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Major epidemics, including some that qualify as pandemics, such as severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), HIV, influenza A (H1N1)pdm/09 and most recently COVID-19, affect the lung. Tuberculosis (TB) remains the top infectious disease killer, but apart from syndemic TB/HIV little is known regarding the interaction of viral epidemics and pandemics with TB. The aim of this consensus-based document is to describe the effects of viral infections resulting in epidemics and pandemics that affect the lung (MERS, SARS, HIV, influenza A (H1N1)pdm/09 and COVID-19) and their interactions with TB. A search of the scientific literature was performed. A writing committee of international experts including the European Centre for Disease Prevention and Control Public Health Emergency (ECDC PHE) team, the World Association for Infectious Diseases and Immunological Disorders (WAidid), the Global Tuberculosis Network (GTN), and members of the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) Study Group for Mycobacterial Infections (ESGMYC) was established. Consensus was achieved after multiple rounds of revisions between the writing committee and a larger expert group. A Delphi process involving the core group of authors (excluding the ECDC PHE team) identified the areas requiring review/consensus, followed by a second round to refine the definitive consensus elements. The epidemiology and immunology of these viral infections and their interactions with TB are discussed with implications for diagnosis, treatment and prevention of airborne infections (infection control, viral containment and workplace safety). This consensus document represents a rapid and comprehensive summary on what is known on the topic.
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Infecciones del Sistema Respiratorio/epidemiología , Tuberculosis/epidemiología , Virosis/epidemiología , Vacuna BCG/uso terapéutico , Betacoronavirus , COVID-19 , Infecciones por Coronavirus/diagnóstico , Infecciones por Coronavirus/tratamiento farmacológico , Infecciones por Coronavirus/epidemiología , Infecciones por Coronavirus/inmunología , Epidemias , Infecciones por VIH/diagnóstico , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/epidemiología , Infecciones por VIH/inmunología , Humanos , Subtipo H1N1 del Virus de la Influenza A , Gripe Humana/diagnóstico , Gripe Humana/tratamiento farmacológico , Gripe Humana/epidemiología , Gripe Humana/inmunología , Pulmón/inmunología , Coronavirus del Síndrome Respiratorio de Oriente Medio , Pandemias , Neumonía Viral/diagnóstico , Neumonía Viral/tratamiento farmacológico , Neumonía Viral/epidemiología , Neumonía Viral/inmunología , Salud Pública , Infecciones del Sistema Respiratorio/diagnóstico , Infecciones del Sistema Respiratorio/tratamiento farmacológico , Infecciones del Sistema Respiratorio/inmunología , SARS-CoV-2 , Síndrome Respiratorio Agudo Grave/diagnóstico , Síndrome Respiratorio Agudo Grave/tratamiento farmacológico , Síndrome Respiratorio Agudo Grave/epidemiología , Síndrome Respiratorio Agudo Grave/inmunología , Tuberculosis/diagnóstico , Tuberculosis/inmunología , Tuberculosis/prevención & control , Virosis/diagnóstico , Virosis/tratamiento farmacológico , Virosis/inmunologíaRESUMEN
Hepatitis C virus (HCV) is highly efficient in establishing a chronic infection, having evolved multiple strategies to suppress the host antiviral responses. The HCV nonstructural 5A (NS5A) protein, in addition to its role in viral replication and assembly, has long been known to hamper the interferon (IFN) response. However, the mechanism of this inhibitory activity of NS5A remains partly characterized. In a functional proteomic screening carried out in HCV replicon cells, we identified the mitochondrial protein LRPPRC as an NS5A binding factor. Notably, we found that downregulation of LRPPRC expression results in a significant inhibition of HCV infection, which is associated with an increased activation of the IFN response. Moreover, we showed that LRPPRC acts as a negative regulator of the mitochondrial-mediated antiviral immunity, by interacting with mitochondrial antiviral signaling protein (MAVS) and inhibiting its association with TRAF3 and TRAF6. Finally, we demonstrated that NS5A is able to interfere with MAVS activity in a LRPPRC-dependent manner. Conclusion: Overall, our results indicate that NS5A contributes to the inhibition of innate immune pathways during HCV infection by exploiting the ability of LRPPRC to inhibit MAVS-regulated antiviral signaling.
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Proteínas Adaptadoras Transductoras de Señales/fisiología , Hepatitis C Crónica/virología , Proteínas Mitocondriales/fisiología , Proteínas de Neoplasias/fisiología , Células Cultivadas , Hepacivirus/fisiología , Humanos , Transducción de Señal , Proteínas no Estructurales Virales/fisiologíaRESUMEN
Antibiotic treatment of tuberculosis takes ≥6â months, putting a major burden on patients and health systems in large parts of the world. Treatment beyond 2â months is needed to prevent tuberculosis relapse by clearing remaining, drug-tolerant Mycobacterium tuberculosis bacilli. However, the majority of patients treated for only 2-3â months will cure without relapse and do not need prolonged treatment. Assays that can identify these patients at an early stage of treatment may significantly help reduce the treatment burden, while a test to identify those patients who will fail treatment may help target host-directed therapies.In this review we summarise the state of the art with regard to discovery of biomarkers that predict relapse-free cure for pulmonary tuberculosis. Positron emission tomography/computed tomography scanning to measure pulmonary inflammation enhances our understanding of "cure". Several microbiological and immunological markers seem promising; however, they still need a formal validation. In parallel, new research strategies are needed to generate reliable tests.
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Biomarcadores/análisis , Pulmón/diagnóstico por imagen , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/aislamiento & purificación , Tuberculosis Pulmonar/diagnóstico por imagen , Antituberculosos/uso terapéutico , Humanos , Pulmón/microbiología , Tomografía Computarizada por Tomografía de Emisión de Positrones , Recurrencia , Tuberculosis Pulmonar/tratamiento farmacológicoRESUMEN
The complex spatial and paracrine relationships between the various liver histotypes are essential for proper functioning of the hepatic parenchymal cells. Only within a correct tissue organization, in fact, they stably maintain their identity and differentiated phenotype. The loss of histotype identity, which invariably occurs in the primary hepatocytes in culture, or in vivo in particular pathological conditions (fibrosis and tumours), is mainly because of the phenomenon of epithelial-to-mesenchymal transition (EMT). The EMT process, that occurs in the many epithelial cells, appears to be driven by a number of general, non-tissue-specific, master transcriptional regulators. The reverse process, the mesenchymal-to-epithelial transition (MET), as yet much less characterized at a molecular level, restores specific epithelial identities, and thus must include tissue-specific master elements. In this review, we will summarize the so far unveiled events of EMT/MET occurring in liver cells. In particular, we will focus on hepatocyte and describe the pivotal role in the control of EMT/MET dynamics exerted by a tissue-specific molecular mini-circuitry. Recent evidence, indeed, highlighted as two transcriptional factors, the master gene of EMT Snail, and the master gene of hepatocyte differentiation HNF4α, exhorting a direct reciprocal repression, act as pivotal elements in determining opposite cellular outcomes. The different balances between these two master regulators, further integrated by specific microRNAs, in fact, were found responsible for the EMT/METs dynamics as well as for the preservation of both hepatocyte and stem/precursor cells identity and differentiation. Overall, these findings impact the maintenance of stem cells and differentiated cells both in in vivo EMT/MET physio-pathological processes as well as in culture.
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Transición Epitelial-Mesenquimal/fisiología , Regulación de la Expresión Génica/fisiología , Factor Nuclear 4 del Hepatocito/metabolismo , Hepatocitos/citología , Modelos Biológicos , Fenotipo , Factores de Transcripción/metabolismo , Factor Nuclear 4 del Hepatocito/uso terapéutico , Hepatocitos/fisiología , Humanos , MicroARNs/metabolismo , Factores de Transcripción de la Familia SnailRESUMEN
Hepatitis C virus (HCV)-induced iron overload has been shown to promote liver fibrosis, steatosis, and hepatocellular carcinoma. The zonal-restricted histological distribution of pathological iron deposits has hampered the attempt to perform large-scale in vivo molecular investigations on the comorbidity between iron and HCV. Diagnostic and prognostic markers are not yet available to assess iron overload-induced liver fibrogenesis and progression in HCV infections. Here, by means of Spike-in SILAC proteomic approach, we first unveiled a specific membrane protein expression signature of HCV cell cultures in the presence of iron overload. Computational analysis of proteomic dataset highlighted the hepatocytic vitronectin expression as the most promising specific biomarker for iron-associated fibrogenesis in HCV infections. Next, the robustness of our in vitro findings was challenged in human liver biopsies by immunohistochemistry and yielded two major results: (i) hepatocytic vitronectin expression is associated to liver fibrogenesis in HCV-infected patients with iron overload; (ii) hepatic vitronectin expression was found to discriminate also the transition between mild to moderate fibrosis in HCV-infected patients without iron overload.
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Biomarcadores/metabolismo , Hepatitis C/metabolismo , Sobrecarga de Hierro/metabolismo , Cirrosis Hepática/metabolismo , Vitronectina/metabolismo , Biomarcadores/análisis , Línea Celular , Humanos , Marcaje Isotópico , Masculino , Proteínas de la Membrana/análisis , Proteínas de la Membrana/metabolismo , Proteómica , Regulación hacia Arriba , Vitronectina/análisisRESUMEN
BACKGROUND: Antiretroviral therapy has considerably reduced HIV disease progression, but complete eradication of HIV cannot actually be achieved. Moreover, prolonged use of protease inhibitors (PIs) causes profound changes in lipid metabolism with an increased risk of cardiovascular diseases. P-glycoprotein (P-gp) is expressed on many cell types, playing an important role in the efflux of drugs including PIs, limiting their intracellular concentration. Furthermore, several studies showed that P-gp is involved in lipid homeostasis and its activity is regulated by cholesterol. METHODS: THP-1 monocytes were used to study: (i) the influence of low-density lipoprotein (LDL) on P-gp expression and function, assessed by flow cytometry and quantitative real-time PCR analysis and measuring ritonavir and rhodamine-123 dye efflux, respectively; and (ii) the influence of ritonavir on cholesterol mobilization. The intracellular levels of ritonavir or cholesterol were measured by HPLC-UV and filipin staining, respectively. RESULTS: In THP-1 cells, LDL was able to yield an increase in both P-gp expression and activity. THP-1 cells treated with LDL showed a decrease in the intracellular ritonavir concentration in a dose-dependent manner. Notably, ritonavir induced reduced cholesterol mobilization in THP-1 cells, probably due to its inhibitory action on P-gp activity. CONCLUSIONS: Our data indicate a potential interplay between LDL and ritonavir mediated by P-gp. This interaction could influence both therapy effectiveness and cellular lipid metabolism, with important implications in the management of HIV patients treated with boosted PIs.
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Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Fármacos Anti-VIH/metabolismo , Fármacos Anti-VIH/farmacocinética , Lipoproteínas LDL/metabolismo , Ritonavir/metabolismo , Ritonavir/farmacocinética , Línea Celular , Cromatografía Líquida de Alta Presión , Citosol/química , Citometría de Flujo , Perfilación de la Expresión Génica , Humanos , Monocitos , Unión Proteica , Reacción en Cadena en Tiempo Real de la Polimerasa , Espectrofotometría UltravioletaRESUMEN
Infectious diseases are a major threat to global health and cause millions of deaths every year, particularly in developing countries. The emergence of multidrug resistance challenges current antimicrobial treatments, inducing uncertainty in therapeutic protocols. New compounds are therefore necessary. A drug repurposing approach could play a critical role in developing new treatments used either alone or in combination with standard therapy regimens. Herein, we focused on cysteamine, an aminothiol endogenously synthesized by human cells during the degradation of coenzyme-A, which is a drug approved for the treatment of nephropathic cystinosis. Cysteamine influences many biological processes due to the presence of the highly reactive thiol group. This review provides an overview of cysteamine-mediated effects on different viruses, bacteria and parasites, with a particular focus on infections caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), Mycobacterium tuberculosis, non-tuberculous mycobacteria (NTM), and Pseudomonas aeruginosa. Evidences for a potential use of cysteamine as a direct antimicrobial agent and/or a host-directed therapy, either alone or in combination with other antimicrobial drugs, are described.
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OBJECTIVES: To investigate whether Mycobacterium tuberculosis (Mtb) DNA is detected in peripheral blood mononuclear cells (PBMC) of subjects with tuberculosis (TB) or TB infection (TBI) living in a low-burden country. METHODS: We prospectively enrolled 57 patients with TB, 41 subjects with TBI, and 39 controls in Rome, Italy. PBMC were isolated, cluster of differentiation (CD)34+ and CD34- cells were immunomagnetic separated, DNA was extracted, and digital polymerase chain reaction for IS6110 and rpoB sequences was used to detect Mtb DNA in PBMC subsets and unfractionated PBMC. RESULTS: We detected Mtb DNA at a low copy number in CD34+ cells in 4o f 30 (13%) patients with TB, 2 of 24 (8%) subjects with TBI, and 1 of 24 (4%) controls. Mtb DNA was detected in unfractionated PBMC in 3 of 51 (6%) patients with TB, 2 of 38 (5%) subjects with TBI, and 2 of 36 (6%) controls. In CD34- cells, only 1 of 31 (3%) subjects with TBI tested positive for Mtb DNA. CONCLUSIONS: Mtb DNA was detected at low frequencies and levels in the PBMC of subjects with TBI and donors with TB living in a low-burden country. In particular, Mtb DNA was detected more frequently in CD34+ cells, supporting the hypothesis that these cells may represent a Mtb niche. This finding informs biological understanding of Mtb pathogenesis and may support the development of a microbial blood biomarker for Mtb infection.
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Tuberculosis Latente , Mycobacterium tuberculosis , Tuberculosis , Adulto , Humanos , Leucocitos Mononucleares , Mycobacterium tuberculosis/genética , ADN BacterianoRESUMEN
BACKGROUND & AIMS: The tumor fate derives from cell autonomous properties and niche microenvironmental cues. The transforming growth factor ß (TGFß) is a major microenvironmental factor for hepatocellular carcinoma (HCC) influencing tumor dedifferentiation, induction of epithelial-to-mesenchymal transition (EMT) and acquisition of metastatic properties. The loss of the transcriptional factor HNF4α is a predominant mechanism through which HCCs progress to a more aggressive phenotype; its re-expression, reducing tumor formation and repressing EMT program, has been suggested as a therapeutic tool for HCC gene therapy. We investigated the influence of TGFß on the anti-EMT and tumor suppressor HNF4α activity. METHODS: Cell motility and invasion were analyzed by wound healing and invasion assays. EMT was evaluated by RT-qPCR and immunofluorescence. ChIP and EMSA assays were utilized for investigation of the HNF4α DNA binding activity. HNF4α post-translational modifications (PTMs) were assessed by 2-DE analysis. GSK3ß activity was modulated by chemical inhibition and constitutive active mutant expression. RESULTS: We demonstrated that the presence of TGFß impairs the efficiency of HNF4α as tumor suppressor. We found that TGFß induces HNF4α PTMs that correlate with the early loss of HNF4α DNA binding activity on target gene promoters. Furthermore, we identified the GSK3ß kinase as one of the TGFß targets mediating HNF4α functional inactivation: GSK3ß chemical inhibition results in HNF4α DNA binding impairment while a constitutively active GSK3ß mutant impairs the TGFß-induced inhibitory effect on HNF4α tumor suppressor activity. CONCLUSIONS: Our data identify in the dominance of TGFß a limit for the HNF4α-mediated gene therapy of HCC.
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Carcinoma Hepatocelular , Terapia Genética , Glucógeno Sintasa Quinasa 3/metabolismo , Factor Nuclear 4 del Hepatocito/metabolismo , Neoplasias Hepáticas , Factor de Crecimiento Transformador beta/metabolismo , Animales , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/secundario , Carcinoma Hepatocelular/terapia , Línea Celular Transformada , Línea Celular Tumoral , Movimiento Celular/fisiología , Transición Epitelial-Mesenquimal/genética , Transición Epitelial-Mesenquimal/fisiología , Regulación Neoplásica de la Expresión Génica/fisiología , Genes Supresores de Tumor/fisiología , Glucógeno Sintasa Quinasa 3 beta , Células Hep G2 , Factor Nuclear 4 del Hepatocito/genética , Hepatocitos/citología , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/terapia , Ratones , Factor de Crecimiento Transformador beta/genética , Microambiente Tumoral/genética , Microambiente Tumoral/fisiologíaRESUMEN
BACKGROUND & AIMS: Autophagy is a lysosome-mediated catabolic process that mediates degradation and recycling of all major components of eukaryotic cells. Different stresses, including viral and bacterial infection, induce autophagy, which can promote cell survival by removing the stress inducer or by attenuating its dangerous effects. High levels of autophagy occur during infection of cells with hepatitis C virus (HCV), but the clinical relevance of this process is not clear. METHODS: Levels of autophagy were analyzed in liver biopsy samples from 22 patients with HCV infection using microtubule-associated protein-1 light chain 3 immunoblotting; associations with histological and metabolic parameters were evaluated by Pearson correlation analysis. We investigated the role of HCV-induced autophagy in lipid degradation in cells infected with the virus or replicons, and analyzed autophagosome contents by confocal microscopy and by measuring lipid levels after inhibition of autophagy by Beclin 1 knockdown or lysosome inhibitors. RESULTS: In liver biopsy samples from patients with HCV, there was an inverse correlation between microvesicular steatosis and level of autophagy (r = -0.617; P = .002). HCV selectively induced autophagy of lipids in virus-infected and replicon cells. In each system, autophagosomes frequently colocalized with lipid deposits, mainly formed by unesterified cholesterol. Inhibition of the autophagic process in these cells significantly increased the induction of cholesterol accumulation by HCV. CONCLUSIONS: Autophagy counteracts the alterations in lipid metabolism induced by HCV. Disruption of the autophagic process might contribute to development of steatosis in patients with HCV.
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Autofagia , Colesterol/metabolismo , Hígado Graso/prevención & control , Hepacivirus/patogenicidad , Hepatitis C/complicaciones , Hígado/virología , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Beclina-1 , Biopsia , Western Blotting , Línea Celular Tumoral , Colesterol/genética , Citoprotección , Hígado Graso/metabolismo , Hígado Graso/patología , Hígado Graso/virología , Hepatitis C/diagnóstico , Hepatitis C/metabolismo , Hepatitis C/patología , Humanos , Italia , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Lisosomas/virología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Microscopía Confocal , Proteínas Asociadas a Microtúbulos/metabolismo , Enfermedad del Hígado Graso no Alcohólico , Replicón , Estudios RetrospectivosRESUMEN
INTRODUCTION: Despite huge efforts, tuberculosis (TB) is still a major public health threat worldwide, it is estimated that a quarter of the global population is infected by Mycobacterium tuberculosis (Mtb). For controlling TB and reducing Mtb transmission it is fundamental to diagnose TB infection (TBI) as well as the progressors from TBI to disease to identify those requiring preventive therapy. At present, there is no gold standard test for TBI diagnosis although several new methodologies have been attempted. AREAS COVERED: This review provides an update on the most recent approaches to develop reliable tests to diagnose TBI and progressors from infection to disease. Experimental tests are based on either the direct identification of Mtb (i.e., Mtb DNA upon host cells isolation; Mtb proteins or peptides) or host response (i.e., levels and quality of specific anti-Mtb antibodies; host blood transcriptome signatures). EXPERT OPINION: The experimental tests described are very interesting. However, further investigation and randomized clinical trials are needed to improve the sensitivity and specificity of these new research-based tests. More reliable proofs-of-concept and simplification of technical procedures are necessary to develop new diagnostic tools for identifying TBI patients and those that will progress from infection to TB disease.
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Mycobacterium tuberculosis , Tuberculosis , Humanos , Tuberculosis/diagnóstico , Tuberculosis/microbiología , Mycobacterium tuberculosis/genética , Sensibilidad y EspecificidadRESUMEN
Knowledge of aging biology needs to be expanded due to the continuously growing number of elderly people worldwide. Aging induces changes that affect all systems of the body. The risk of cardiovascular disease and cancer increases with age. In particular, the age-induced adaptation of the immune system causes a greater susceptibility to infections and contributes to the inability to control pathogen growth and immune-mediated tissue damage. Since the impact of aging on immune function, is still to be fully elucidated, this review addresses some of the recent understanding of age-related changes affecting key components of immunity. The emphasis is on immunosenescence and inflammaging that are impacted by common infectious diseases that are characterized by a high mortality, and includes COVID-19, HIV and tuberculosis.
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COVID-19 , Infecciones por VIH , Tuberculosis , Humanos , Anciano , Inflamación , EnvejecimientoRESUMEN
Background: Despite the significant progress achieved in understanding the pathology and clinical management of SARS-CoV-2 infection, still pathogenic and clinical issues need to be clarified. Treatment with modulators of epigenetic targets, i.e., epidrugs, is a current therapeutic option in several cancers and could represent an approach in the therapy of viral diseases. Results: Aim of this study was the analysis of the role of histone deacetylase (HDAC) inhibition in the modulation of SARS-CoV-2 infection of mesothelial cells (MCs).MeT5A cells, a pleura MC line, were pre-treated with different specific class I and IIb HDAC inhibitors. Unexpectedly, treatment with HDAC1-3 inhibitors significantly increased ACE2/TMPRSS2 expression, suggesting a role in favoring SARS-CoV-2 infection. We focused our analysis on the most potent ACE2/TMPRSS2 inducer among the inhibitors analysed, MS-275, a HDAC1-3 inhibitor. ACE2/TMPRSS2 expression was validated by Western Blot (WB) and immunofluorescence. The involvement of HDAC inhibition in receptor induction was confirmed by HDAC1/HDAC2 silencing. In accordance to the ACE2/TMPRSS2 expression data, MS-275 increased SARS-CoV-2 replication and virus propagation in Vero E6 cells.Notably, MS-275 was able to increase ACE2/TMPRSS2 expression and SARS-CoV-2 production, although to a lesser extent, also in the lung adenocarcinoma cell line Calu-3 cells.Mechanistically, treatment with MS-275 increased H3 and H4 histone acetylation at ACE2/TMPRSS2 promoters, increasing their transcription. Conclusion: This study highlights a previously unrecognized effect of HDAC1-3 inhibition in increasing SARS-CoV-2 cell entry, replication and productive infection correlating with increased expression of ACE2 and TMPRSS2. These data, while adding basic insight into COVID-19 pathogenesis, warn for the use of HDAC inhibitors in SARS-CoV-2 patients.
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COVID-19 , SARS-CoV-2 , Humanos , COVID-19/metabolismo , Enzima Convertidora de Angiotensina 2/metabolismo , Inhibidores de Histona Desacetilasas/farmacología , Inhibidores de Histona Desacetilasas/metabolismo , Pulmón/metabolismo , Células Epiteliales , Histona Desacetilasa 1/metabolismoRESUMEN
Hepatic fat export occurs by apolipoprotein B-100-containing lipoprotein production, whereas impaired production leads to liver steatosis. Hepatitis C virus (HCV) infection is associated to dysregulation of apoB-100 secretion and steatosis; however, the molecular mechanism by which HCV affects the apoB-100 secretion is not understood. Here, combining quantitative proteomics and computational biology, we propose ferritin heavy chain (Fth) as being the cellular determinant of apoB-100 production inhibition. By means of molecular analyses, we found that HCV nonstructural proteins and NS5A appear to be sufficient for inducing Fth up-regulation. Fth in turn was found to inhibit apoB-100 secretion leading to increased intracellular degradation via proteasome. Notably, intracellular Fth down-regulation by siRNA restores apoB-100 secretion. The inverse correlation between ferritin and plasma apoB-100 concentrations was also found in JFH-1 HCV cell culture systems (HCVcc) and HCV-infected patients. Finally, Fth expression was found to be required for robust HCV infection. These observations provide a further molecular explanation for the onset of liver steatosis and allow for hypothesizing on new therapeutic and antiviral strategies.
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Apoferritinas/metabolismo , Apolipoproteína B-100/antagonistas & inhibidores , Regulación Viral de la Expresión Génica , Hepacivirus/patogenicidad , Apolipoproteína B-100/sangre , Línea Celular Tumoral , Biología Computacional , Hígado Graso/metabolismo , Hígado Graso/patología , Hígado Graso/virología , Hepacivirus/genética , Hepacivirus/metabolismo , Hepatitis C/patología , Hepatitis C/virología , Hepatocitos/metabolismo , Hepatocitos/patología , Hepatocitos/virología , Interacciones Huésped-Patógeno , Humanos , Marcaje Isotópico , Complejo de la Endopetidasa Proteasomal/metabolismo , Mapas de Interacción de Proteínas , Proteolisis , Proteómica/métodos , Transfección , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismoRESUMEN
UNLABELLED: The concept that cellular terminal differentiation is stably maintained once development is complete has been questioned by numerous observations showing that differentiated epithelium may undergo an epithelial-to-mesenchymal transition (EMT) program. EMT and the reverse process, mesenchymal-to-epithelial transition (MET), are typical events of development, tissue repair, and tumor progression. In this study, we aimed to clarify the molecular mechanisms underlying these phenotypic conversions in hepatocytes. Hepatocyte nuclear factor 4α (HNF4α) was overexpressed in different hepatocyte cell lines and the resulting gene expression profile was determined by real-time quantitative polymerase chain reaction. HNF4α recruitment on promoters of both mesenchymal and EMT regulator genes was determined by way of electrophoretic mobility shift assay and chromatin immunoprecipitation. The effect of HNF4α depletion was assessed in silenced cells and in the context of the whole liver of HNF4 knockout animals. Our results identified key EMT regulators and mesenchymal genes as new targets of HNF4α. HNF4α, in cooperation with its target HNF1α, directly inhibits transcription of the EMT master regulatory genes Snail, Slug, and HMGA2 and of several mesenchymal markers. HNF4α-mediated repression of EMT genes induces MET in hepatomas, and its silencing triggers the mesenchymal program in differentiated hepatocytes both in cell culture and in the whole liver. CONCLUSION: The pivotal role of HNF4α in the induction and maintenance of hepatocyte differentiation should also be ascribed to its capacity to continuously repress the mesenchymal program; thus, both HNF4α activator and repressor functions are necessary for the identity of hepatocytes.
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Diferenciación Celular/fisiología , Células Epiteliales/patología , Factor Nuclear 4 del Hepatocito/fisiología , Hepatocitos/patología , Mesodermo/patología , Animales , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Factor Nuclear 1-alfa del Hepatocito/fisiología , Factor Nuclear 4 del Hepatocito/genética , Humanos , Neoplasias Hepáticas/patología , Ratones , Ratones Noqueados , Modelos Animales , Fenotipo , Factores de Transcripción de la Familia Snail , Factores de Transcripción/fisiologíaRESUMEN
BACKGROUND/AIMS: The life cycle of hepatitis C virus (HCV) is intimately linked to the lipid metabolism of the host. In particular, HCV exploits the metabolic machinery of the lipoproteins in several steps of its life cycle such as circulation in the bloodstream, cell attachment and entry, assembly and release of viral particles. However, the details of how HCV interacts with and influences the metabolism of the host lipoproteins are not well understood. A study was undertaken to investigate whether HCV directly affects the protein composition of host circulating lipoproteins. METHODS: A proteomic analysis of circulating very low-, low- and high-density lipoproteins (VLDL, LDL and HDL), isolated from either in-treatment naïve HCV-infected patients or healthy donors (HD), was performed using two-dimensional gel electrophoresis and tandem mass spectrometry (MALDI-TOF/TOF). The results obtained were further investigated using in vitro models of HCV infection and replication. RESULTS: A decreased level of apolipoprotein A-I (apoA-I) was found in the LDL fractions of HCV-infected patients. This result was confirmed by western blot and ELISA analysis. HCV cellular models (JFH1 HCV cell culture system (HCVcc) and HCV subgenomic replicons) showed that the decreased apoA-I/LDL association originates from hepatic biogenesis rather than lipoprotein catabolism occurring in the circulation, and is not due to a downregulation of the apoA-I protein concentration. The sole non-structural viral proteins were sufficient to impair the apoA-I/LDL association. Functional evidence was obtained for involvement of apoA-I in the viral life cycle such as RNA replication and virion production. The specific siRNA-mediated downregulation of apoA-I led to a reduction in both HCV RNA and viral particle levels in culture. CONCLUSIONS: This study shows that HCV induces lipoprotein structural modification and that its replication and production are linked to the host lipoprotein metabolism, suggesting apoA-I as a new possible target for antiviral therapy.
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Apolipoproteína A-I/sangre , Hepacivirus/fisiología , Hepatitis C/sangre , Lipoproteínas LDL/sangre , Adulto , Estudios de Casos y Controles , Células Cultivadas , Regulación hacia Abajo/fisiología , Electroforesis en Gel Bidimensional/métodos , Femenino , Hepatitis C/virología , Humanos , Masculino , Persona de Mediana Edad , Proteómica , Virión/fisiología , Replicación Viral/fisiologíaRESUMEN
The novel SARS-CoV-2 variants of concern (VOC) represent a considerable global alarm because their mutations are known to affect transmissibility and cause immune escape. While preventing severe disease and deaths, the available vaccines do not avoid infection; therefore, COVID-19 disease management still requires effective therapies. We have recently reported that the aminothiol cysteamine, a drug already applied to humans, exerts direct antiviral activity against SARS-CoV-2 and has in vitro immunomodulatory effect. To evaluate whether this compound exerts antiviral effects also against SARS-CoV-2 variants, we performed different infected cell-based assays using Wild type, Delta, or Omicron VOC. We found that cysteamine significantly reduces the cytopathic effect induced by SARS-CoV-2 Wild type strain and Delta variant in Vero E6 cells. On the other hand, cysteamine had no effects on the survival of cells infected with the Omicron variant, due to the lack of cytotoxicity on Vero E6 cells, at least when infected at MOI = 0.001 for 72 h. Moreover, cysteamine significantly reduced the production of Wild type, Delta, and Omicron variants as measured by the virus released in the culture media (Vero E6 and Calu-3 cells) and by transmission electron microscopy analysis (Vero E6 cells). Notably, cysteamine is more effective in inhibiting the Omicron rather than Delta or Wild type viruses, with an 80% inhibition of Omicron production compared to 40% of Wild type and Delta variant. Overall, our findings demonstrate that cysteamine exerts direct antiviral actions against SARS-CoV-2 Delta and Omicron variants, in addition to the Wild type virus. Our data further demonstrate that cysteamine is a good candidate as repurposing drug for the treatment of SARS-CoV-2 infection for the present and, likely, the future VOC and, therefore, it would be important to investigate its clinical relevance in randomized clinical trials.
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Rationale: Circulating pathogen-derived proteins can serve as useful biomarkers for infections but may be detected with poor sensitivity and specificity by standard immunoassays due to masking effects and cross-reactivity. Mass spectrometry (MS)-read immunoassays for biomarker-derived peptides can resolve these issues, but lack standard workflows to select species-specific peptides with strong MS signal that are suitable for antibody generation. Methods:Using a Mycobacterium tuberculosis (Mtb) protein as an example, candidate peptides were selected by length, species-specificity, MS intensity, and antigenicity score. MS data from spiked healthy serum was employed to define MS feature thresholds, including a novel measure of internal MS data correlation, to produce a peak detection algorithm. Results: This algorithm performed better in rejecting false positive signal than each of its criteria, including those currently employed for this purpose. Analysis of an Mtb peptide biomarker (CFP-10pep) by this approach identified tuberculosis cases not detected by microbiologic assays, including extrapulmonary tuberculosis and tuberculosis cases in children infected with HIV-1. Circulating CFP-10pep levels measured in a non-human primate model of tuberculosis distinguished disease from asymptomatic infection and tended to correspond with Mtb granuloma size, suggesting that it could also serve as a surrogate marker for Mtb burden and possibly treatment response. Conclusions: These biomarker selection and analysis approach appears to have strong potential utility for infectious disease diagnosis, including cryptic infections, and possibly to monitor changes in Mtb burden that may reflect disease progression or a response to treatment, which are critical needs for more effective disease control.
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Mycobacterium tuberculosis , Tuberculosis , Animales , Biomarcadores , Péptidos , Sensibilidad y Especificidad , Tuberculosis/diagnóstico , Tuberculosis/microbiologíaRESUMEN
The effects of indole-3-carbinol (I3C) compound have been described deeply as antitumor drug in multiple cancers. Herein, I3C compound was tested for toxicity and antiviral activity against SARS-CoV-2 infection. Antiviral activity was assessed in vitro in both in VeroE6 cell line and human Lung Organoids (hLORGs) where I3C exhibited a direct anti-SARS-CoV-2 replication activity with an antiviral effect and a modulation of the expression of genes implicated in innate immunity and inflammatory response was observed at 16.67 µM. Importantly, we further show the I3C is also effective against the SARS-CoV-2 Omicron variant. In mouse model, instead, we assessed possible toxicity effects of I3C through two different routes of administration: intragastrically (i.g.) and intraperitoneally (i.p.). The LD50 (lethal dose 50%) values in mice were estimated to be: 1410 and 1759 mg/kg i.g.; while estimated values for i.p. administration were: 444.5 mg/kg and 375 mg/kg in male and female mice, respectively. Below these values, I3C (in particular at 550 mg/kg for i.g. and 250 mg/kg for i.p.) induces neither death, nor abnormal toxic symptoms as well as no histopathological lesions of the tissues analysed. These tolerated doses are much higher than those already proven effective in pre-clinical cancer models and in vitro experiments. In conclusion, I3C exhibits a significant antiviral activity, and no toxicity effects were recorded for this compound at the indicated doses, characterizing it as a safe and potential antiviral compound. The results presented in this study could provide experimental pre-clinical data necessary for the start of human clinical trials with I3C for the treatment of SARS-CoV-2 and beyond.
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Objective: Several therapies with immune-modulatory functions have been proposed to reduce the overwhelmed inflammation associated with COVID-19. Here we investigated the impact of IL-10 in COVID-19, through the ex-vivo assessment of the effects of exogenous IL-10 on SARS-CoV-2-specific-response using a whole-blood platform. Methods: Two cohorts were evaluated: in "study population A", plasma levels of 27 immune factors were measured by a multiplex (Luminex) assay in 39 hospitalized "COVID-19 patients" and 29 "NO COVID-19 controls" all unvaccinated. In "study population B", 29 COVID-19 patients and 30 NO COVID-19-Vaccinated Controls (NO COVID-19-VCs) were prospectively enrolled for the IL-10 study. Whole-blood was stimulated overnight with SARS-COV-2 antigens and then treated with IL-10. Plasma was collected and used for ELISA and multiplex assay. In parallel, whole-blood was stimulated and used for flow cytometry analysis. Results: Baseline levels of several immune factors, including IL-10, were significantly elevated in COVID-19 patients compared with NO COVID-19 subjects in "study population A". Among them, IL-2, FGF, IFN-γ, and MCP-1 reached their highest levels within the second week of infection and then decreased. To note that, MCP-1 levels remained significantly elevated compared with controls. IL-10, GM-CSF, and IL-6 increased later and showed an increasing trend over time. Moreover, exogenous addition of IL-10 significantly downregulated IFN-γ response and several other immune factors in both COVID-19 patients and NO COVID-19-VCs evaluated by ELISA and a multiplex analysis (Luminex) in "study population B". Importantly, IL-10 did not affect cell survival, but decreased the frequencies of T-cells producing IFN-γ, TNF-α, and IL-2 (p<0.05) and down-modulated HLA-DR expression on CD8+ and NK cells. Conclusion: This study provides important insights into immune modulating effects of IL-10 in COVID-19 and may provide valuable information regarding the further in vivo investigations.