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Expansion in production and commercial use of nanomaterials increases the potential human exposure during the lifecycle of these materials (production, use, and disposal). Inhalation is a primary route of exposure to nanomaterials; therefore it is critical to assess their potential respiratory hazard. Herein, we developed a three-dimensional alveolar model (EpiAlveolar) consisting of human primary alveolar epithelial cells, fibroblasts, and endothelial cells, with or without macrophages for predicting long-term responses to aerosols. Following thorough characterization of the model, proinflammatory and profibrotic responses based on the adverse outcome pathway concept for lung fibrosis were assessed upon repeated subchronic exposures (up to 21 days) to two types of multiwalled carbon nanotubes (MWCNTs) and silica quartz particles. We simulate occupational exposure doses for the MWCNTs (1-30 µg/cm2) using an air-liquid interface exposure device (VITROCELL Cloud) with repeated exposures over 3 weeks. Specific key events leading to lung fibrosis, such as barrier integrity and release of proinflammatory and profibrotic markers, show the responsiveness of the model. Nanocyl induced, in general, a less pronounced reaction than Mitsui-7, and the cultures with human monocyte-derived macrophages (MDMs) showed the proinflammatory response at later time points than those without MDMs. In conclusion, we present a robust alveolar model to predict inflammatory and fibrotic responses upon exposure to MWCNTs.
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BACKGROUND: Although lung volume reduction surgery (LVRS) improves survival in appropriately selected patients with COPD, few procedures are performed. The National Institute for Health and Care Excellence has recommended a more systematic approach to identifying potential candidates. We investigated LVRS referrals from a UK primary care population and aimed to establish an accurate estimate of eligible patients and determine a strategy for identifying potential candidates systematically. METHODS: Clinical Practice Research Datalink GOLD (a primary care database) and the linked Hospital Episode Statistics inpatient and Diagnostic Imaging Dataset were used. Patients with COPD who had undergone LVRS, patients who met basic eligibility criteria for further screening for LVRS, and patients meeting a more stringent eligibility criteria were identified from April 2012 to September 2015. Thoracic CT scan, pulmonary rehabilitation status, referral to respiratory outpatient clinics, and acute exacerbation of COPD requiring hospitalization were compared between actual LVRS recipients and potentially eligible patients. RESULTS: Among the 73,697 patients with COPD included, 36 (0.05%) received LVRS, 5,984 (8.1%) met basic eligibility criteria, and 159 (0.2%) met more stringent eligibility criteria. LVRS recipients were younger (mean age ± SD, 64 ± 9.2 years) than the stringently eligible patients (mean age ± SD, 69 ± 8.9 years; P = .01). Few patients meeting stringent eligibility criteria (6.9%) had a CT scan of the thorax in the preceding 3 years or had been referred for assessment in secondary care. CONCLUSIONS: A substantial unmet need exists among patients with COPD who could potentially benefit from a lung volume reduction procedure but who are not being investigated or referred to consider this possibility.
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Neumonectomía , Atención Primaria de Salud , Enfermedad Pulmonar Obstructiva Crónica/cirugía , Derivación y Consulta/estadística & datos numéricos , Tomografía Computarizada por Rayos X/estadística & datos numéricos , Anciano , Anciano de 80 o más Años , Determinación de la Elegibilidad , Femenino , Necesidades y Demandas de Servicios de Salud , Humanos , Masculino , Persona de Mediana Edad , Selección de Paciente , Enfermedad Pulmonar Obstructiva Crónica/diagnóstico por imagen , Reino UnidoRESUMEN
RATIONALE: Previous studies have suggested that acute exacerbations of chronic obstructive pulmonary disease (COPD) may be associated with increased risk of myocardial infarction and ischemic stroke. OBJECTIVES: We aimed to quantify the increased risks of myocardial infarction and ischemic stroke risk associated with both moderate and severe acute exacerbation, and to investigate factors that may modify these risks. METHODS: We performed a self-controlled case series to investigate the rates of myocardial infarction and ischemic stroke after acute exacerbation compared with stable time, within individuals. The participants were 5,696 adults with COPD with a first myocardial infarction (n = 2,850) or ischemic stroke (n = 3,010) and at least one acute exacerbation from the UK Clinical Practice Research Datalink with linked Hospital Episodes Statistics data. RESULTS: The risks of both myocardial infarction and ischemic stroke were increased in the 91 days after an acute exacerbation. The risks were greater after a severe exacerbation (incidence rate ratio [IRR], 2.58; 95% confidence interval [CI], 2.26-2.95 for myocardial infarction; and IRR, 1.97; 95% CI, 1.66-2.33 for ischemic stroke) than after a moderate exacerbation (IRR, 1.58; 95% CI, 1.46-1.71 for myocardial infarction; and IRR, 1.45; 95% CI, 1.33-1.57 for ischemic stroke). The relative risks of myocardial infarction and ischemic stroke associated with acute exacerbation were lower among those with more frequent exacerbations (IRR, 1.42; 95% CI, 1.24-1.62 vs. IRR, 1.69; 95% CI, 1.50-1.91 for myocardial infarction; and IRR, 1.30; 95% CI, 1.15-1.48 vs. IRR, 1.68; 95% CI, 1.50-1.89 for ischemic stroke). Higher GOLD stage was associated with a lower rate of myocardial infarction (IRR, 1.98; 95% CI, 1.61-2.05 vs. IRR, 1.69; 95% CI, 1.45-1.98) but not for ischemic stroke. Aspirin use at baseline was associated with a lower risk of ischemic stroke (IRR, 1.28; 95% CI, 1.10-1.50 vs. IRR, 1.63; 95% CI, 1.47-1.80) but not with myocardial infarction. CONCLUSIONS: Acute exacerbations of COPD are associated with an increased risk of myocardial infarction and ischemic stroke within 28 days of their onset. Several patient characteristics were identified that are associated with these events.
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Isquemia Encefálica/epidemiología , Infarto del Miocardio/epidemiología , Enfermedad Pulmonar Obstructiva Crónica/epidemiología , Accidente Cerebrovascular/epidemiología , Anciano , Anciano de 80 o más Años , Aspirina/uso terapéutico , Progresión de la Enfermedad , Femenino , Humanos , Masculino , Inhibidores de Agregación Plaquetaria/uso terapéutico , Factores de Riesgo , Índice de Severidad de la Enfermedad , Reino Unido/epidemiologíaRESUMEN
Despite combined antiretroviral therapy (cART), HIV+ patients still develop neurological disorders, which may be due to persistent HIV infection and selective evolution in brain tissues. Single-molecule real-time (SMRT) sequencing technology offers an improved opportunity to study the relationship among HIV isolates in the brain and lymphoid tissues because it is capable of generating thousands of long sequence reads in a single run. Here, we used SMRT sequencing to generate ~ 50,000 high-quality full-length HIV envelope sequences (> 2200 bp) from seven autopsy tissues from an HIV+/cART+ subject, including three brain and four non-brain sites. Sanger sequencing was used for comparison with SMRT data and to clone functional pseudoviruses for in vitro tropism assays. Phylogenetic analysis demonstrated that brain-derived HIV was compartmentalized from HIV outside the brain and that the variants from each of the three brain tissues grouped independently. Variants from all peripheral tissues were intermixed on the tree but independent of the brain clades. Due to the large number of sequences, a clustering analysis at three similarity thresholds (99, 99.5, and 99.9%) was also performed. All brain sequences clustered exclusive of any non-brain sequences at all thresholds; however, frontal lobe sequences clustered independently of occipital and parietal lobes. Translated sequences revealed potentially functional differences between brain and non-brain sequences in the location of putative N-linked glycosylation sites (N-sites), V1 length, V3 charge, and the number of V4 N-sites. All brain sequences were predicted to use the CCR5 co-receptor, while most non-brain sequences were predicted to use CXCR4 co-receptor. Tropism results were confirmed by in vitro infection assays. The study is the first to use a SMRT sequencing approach to study HIV compartmentalization in tissues and supports other reports of limited trafficking between brain and non-brain sequences during cART. Due to the long sequence length, we could observe changes along the entire envelope gene, likely caused by differential selective pressure in the brain that may contribute to neurological disease.
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Encéfalo/virología , Infecciones por VIH/virología , VIH-1/fisiología , Tropismo Viral/genética , Productos del Gen env del Virus de la Inmunodeficiencia Humana/genética , Adulto , Infecciones por VIH/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Macrófagos/virología , Masculino , Filogenia , Provirus/genética , Receptores CXCR4RESUMEN
HIV-1 R5 variants exploit CCR5 as a coreceptor to infect both T cells and macrophages. R5 viruses that are transmitted or derived from immune tissue and peripheral blood are mainly inefficient at mediating infection of macrophages. In contrast, highly macrophage-tropic (mac-tropic) R5 viruses predominate in brain tissue and can be detected in cerebrospinal fluid but are infrequent in immune tissue or blood even in late disease. These mac-tropic R5 variants carry envelope glycoproteins (Envs) adapted to exploit low levels of CD4 on macrophages to induce infection. However, it is unclear whether this adaptation is conferred by an increased affinity of the Env trimer for CD4 or is mediated by postbinding structural rearrangements in the trimer that enhance the exposure of the coreceptor binding site and facilitate events leading to fusion and virus entry. In this study, we investigated CD4 binding to mac-tropic and non-mac-tropic Env trimers and showed that CD4-IgG binds efficiently to mac-tropic R5 Env trimers, while binding to non-mac-tropic trimers was undetectable. Our data indicated that the CD4 binding site (CD4bs) is highly occluded on Env trimers of non-mac-tropic R5 viruses. Such viruses may therefore infect T cells via viral synapses where Env and CD4 become highly concentrated. This environment will enable high-avidity interactions that overcome extremely low Env-CD4 affinities.IMPORTANCE HIV R5 variants bind to CD4 and CCR5 receptors on T cells and macrophages to initiate infection. Transmitted HIV variants infect T cells but not macrophages, and these viral strains persist in immune tissue even in late disease. Here we show that the binding site for CD4 present on HIV's envelope protein is occluded on viruses replicating in immune tissue. This occlusion likely prevents antibody binding to this site and neutralization of the virus, but it makes it difficult for virus-CD4 interactions to occur. Such viruses probably pass from T cell to T cell via cell contacts where CD4 is highly concentrated and allows infection via inefficient envelope-CD4 binding. Our data are highly relevant for vaccines that aim to induce antibodies targeting the CD4 binding site on the envelope protein.
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Antígenos CD4/metabolismo , VIH-1/fisiología , Macrófagos/metabolismo , Macrófagos/virología , Receptores CCR5/metabolismo , Tropismo Viral , Productos del Gen env del Virus de la Inmunodeficiencia Humana/metabolismo , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/metabolismo , Antígenos CD4/genética , Línea Celular , Epítopos de Linfocito T/inmunología , Citometría de Flujo , Expresión Génica , Anticuerpos Anti-VIH/inmunología , Anticuerpos Anti-VIH/metabolismo , Proteína gp120 de Envoltorio del VIH/genética , Proteína gp120 de Envoltorio del VIH/inmunología , Proteína gp120 de Envoltorio del VIH/metabolismo , Infecciones por VIH/inmunología , Infecciones por VIH/metabolismo , Infecciones por VIH/virología , Humanos , Inmunoglobulina G/inmunología , Inmunoglobulina G/metabolismo , Macrófagos/inmunología , Pruebas de Neutralización , Fragmentos de Péptidos/inmunología , Unión Proteica , Multimerización de Proteína , Productos del Gen env del Virus de la Inmunodeficiencia Humana/química , Productos del Gen env del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
Untreated HIV-positive (HIV-1+) individuals frequently suffer from HIV-associated neurocognitive disorders (HAND), with about 30% of AIDS patients suffering severe HIV-associated dementias (HADs). Antiretroviral therapy has greatly reduced the incidence of HAND and HAD. However, there is a continuing problem of milder neurocognitive impairments in treated HIV+ patients that may be increasing with long-term therapy. In the present study, we investigated whether envelope (env) genes could be amplified from proviral DNA or RNA derived from brain tissue of 12 individuals with normal neurology or minor neurological conditions (N/MC individuals). The tropism and characteristics of the brain-derived Envs were then investigated and compared to those of Envs derived from immune tissue. We showed that (i) macrophage-tropic R5 Envs could be detected in the brain tissue of 4/12 N/MC individuals, (ii) macrophage-tropic Envs in brain tissue formed compartmentalized clusters distinct from non-macrophage-tropic (non-mac-tropic) Envs recovered from the spleen or brain, (iii) the evidence was consistent with active viral expression by macrophage-tropic variants in the brain tissue of some individuals, and (iv) Envs from immune tissue of the N/MC individuals were nearly all tightly non-mac-tropic, contrasting with previous data for neuro-AIDS patients where immune tissue Envs mediated a range of macrophage infectivities, from background levels to modest infection, with a small number of Envs from some patients mediating high macrophage infection levels. In summary, the data presented here show that compartmentalized and active macrophage-tropic HIV-1 variants are present in the brain tissue of individuals before neurological disease becomes overt or serious.IMPORTANCE The detection of highly compartmentalized macrophage-tropic R5 Envs in the brain tissue of HIV patients without serious neurological disease is consistent with their emergence from a viral population already established there, perhaps from early disease. The detection of active macrophage-tropic virus expression, and probably replication, indicates that antiretroviral drugs with optimal penetration through the blood-brain barrier should be considered even for patients without neurological disease (neuro-disease). Finally, our data are consistent with the brain forming a sanctuary site for latent virus and low-level viral replication in the absence of neuro-disease.
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Síndrome de Inmunodeficiencia Adquirida/virología , Encéfalo/virología , VIH-1/aislamiento & purificación , VIH-1/fisiología , Macrófagos/virología , Tropismo Viral , Síndrome de Inmunodeficiencia Adquirida/complicaciones , Síndrome de Inmunodeficiencia Adquirida/tratamiento farmacológico , Barrera Hematoencefálica , Genes env , VIH-1/genética , Humanos , Virión/genética , Replicación ViralRESUMEN
Glucan particles (GPs) are hollow, porous 3-5 µm microspheres derived from the cell walls of Baker's yeast (Saccharomyces cerevisiae). The 1,3-ß-glucan outer shell provides for receptor-mediated uptake by phagocytic cells expressing ß-glucan receptors. GPs have been used for macrophage-targeted delivery of a wide range of payloads (DNA, siRNA, protein, small molecules, and nanoparticles) encapsulated inside the hollow GPs or bound to the surface of chemically derivatized GPs. Gallium nanoparticles have been proposed as an inhibitory agent against HIV infection. Here, macrophage targeting of gallium using GPs provides for more efficient delivery of gallium and inhibition of HIV infection in macrophages compared to free gallium nanoparticles.
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BACKGROUND: Non-mac-tropic HIV-1 R5 viruses are predominantly transmitted and persist in immune tissue even in AIDS patients who carry highly mac-tropic variants in the brain. Non-mac-tropic R5 envelopes (Envs) require high CD4 levels for infection contrasting with highly mac-tropic Envs, which interact more efficiently with CD4 and mediate infection of macrophages that express low CD4. Non-mac-tropic R5 Envs predominantly target T-cells during transmission and in immune tissue where they must outcompete mac-tropic variants. Here, we investigated whether Env+ pseudoviruses bearing transmitted/founder (T/F), early and late disease non-mac-tropic R5 envelopes mediated more efficient infection of CD4+ T-cells compared to those with highly mac-tropic Envs. RESULTS: Highly mac-tropic Envs mediated highest infectivity for primary T-cells, Jurkat/CCR5 cells, myeloid dendritic cells, macrophages, and HeLa TZM-bl cells, although this was most dramatic on macrophages. Infection of primary T-cells mediated by all Envs was low. However, infection of T-cells was greatly enhanced by increasing virus attachment with DEAE dextran and spinoculation, which enhanced the three Env+ virus groups to similar extents. Dendritic cell capture of viruses and trans-infection also greatly enhanced infection of primary T-cells. In trans-infection assays, non-mac-tropic R5 Envs were preferentially enhanced and those from late disease mediated levels of T-cell infection that were equivalent to those mediated by mac-tropic Envs. CONCLUSIONS: Our results demonstrate that T/F, early or late disease non-mac-tropic R5 Envs do not preferentially mediate infection of primary CD4+ T-cells compared to highly mac-tropic Envs from brain tissue. We conclude that non-macrophage-tropism of HIV-1 R5 Envs in vitro is determined predominantly by a reduced capacity to target myeloid cells via low CD4 rather than a specific adaptation for T-cells entry that precludes macrophage infection.
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Linfocitos T CD4-Positivos/virología , VIH-1/fisiología , Macrófagos/virología , Tropismo Viral , Internalización del Virus , Productos del Gen env del Virus de la Inmunodeficiencia Humana/metabolismo , Células Cultivadas , HumanosRESUMEN
HIV-1 R5 viruses vary extensively in their capacity to infect macrophages. R5 viruses that confer efficient infection of macrophages are able to exploit low levels of CD4 for infection and predominate in brain tissue, where macrophages are a major target for infection. HIV-1 R5 founder viruses that are transmitted were reported to be non-macrophage-tropic. Here, we investigated the sensitivities of macrophage-tropic and non-macrophage-tropic R5 envelopes to neutralizing antibodies. We observed striking differences in the sensitivities of Env(+) pseudovirions to soluble CD4 (sCD4) and to neutralizing monoclonal antibodies (MAbs) that target the CD4 binding site. Macrophage-tropic R5 Envs were sensitive to sCD4, while non-macrophage-tropic Envs were significantly more resistant. In contrast, all Envs were sensitive to VRC01 regardless of tropism, while MAb b12 conferred an intermediate neutralization pattern where all the macrophage-tropic and about half of the non-macrophage-tropic Envs were sensitive. CD4, b12, and VRC01 share binding specificities on the outer domain of gp120. However, these antibodies differ in their ability to induce conformational changes on the trimeric envelope and in specificity for residues on the V1V2 loop stem and ß20-21 junction that are targets for CD4 in recruiting the bridging sheet. These distinct specificities of CD4, b12, and VRC01 likely explain the observed differences in Env sensitivity to inhibition by these reagents and provide an insight into the envelope mechanisms that control macrophage tropism. We present a model where the efficiency of bridging-sheet recruitment by CD4 is a major determinant of HIV-1 R5 envelope sensitivity to soluble CD4 and macrophage tropism.
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Antígenos CD4/metabolismo , Proteína gp120 de Envoltorio del VIH/metabolismo , VIH-1/fisiología , Macrófagos/virología , Receptores del VIH/metabolismo , Tropismo Viral , Acoplamiento Viral , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Anti-VIH/inmunología , Humanos , Modelos Biológicos , Proteínas Recombinantes/metabolismoRESUMEN
BACKGROUND: Transmitted HIV-1 clade B or C R5 viruses have been reported to infect macrophages inefficiently, while other studies have described R5 viruses in late disease with either an enhanced macrophage-tropism or carrying envelopes with an increased positive charge and fitness. In contrast, our previous data suggested that viruses carrying non-macrophage-tropic R5 envelopes were still predominant in immune tissue of AIDS patients. To further investigate the tropism and charge of HIV-1 viruses in late disease, we evaluated the properties of HIV-1 envelopes amplified from immune and brain tissues of AIDS patients with neurological complications. RESULTS: Almost all envelopes amplified were R5. There was clear compartmentalization of envelope sequences for four of the five subjects. However, strong compartmentalization of macrophage-tropism in brain was observed even when brain and immune tissue envelope sequences were not segregated. R5 envelopes from immune tissue of four subjects carried a higher positive charge compared to brain envelopes. We also confirm a significant correlation between macrophage tropism and sensitivity to soluble CD4, a weak association with sensitivity to the CD4 binding site antibody, b12, but no clear relationship with maraviroc sensitivity. CONCLUSIONS: Our study shows that non-macrophage-tropic R5 envelopes carrying gp120s with an increased positive charge were predominant in immune tissue in late disease. However, highly macrophage-tropic variants with lower charged gp120s were nearly universal in the brain. These results are consistent with HIV-1 R5 envelopes evolving gp120s with an increased positive charge in immune tissue or sites outside the brain that likely reflect an adaptation for increased replication or fitness for CD4+ T-cells. Our data are consistent with the presence of powerful pressures in brain and in immune tissues selecting for R5 envelopes with very different properties; high macrophage-tropism, sCD4 sensitivity and low positive charge in brain and non-macrophage-tropism, sCD4 resistance and high positive charge in immune tissue.
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Síndrome de Inmunodeficiencia Adquirida/virología , Encéfalo/inmunología , Encéfalo/virología , Proteína gp120 de Envoltorio del VIH/química , VIH-1/fisiología , Macrófagos/virología , Tropismo Viral , Síndrome de Inmunodeficiencia Adquirida/inmunología , Adaptación Biológica , Proteína gp120 de Envoltorio del VIH/genética , VIH-1/genética , VIH-1/crecimiento & desarrollo , VIH-1/patogenicidad , Humanos , Datos de Secuencia Molecular , Selección Genética , Análisis de Secuencia de ADNRESUMEN
BACKGROUND: The conserved CD4 binding site (CD4bs) on HIV-1 gp120 is a major target for vaccines. It is a priority to determine sites and structures within the CD4bs that are important for inclusion in vaccines. We studied a gp120 pocket penetrated by W100 of the potent CD4bs monoclonal antibody (mab), b12. We compared HIV-1 envelopes and corresponding mutants that carried blocked W100 pockets to evaluate whether other CD4bs mabs target this site. FINDINGS: All CD4bs mabs tested blocked soluble CD4 binding to gp120 consistent with their designation as CD4bs directed antibodies. All CD4bs mabs tested neutralized pseudovirions carrying NL4.3 wild type (wt) envelope. However, only b12 failed to neutralize pseudoviruses carrying mutant envelopes with a blocked W100 pocket. In addition, for CD4bs mabs that neutralized pseudovirions carrying primary envelopes, mutation of the W100 pocket had little or no effect on neutralization sensitivity. CONCLUSIONS: Our data indicate that the b12 W100 pocket on gp120 is infrequently targeted by CD4bs mabs. This site is therefore not a priority for preservation in vaccines aiming to elicit antibodies targeting the CD4bs.