RESUMO
BACKGROUND: A very pure multi-walled carbon nanotube (MWCNT) that was shown to have very low toxicity in vitro, was evaluated for lung and systemic effects and distribution following inhalation exposure. METHODS: B6C3F1/N mice were exposed to varying doses (0, 0.06, 0.2, and 0.6 mg/m3) of the (99.1% carbon) MWCNT by inhalation for 30 days (excluding weekends). Ten days following the last exposure, the lungs and spleen were harvested and processed for histology and immune cell population assessment. In addition, lung lavage cells and fluid were analyzed. Stimulated Raman scattering (SRS) was used to identify particles in the lungs, spleen, kidneys, liver, mediastinal and brachial lymph nodes, and olfactory bulb. Splenic tissue sections were stained with hematoxylin and eosin (H&E) for light microscopic histopathology assessment. Blood plasma was analyzed for cytokines and cathepsins. A section of the spleen was processed for RNA isolation and relative gene expression for 84 inflammation-related cytokines/chemokines. RESULTS: Following MWCNT exposure, particles were clearly evident in the lungs, spleens, lymph nodes and olfactory bulbs, (but not livers or kidneys) of exposed mice in a dose-dependent manner. Examination of the lavaged lung cells was unremarkable with no significant inflammation indicated at all particle doses. In contrast, histological examination of the spleen indicated the presence of apoptotic bodies within T cells regions of the white pulp area. Isolated splenic leukocytes had significant changes in various cells including an increased number of proinflammatory CD11b+Ly6C+ splenic cells. The gene expression studies confirmed this observation as several inflammation-related genes were upregulated particularly in the high dose exposure (0.6 mg/m3). Blood plasma evaluations showed a systemic down-regulation of inflammatory cytokines and a dose-dependent up-regulation of lysosomal cathepsins. CONCLUSIONS: The findings in the lungs were consistent with our hypothesis that this MWCNT exposure would result in minimal lung inflammation and injury. However, the low toxicity of the MWCNT to lung macrophages may have contributed to enhanced migration of the MWCNT to the spleen through the lymph nodes, resulting in splenic toxicity and systemic changes in inflammatory mediators.
Assuntos
Exposição por Inalação , Nanotubos de Carbono , Material Particulado/toxicidade , Pneumonia , Animais , Líquido da Lavagem Broncoalveolar , Pulmão , Camundongos , Camundongos EndogâmicosRESUMO
Extreme wildfire events are becoming more common and while the immediate risks of particulate exposures to susceptible populations (i.e., elderly, asthmatics) are appreciated, the long-term health effects are not known. In 2017, the Seeley Lake (SL), MT area experienced unprecedented levels of wildfire smoke from July 31 to September 18, with a daily average of 220.9 µg/m3. The aim of this study was to conduct health assessments in the community and evaluate potential adverse health effects. The study resulted in the recruitment of a cohort (n = 95, average age: 63 years), for a rapid response screening activity following the wildland fire event, and two follow-up visits in 2018 and 2019. Analysis of spirometry data found a significant decrease in lung function (FEV1/FVC ratio: forced expiratory volume in first second/forced vital capacity) and a more than doubling of participants that fell below the lower limit of normal (10.2% in 2017 to 45.9% in 2018) one year following the wildfire event, and remained decreased two years (33.9%) post exposure. In addition, observed FEV1 was significantly lower than predicted values. These findings suggest that wildfire smoke can have long-lasting effects on human health. As wildfires continue to increase both here and globally, understanding the health implications is vital to understanding the respiratory impacts of these events as well as developing public health strategies to mitigate the effects.
RESUMO
Epidemiological studies have shown a correlation between chronic biomass smoke exposure and increased respiratory infection. Pulmonary macrophages are instrumental in both the innate and the adaptive immune responses to respiratory infection. In the present study, in vitro systems were utilized where alveolar macrophages (AM) and bone marrow-derived macrophages (BMdM) were exposed to concentrated wood smoke-derived particulate matter (WS-PM) and mice were exposed in vivo to either concentrated WS-PM or inhaled WS. In vivo studies demonstrated that WS-exposed mice inoculated with Streptococcus pneumoniae had a higher bacterial load 24 h post-exposure, and corresponding AM were found to have decreased lymphocyte activation activity. Additionally, while classic markers of inflammation (cellular infiltration, total protein, neutrophils) were not affected, there were changes in pulmonary macrophages populations, including significant decreases in macrophages expressing markers of activation in WS-exposed mice. The lymphocyte activation activity of WS-PM-exposed AM was significantly suppressed, but the phagocytic activity appeared unchanged. In an effort to determine a pathway for WS-induced suppression, RelB activation, assessed by nuclear translocation, was observed in AM exposed to either inhaled WS or instilled WS-PM. Finally, an analysis of WS-PM fractions determined the presence of 4-5 polycyclic aromatic hydrocarbons (PAHs), and preliminary work suggests a potential role for these PAHs to alter macrophage functions. These studies show a decreased ability of WS-exposed pulmonary macrophages to effectively mount a defense against infection, the effect lasts at least a week post-exposure, and appears to be mediated via RelB activation.
Assuntos
Macrófagos/efeitos dos fármacos , Fumaça/efeitos adversos , Madeira , Animais , Apresentação de Antígeno , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/citologia , Citocromo P-450 CYP1A1/metabolismo , Citocinas/metabolismo , Macrófagos/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Fagocitose/efeitos dos fármacos , Infecções Pneumocócicas/microbiologia , Hidrocarbonetos Policíclicos Aromáticos/análise , Hidrocarbonetos Policíclicos Aromáticos/farmacologia , Receptores de Hidrocarboneto Arílico/metabolismo , Fator de Transcrição RelB/metabolismoRESUMO
Allergic asthma is a chronic inflammatory disorder of the airway associated with bronchial obstruction, airway hyper-reactivity (AHR), and mucus production. The epithelium may direct and propagate asthmatic-like responses. Central to this theory is the observation that viruses, air pollution, and allergens promote epithelial damage and trigger the generation of IL-25, IL-33, and TSLP via innate pathways such as TLRs and purinergic receptors. Similarly, engineered nanomaterials promote a Th2-associated pathophysiology. In this study, we tested the hypothesis that instillation of multi-walled carbon nanotubes (MWCNT) impair pulmonary function in C57Bl/6 mice due to the development of IL-33-dependent Th2-associated inflammation. MWCNT exposure resulted in elevated levels of IL-33 in the lavage fluid (likely originating from airway epithelial cells), enhanced AHR, eosinophil recruitment, and production of Th2-associated cytokines and chemokines. Moreover, these events were dependent on IL-13 signaling and the IL-33/ST2 axis, but independent of T and B cells. Finally, MWCNT exposure resulted in the recruitment of innate lymphoid cells. Collectively, our data suggest that MWCNT induce epithelial damage that results in release of IL-33, which in turn promotes innate lymphoid cell recruitment and the development of IL-13-dependent inflammatory response.
Assuntos
Imunidade Inata/efeitos dos fármacos , Interleucinas/metabolismo , Pulmão/citologia , Pulmão/efeitos dos fármacos , Nanotubos de Carbono/toxicidade , Hipersensibilidade Respiratória/induzido quimicamente , Animais , Linhagem Celular , Células Epiteliais/efeitos dos fármacos , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Inflamação/metabolismo , Interleucina-13/genética , Interleucina-13/metabolismo , Interleucina-33 , Interleucinas/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nanotubos de Carbono/química , Alvéolos Pulmonares/citologia , Alvéolos Pulmonares/fisiologia , Linfócitos T Auxiliares-Indutores/fisiologiaRESUMO
The lung is constantly exposed to potentially pathogenic particles and microorganisms. It has become evident recently that not only innate but also adaptive immune responses to particulates, such as SiO(2) entering the respiratory tract, are complex and dynamic events. Although the cellular mechanisms and anatomical consequences involved in the development of silicosis have been studied extensively, they still remain poorly understood. Based on their capacity for immune regulation, lymphocytes may play a key role in the respiratory response to environmental challenge by SiO(2). The objective of this study was to characterize the impact of SiO(2) exposure on respiratory immune processes, with particular emphasis on evaluating the importance of lymphocytes in the murine silicosis model. Therefore, lymphopenic mice, including NK-deficient, Rag1(-/-), or a combination (Rag1(-/-) NK-depleted), were used and demonstrated that SiO(2)-induced fibrosis and inflammation can occur independently of T, B, NK T, and NK cells. Studies in Rag1(-/-) mice suggest further that lymphocytes may participate in the regulation of SiO(2)-induced inflammation through modulation of the Nalp3 inflammasome. This observation may have clinical relevance in the treatment of inflammatory and fibrotic lung diseases that are refractory or respond suboptimally to current therapeutics.
Assuntos
Imunidade Inata/imunologia , Silicose/imunologia , Silicose/patologia , Administração Intranasal , Animais , Linfócitos B/imunologia , Linfócitos B/patologia , Contagem de Células , Citocinas/metabolismo , Regulação da Expressão Gênica , Mediadores da Inflamação/metabolismo , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/patologia , Pulmão/imunologia , Pulmão/patologia , Ativação Linfocitária/imunologia , Depleção Linfocítica , Camundongos , Camundongos Endogâmicos C57BL , Análise de Sequência com Séries de Oligonucleotídeos , Tamanho do Órgão , Fibrose Pulmonar/complicações , Fibrose Pulmonar/imunologia , Fibrose Pulmonar/patologia , Dióxido de Silício/administração & dosagem , Silicose/complicações , Linfócitos T/imunologia , Linfócitos T/patologia , Fatores de TempoRESUMO
The International Biomass Smoke Health Effects (IBSHE) conference was convened in Missoula, MT, to define our current knowledge of smoke exposure and the potential health effects. In an effort to ascertain the relative health effects of an exposure to biomass smoke, numerous studies have utilized either animal or in vitro systems. A wide variety of systems that have been employed ranged from more mainstream animal models (i.e., rodents) and transformed cell lines to less common animal (piglets and dogs) and explant models. The Toxicology and Animal Study Design Workgroup at IBSHE was tasked with an analysis of the use of animal models in the assessment of the health effects of biomass smoke exposure. The present article contains a mini-review of models utilized historically, in addition to the adverse health effects assessed, and an overview of the discussion within the breakout session. The most common question that arose in discussions at the IBSHE conference was from local and federal health departments: What level of smoke is unhealthy? The present workgroup determined categories of exposure, common health concerns, and the availability of animal models to answer key health questions.
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Poluentes Atmosféricos/análise , Poluentes Atmosféricos/toxicidade , Biomassa , Fumaça/efeitos adversos , Fumaça/análise , Animais , Modelos Animais de Doenças , Saúde , Humanos , Projetos de PesquisaRESUMO
Urinary levoglucosan was investigated as a potential biomarker of wood smoke exposure in two different controlled experimental settings. Nine subjects were exposed to smoke from a campfire in a controlled setting, and four were exposed to smoke from an older-model wood stove. All subjects were asked to provide urine samples before and after exposure, and to wear personal particulate matter with a diameter of < or =2.5 microm (PM(2.5)) monitors during exposure. Urinary levoglucosan measurements from both studies showed no consistent response to the smoke exposure. A third experiment was conducted to assess the contribution of dietary factors to urinary levoglucosan levels. Nine subjects were asked to consume caramel and provide urine samples before and after consumption. Urinary levoglucosan levels increased within 2 h of caramel consumption and returned to pre-exposure levels within 24 h. These studies suggest that diet is a major factor in determining urinary levoglucosan levels and that recent dietary history needs to be taken into account for future work involving levoglucosan as a biomarker of wood smoke exposure.
Assuntos
Exposição Ambiental/análise , Glucose/análogos & derivados , Fumaça , Madeira , Adolescente , Adulto , Idoso , Biomarcadores/urina , Dieta , Feminino , Glucose/análise , Humanos , Masculino , Pessoa de Meia-Idade , Material Particulado/análise , Adulto JovemRESUMO
Various techniques have been utilized historically to generate acute pulmonary inflammation in the murine system. Crystalline silica exposure results in acute inflammation followed by pulmonary fibrosis. Methods of exposure are varied in their techniques, as well as types of anesthesia. Therefore, the current study sought to compare the effects of two major anesthesia (isoflurane and ketamine) and three routes of instillation, intranasal (IN), intratracheal (IT), and trans-oral (TO), on markers of inflammation. Mice were anesthetized with isoflurane or ketamine and instilled IN with silica or phosphate-buffered saline (PBS). Mice were sacrificed and lavaged after 3 days. To assess inflammation, alveolar cells were assessed by cytospin and lavage fluid was analyzed for inflammatory cytokines and total protein. While all parameters were increased in silica-exposed groups, regardless of anesthesia type, there were significant increases in neutrophils and total protein in mice anesthetized with ketamine, compared to isoflurane. In comparing instillation techniques, mice were anesthetized with isoflurane and instilled IN, IT, or TO with silica. Increases were observed in all parameters, except tumor necrosis factor-alpha, following IT silica instillation as compared to the IN and TO instillation groups. In addition, fluorescent microsphere uptake by alveolar macrophages supported the notion that all methods of instillation were uniform, but IT had significantly greater dispersion. Taken together, these data show that each method of exposure tested generated significant inflammation among the silica groups, and any differences in parameters or techniques should be taken into consideration when developing an animal model to study pulmonary diseases.
Assuntos
Anestesia por Inalação/métodos , Modelos Animais de Doenças , Doenças Pulmonares Intersticiais/patologia , Dióxido de Silício/toxicidade , Silicose/patologia , Doença Aguda , Anestésicos Inalatórios , Animais , Biomarcadores/metabolismo , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/citologia , Citocinas/metabolismo , Vias de Administração de Medicamentos , Intubação Intratraqueal , Isoflurano , Ketamina , Doenças Pulmonares Intersticiais/etiologia , Doenças Pulmonares Intersticiais/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Proteínas/metabolismo , Alvéolos Pulmonares/efeitos dos fármacos , Alvéolos Pulmonares/metabolismo , Alvéolos Pulmonares/patologia , Dióxido de Silício/administração & dosagem , Silicose/etiologia , Silicose/metabolismoRESUMO
BACKGROUND: Biomass smoke is an important source of particulate matter (PM), and much remains to be discovered with respect to the human health effects associated with this specific PM source. Exposure to biomass smoke can occur in one of two main categories: short-term exposures consist of periodic, seasonal exposures typified by communities near forest fires or intentional agricultural burning, and long-term exposures are chronic and typified by the use of biomass materials for cooking or heating. Levoglucosan (LG), a sugar anhydride released by combustion of cellulose-containing materials, is an attractive candidate as a biomarker of wood smoke exposure. OBJECTIVES: In the present study, Balb/c mice and children were assessed for LG in urine to determine its feasibility as a biomarker. METHODS: We performed urinary detection of LG by gas chromatography/mass spectrometry after intranasal instillations of LG or concentrated PM (mice) or biomass exposure (mice or humans). RESULTS: After instillation, we recovered most of the LG within the first 4 hr. Experiments using glucose instillation proved the specificity of our system, and instillation of concentrated PM from wood smoke, ambient air, and diesel exhaust supported a connection between wood smoke and LG. In addition, LG was detected in the urine of mice exposed to wood smoke. Finally, a pilot human study proved our ability to detect LG in urine of children. CONCLUSIONS: These results demonstrate that LG in the lungs is detectable in the urine of both mice and humans and that it is a good candidate as a biomarker of exposure to biomass smoke.
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Biomarcadores/urina , Exposição Ambiental , Glucanos/urina , Fumaça/efeitos adversos , Madeira , Animais , Criança , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Modelos TeóricosRESUMO
Chronic exposure to crystalline silica can lead to the development of silicosis, an irreversible, inflammatory and fibrotic pulmonary disease. Although, previous studies established the macrophage receptor with collagenous structure (MARCO) as an important receptor for binding and uptake of crystalline silica particles in vitro, the role of MARCO in regulating the inflammatory response following silica exposure in vivo remains unknown. Therefore, we determined the role of MARCO in crystalline silica-induced pulmonary pathology using C57Bl/6 wild-type (WT) and MARCO(-/-) mice. Increased numbers of MARCO(+) pulmonary macrophages were observed following crystalline silica, but not phosphate-buffered saline and titanium dioxide (TiO(2)), instillation in WT mice, highlighting a specific role of MARCO in silica-induced pathology. We hypothesized that MARCO(-/-) mice will exhibit diminished clearance of silica leading to enhanced pulmonary inflammation and exacerbation of silicosis. Alveolar macrophages isolated from crystalline silica-exposed mice showed diminished particle uptake in vivo as compared with WT mice, indicating abnormalities in clearance mechanisms. Furthermore, MARCO(-/-) mice exposed to crystalline silica showed enhanced acute inflammation and lung injury marked by increases in early response cytokines and inflammatory cells compared with WT mice. Similarly, histological examination of MARCO(-/-) lungs at 3 months post-crystalline silica exposure showed increased chronic inflammation compared with WT; however, only a small difference was observed with respect to development of fibrosis as measured by hydroxyproline content. Altogether, these results demonstrate that MARCO is important for clearance of crystalline silica in vivo and that the absence of MARCO results in exacerbations in innate pulmonary immune responses.
Assuntos
Macrófagos/efeitos dos fármacos , Dióxido de Silício/toxicidade , Silicose/patologia , Animais , Separação Celular , Cristalização , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Análise de Sequência com Séries de Oligonucleotídeos , Tamanho do Órgão/efeitos dos fármacos , Pneumonia/patologia , Fibrose Pulmonar/patologia , RNA/biossíntese , RNA/genética , Dióxido de Silício/farmacocinéticaRESUMO
Increasing evidence suggests that lung mechanics and structure are maintained in part by an intimate balance between the L-arginine-metabolizing enzymes nitric oxide synthase (NOS) and arginase. Asymmetric dimethylarginine (ADMA) is a competitive endogenous inhibitor of NOS. The role of ADMA in the regulation of NOS and arginase in the airways has not yet been explored. Our objective was to investigate the role of ADMA in lung physiology. A murine model of continuous subcutaneous ADMA infusion via osmotic minipump was used for assessment of elevated ADMA in vivo, and primary lung fibroblasts were used for in vitro assessments. Two weeks after minipump placement, animals were anesthetized and mechanically ventilated, and lung mechanical responses were evaluated. Lungs were assessed histologically and biochemically for collagen content, arginase activity, and arginase protein levels. Lung lavage fluid was assessed for cellularity, nitrite, urea, and cytokine concentrations. ADMA infusion resulted in significantly enhanced lung resistance and decreased dynamic compliance in response to methacholine. These physiologic changes were associated with significantly increased lung collagen content in the absence of inflammation. Significant decreases in lung fluid nitrite were accompanied by elevated lung fluid urea and arginase activity in lung homogenates. These changes were reversed in mice 4 weeks after completion of ADMA administration. In addition, treatment of primary mouse lung fibroblasts with ADMA stimulated arginase activity and collagen formation in vitro. These data support the idea that ADMA may play a role in airway diseases, including asthma and pulmonary fibrosis, through NOS inhibition and enhancement of arginase activity.
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Arginase/metabolismo , Arginina/análogos & derivados , Colágeno/metabolismo , Fibroblastos/enzimologia , Pulmão/enzimologia , Óxido Nítrico Sintase/antagonistas & inibidores , Animais , Arginina/toxicidade , Asma/induzido quimicamente , Asma/enzimologia , Asma/patologia , Lavagem Broncoalveolar , Células Cultivadas , Fibroblastos/patologia , Pulmão/patologia , Camundongos , Camundongos Endogâmicos BALB C , Óxido Nítrico Sintase/metabolismo , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/enzimologia , Fibrose Pulmonar/patologiaRESUMO
Crystalline silica exposure can result in pulmonary fibrosis, where the pulmonary macrophage is key as a result of its ability to react to silica particles. In the mouse silicosis model, there is initial Th1-type inflammation, characterized by TNF-alpha and IFN-gamma. Previous studies determined that Th2 mediators (i.e., IL-13) are vital to development of pulmonary fibrosis. The present study, using in vivo and in vitro techniques, compares silica exposures between Balb/c and Th2-deficient mice in an effort to determine the link between Th2 immunity and silicosis. In long-term experiments, a significant increase in fibrosis and activated interstitial macrophages was observed in Balb/c but not IL-4Ralpha(-/-) mice. Additionally, a significant increase in Ym1 mRNA levels, a promoter of Th2 immunity, was determined in the interstitial leukocyte population of silica-exposed Balb/c mice. To elucidate the effects of silica on macrophage function, bone marrow-derived macrophages (BMdM) were exposed to particles and assayed for T cell (TC) stimulation activity. As a control, Ym1 mRNA expression in Balb/c BMdM was determined using IL-4 stimulation. In the in vitro assay, a significant increase in TC activation, as defined by surface markers and cytokines, was observed in the cultures containing the silica-exposed macrophages in wild-type and IL-4Ralpha(-/-) mice, with one exception: IL-4Ralpha(-/-) BMdM were unable to induce an increase in IL-13. These results suggest that crystalline silica alters cellular functions of macrophages, including activation of TC, and that the increase in Th2 immunity associated with silicosis is via the IL-4Ralpha-Ym1 pathway.
Assuntos
Macrófagos/fisiologia , Fibrose Pulmonar/fisiopatologia , Receptores de Superfície Celular/fisiologia , Dióxido de Silício/toxicidade , Linfócitos T/fisiologia , Animais , Células Apresentadoras de Antígenos/fisiologia , Divisão Celular , Colágeno/análise , Citometria de Fluxo , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Reação em Cadeia da Polimerase , Fibrose Pulmonar/induzido quimicamente , RNA Mensageiro/genética , Receptores de Superfície Celular/deficiência , Receptores de Superfície Celular/genética , Linfócitos T/citologia , Linfócitos T/patologiaRESUMO
Silica inhalation results in chronic lung inflammation and fibrosis. While the role of the alveolar macrophage (AM) is considered key to the effects of silica on lung pathology, the etiology is not completely understood. Evidence suggests an increase in antigen presenting cell (APC) activity as a contributing factor to this process, as well as potential roles for both AM and interstitial macrophages (IM) in silicosis. In order to study the effects of crystalline silica on the APC activity of pulmonary macrophages, mice were exposed intranasally and changes in pulmonary macrophage populations were assessed using flow cytometry. Following intranasal instillation of silica, a significant increase in the APC activity of AM was observed, as well as a significant increase in a subset of IM expressing classic APC markers (MHC class II, CD11c). In addition, an in vitro system using bone marrow-derived macrophages (BMDM) was generated to assess the effects of silica on the APC activity of macrophages in vitro. Data using BMDM in the in vitro APC assay demonstrated a significant increase in APC activity following silica exposure, but not following exposure to saline or a control particle (TiO(2)). Using a combination of in vivo and in vitro experiments, the current study describes a significant increase in an interstitial macrophage subset with an APC phenotype, as well as an increase in the APC activity of both AM and BMDM, as a direct result of exposure to crystalline silica. These studies suggest a specific mechanism, macrophage subset activation, by which crystalline silica exposure results in chronic pulmonary inflammation and, eventually, fibrosis.
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Células Apresentadoras de Antígenos/efeitos dos fármacos , Exposição por Inalação/efeitos adversos , Macrófagos Alveolares/efeitos dos fármacos , Dióxido de Silício/toxicidade , Animais , Células Apresentadoras de Antígenos/imunologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/imunologia , Líquido Extracelular/citologia , Citometria de Fluxo , Técnicas In Vitro , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos Alveolares/citologia , Macrófagos Alveolares/imunologia , Camundongos , Camundongos Endogâmicos BALB C , FenótipoRESUMO
The polyprotein precursor of the Hepatitis C virus (HCV) contains multiple membrane-spanning domains that define the membrane topology and subsequent maturation of the viral structural proteins. In order to examine the biogenesis of the E1-E2 heterodimeric complex, we inserted an affinity tag (S-peptide) at specific locations within the envelope glycoproteins. In particular, and based on the prediction that the E1 glycoprotein may be able to assume a polytopic topology containing two membrane-spanning domains, we inserted the affinity tag within a putative cytoplasmic loop of the E1 glycoprotein. The HCV structural polyprotein containing this tag (at amino acids 295/296) was highly expressed and able to form a properly processed and noncovalently associated E1-E2 complex. This complex was bound by murine and conformation-dependent human monoclonal antibodies (MAbs) comparably to the native untagged complex. In addition, MAb recognition was retained upon reconstituting the tagged E1-E2 complex in lipid membrane as topologically constrained proteoliposomes. Our findings are consistent with the model of a topologically flexible E1 glycoprotein that is able to adopt a polytopic form. This form of the E1-E2 complex may be important in the HCV life cycle and in pathogenesis.