82 (H3N2), serpinB1(-/-) mice died earlier and in greater numbers than did wild-type mice. Sublethally infected animals suffered increased morbidity, delayed resolution of epithelial injury, and increased immune cell death. Viral clearance and SP-D/SP-A upregulation were unimpaired and so were early virus-induced cytokine and chemokine burst and influx of large numbers of neutrophils and monocytes. Whereas initial cytokines and chemokines rapidly cleared in wild-type mice, TNF-α, IL-6, KC/CXCL1, G-CSF, IL-17A, and MCP-1/CCL2 remained elevated in serpinB1(-/-) mice. Monocyte-derived cells were the dominant immune cells in influenza-infected lungs, and those from serpinB1(-/-) mice produced excessive IL-6 and TNF-α when tested ex vivo. Pulmonary γδ T-cells that produced IL-17A were also increased. CONCLUSIONS: Because viral clearance was unimpaired, the study highlights the critical role of serpinB1 in mitigating inflammation and restricting pro-inflammatory cytokine production in influenza infection.
Assuntos
Inflamação/metabolismo , Pneumopatias/virologia , Pulmão/patologia , Infecções por Orthomyxoviridae/patologia , Serpinas/metabolismo , Animais , Morte Celular , Citocinas/genética , Citocinas/metabolismo , Regulação da Expressão Gênica/fisiologia , Inflamação/patologia , Vírus da Influenza A Subtipo H3N2 , Pneumopatias/patologia , Camundongos , Camundongos Knockout , Infecções por Orthomyxoviridae/imunologia , Serpinas/genética , Fatores de TempoRESUMO
Individuals with metabolic dysregulation of cellular glycosylation often experience severe influenza disease, with a poor immune response to the virus and low vaccine efficacy. Here, we investigate the consequences of aberrant cellular glycosylation for the glycome and the biology of influenza virus. We transiently induced aberrant N-linked glycosylation in cultured cells with an oligosaccharyltransferase inhibitor, NGI-1. Cells treated with NGI-1 produced morphologically unaltered viable influenza virus with sequence-neutral glycosylation changes (primarily reduced site occupancy) in the hemagglutinin and neuraminidase proteins. Hemagglutinin with reduced glycan occupancy required a higher concentration of surfactant protein D (an important innate immunity respiratory tract collectin) for inhibition compared to that with normal glycan occupancy. Immunization of mice with NGI-1-treated virus significantly reduced antihemagglutinin and antineuraminidase titers of total serum antibody and reduced hemagglutinin protective antibody responses. Our data suggest that aberrant cellular glycosylation may increase the risk of severe influenza as a result of the increased ability of glycome-modified influenza viruses to evade the immune response. IMPORTANCE People with disorders such as cancer, autoimmune disease, diabetes, or obesity often have metabolic dysregulation of cellular glycosylation and also have more severe influenza disease, a reduced immune response to the virus, and reduced vaccine efficacy. Since influenza viruses that infect such people do not show consistent genomic variations, it is generally assumed that the altered biology is mainly related to host factors. However, since host cells are responsible for glycosylation of influenza virus hemagglutinin and neuraminidase, and glycosylation is important for interactions of these proteins with the immune system, the viruses may have functional differences that are not reflected by their genomic sequence. Here, we show that imbalanced cellular glycosylation can modify the viral glycome without genomic changes, leading to reduced innate and adaptive host immune responses to infection. Our findings link metabolic dysregulation of host glycosylation to increased risk of severe influenza and reduced influenza virus vaccine efficacy.