Adsorption of Polyethyleneimine on Silver Nanoparticles and Its Interaction with a Plasmid DNA: A Surface-Enhanced Raman Scattering Study

Raman spectroscopy is applied in this work to study the adsorption of poly(ethyleneimine) (PEI) on Ag nanoparticles obtained by reduction with citrate, as well as to the study of the interaction between PEI and a plasmid. The surface-enhanced Raman spectroscopy (SERS) affords important information about the interaction and orientation of the polymer on the particles. In particular we have found that this polymer interacts with the surface through their amino groups in an interaction which also involves a change in the protonation state of amino groups as well as an increase of the chain order. This interaction implies a charge-transfer effect as deduced from the strong resonant effect in Raman spectra obtained at different excitation wavelengths. The complex formed by PEI and a plasmid, obtained by encoding the HBV (hepatitis B virus) genome inside the EcoRI restriction site of pGEM vector, was also studied by SERS. The interaction between both polymers leads to a conformational change affecting both macromolecules that can be detected by Raman at different excitation wavelengths. PEI undergoes a change to a more disordered structure as well as an increase of the number of protonated amino groups. The plasmid undergoes a structural change from A-DNA structure to B-DNA, along with a change in the superhelicity resulting in a more lineal structure when the plasmid interacts with PEI.Peer reviewe

According to Hepatitis C Virus (HCV) Infection Stage, Interleukin-7 Plus 4-1BB Triggering Alone or Combined with PD-1 Blockade Increases TRAF1lowHCV-Specific CD8+Cell Reactivity.

Hepatitis C virus (HCV)-specific CD8+T cells suffer a progressive exhaustion during persistent infection (PI) with HCV. This process could involve the positive immune checkpoint 4-1BB/4-1BBL through the loss of its signal transducer, TRAF1. To address this issue, peripheral HCV-specific CD8+T cells (pentamer-positive [pentamer+]/CD8+T cells) from patients with PI and resolved infection (RI) after treatment were studied. The duration of HCV infection and the liver fibrosis progression rate inversely correlated with the likelihood of detection of peripheral pentamer+/CD8+cells. In PI, pentamer+/CD8+cells had impaired antigen-specific reactivity that worsened when these cells were not detectableex vivoShort/midduration PI was characterized by detectable peripheral PD-1+CD127lowTRAF1lowcells. After triggering of T cell receptors (TCR), the TRAF1 level positively correlated with the levels of CD127, Mcl-1, and CD107a expression and proliferation intensity but negatively with PD-1 expression, linking TRAF1lowto exhaustion.In vitrotreatment with interleukin-7 (IL-7) upregulated TRAF1 expression, while treatment with transforming growth factor-β1 (TGF-β1) did the opposite, suggesting that the IL-7/TGF-β1 balance, besides TCR stimulation, could be involved in TRAF1 regulation. In fact, the serum TGF-β1 concentration was higher in patients with PI than in patients with RI, and it negatively correlated with TRAF1 expression. In line with IL-7 increasing the level of TRAF1 expression, IL-7 plus 4-1BBL treatmentin vitroenhanced T cell reactivity in patients with short/midduration infection. However, in patients with long-lasting PI, anti-PD-L1, in addition to the combination of IL-7 and 4-1BBL, was necessary to reestablish T cell proliferation in individuals with slowly progressing liver fibrosis (slow fibrosers) but had no effect in rapid fibrosers. In conclusion, a peripheral hyporeactive TRAF1lowHCV-specific CD8+T cell response, restorable by IL-7 plus 4-1BBL treatment, characterizes short/midduration PI. In long-lasting disease, HCV-specific CD8+T cells are rarely detectableex vivo, but treatment with IL-7, 4-1BBL, and anti-PD-L1 recovers their reactivityin vitroin slow fibrosers.IMPORTANCEHepatitis C virus (HCV) infects 71 million people worldwide. Two-thirds develop a chronic disease that can lead to cirrhosis and hepatocellular carcinoma. Direct-acting antivirals clear the infection, but there are still patients who relapse. In these cases, additional immunotherapy could play a vital role. A successful anti-HCV immune response depends on virus-specific CD8+T cells. During chronic infection, these cells are functionally impaired, which could be due to the failure of costimulation. This study describes exhausted specific T cells, characterized by low levels of expression of the signal transducer TRAF1 of the positive costimulatory pathway 4-1BB/4-1BBL. IL-7 upregulated TRAF1 expression and improved T cell reactivity in patients with short/midduration disease, while in patients with long-lasting infection, it was also necessary to block the negative PD-1/PD-L1 checkpoint. When the results are taken together, this work supports novel ways of restoring the specific CD8+T cell response, shedding light on the importance of TRAF1 signaling. This could be a promising target for future immunotherapy

Serological response and breakthrough infection after COVID-19 vaccination in patients with cirrhosis and post-liver transplant

BACKGROUND: Vaccine hesitancy and lack of access remain major issues in disseminating COVID-19 vaccination to liver patients globally. Factors predicting poor response to vaccination and risk of breakthrough infection are important data to target booster vaccine programs. The primary aim of the current study was to measure humoral responses to 2 doses of COVID-19 vaccine. Secondary aims included the determination of factors predicting breakthrough infection. METHODS: COVID-19 vaccination and Biomarkers in cirrhosis And post-Liver Transplantation is a prospective, multicenter, observational case-control study. Participants were recruited at 4-10 weeks following first and second vaccine doses in cirrhosis [n = 325; 94% messenger RNA (mRNA) and 6% viral vaccine], autoimmune liver disease (AILD) (n = 120; 77% mRNA and 23% viral vaccine), post-liver transplant (LT) (n = 146; 96% mRNA and 3% viral vaccine), and healthy controls (n = 51; 72% mRNA, 24% viral and 4% heterologous combination). Serological end points were measured, and data regarding breakthrough SARS-CoV-2 infection were collected. RESULTS: After adjusting by age, sex, and time of sample collection, anti-Spike IgG levels were the lowest in post-LT patients compared to cirrhosis (p < 0.0001), AILD (p < 0.0001), and control (p = 0.002). Factors predicting reduced responses included older age, Child-Turcotte-Pugh B/C, and elevated IL-6 in cirrhosis; non-mRNA vaccine in AILD; and coronary artery disease, use of mycophenolate and dysregulated B-call activating factor, and lymphotoxin-α levels in LT. Incident infection occurred in 6.6%, 10.6%, 7.4%, and 15.6% of cirrhosis, AILD, post-LT, and control, respectively. The only independent factor predicting infection in cirrhosis was low albumin level. CONCLUSIONS: LT patients present the lowest response to the SARS-CoV-2 vaccine. In cirrhosis, the reduced response is associated with older age, stage of liver disease and systemic inflammation, and breakthrough infection with low albumin level

Deep-sequencing reveals broad subtype-specific HCV resistance mutations associated with treatment failure

A percentage of hepatitis C virus (HCV)-infected patients fail direct acting antiviral (DAA)-based treatment regimens, often because of drug resistance-associated substitutions (RAS). The aim of this study was to characterize the resistance profile of a large cohort of patients failing DAA-based treatments, and investigate the relationship between HCV subtype and failure, as an aid to optimizing management of these patients. A new, standardized HCV-RAS testing protocol based on deep sequencing was designed and applied to 220 previously subtyped samples from patients failing DAA treatment, collected in 39 Spanish hospitals. The majority had received DAA-based interferon (IFN) a-free regimens; 79% had failed sofosbuvir-containing therapy. Genomic regions encoding the nonstructural protein (NS) 3, NS5A, and NS5B (DAA target regions) were analyzed using subtype-specific primers. Viral subtype distribution was as follows: genotype (G) 1, 62.7%; G3a, 21.4%; G4d, 12.3%; G2, 1.8%; and mixed infections 1.8%. Overall, 88.6% of patients carried at least 1 RAS, and 19% carried RAS at frequencies below 20% in the mutant spectrum. There were no differences in RAS selection between treatments with and without ribavirin. Regardless of the treatment received, each HCV subtype showed specific types of RAS. Of note, no RAS were detected in the target proteins of 18.6% of patients failing treatment, and 30.4% of patients had RAS in proteins that were not targets of the inhibitors they received. HCV patients failing DAA therapy showed a high diversity of RAS. Ribavirin use did not influence the type or number of RAS at failure. The subtype-specific pattern of RAS emergence underscores the importance of accurate HCV subtyping. The frequency of “extra-target” RAS suggests the need for RAS screening in all three DAA target regions

Inhibición de la replicación del virus delta de la hepatitis mediada por interferon, trans-ribozimas y sondas antisense

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Bioquímica. Fecha de lectura: 25-6-199

Clinical significance of occult hepatitis B virus infection

Occult hepatitis B virus (HBV) infection (OBI) is defined as the presence of HBV DNA in the liver (with or without detectable HBV DNA in serum) for individuals testing HBV surface antigen negative. Until recently, the clinical effect of OBI was unclear on the progression of liver disease; on the development of hepatocellular carcinoma; and on the risk for reactivation or transmission of HBV infection. Several studies suggest a high prevalence of OBI among patients with cryptogenic chronic liver disease, but its role in the progression to cirrhosis remains unclear. Although OBI has been well documented in human immunodeficiency virus (HIV)-positive patients, especially among those coinfected with hepatitis C virus, further studies are needed to determine its current clinical impact in HIV setting

La inhibición de la actividad Aurora quinasa B mediada por VHC regula la respuesta antiinflamatoria en las fases iniciales de la infección

Trabajo presentado en el XXXVII Congreso de la Sociedad Española de Bioquímica y Biología Molecular (SEBBM), celbrado en Granada del 9 al 12 de septiembre de 2014.[Introducción] Los cambios epigenéticos, entre los que se encuentran las modificaciones covalentes de histonas, son cruciales en la inducción de inestabilidad genética asociada a enfermedades en humanos, incluyendo el desarrollo de fibrosis hepática y carcinoma hepatocelular. Sin embargo, se desconoce el papel que este tipo de modificaciones pueda tener en la evolución de la enfermedad hepática producida por el virus de la hepatitis C (VHC).[Objetivos] Estudiar las modificaciones covalentes de histonas inducidas por la infección por VHC y determinar los mecanismos moleculares implicados.[Métodos] Para determinar el papel del VHC en la inducción de cambios epigenéticos se utilizó un sistema de infección in vitro de VHC (VHCcc) sobre la línea celular Huh7.5. Alternativamente la región codificante de la proteína del core viral (genotipos 1a, 1b y 2a) se clonó en un vector de expresión eucariota con el que se realizaron ensayos de transfección transitoria sobre la línea celular Huh7.5 y en cultivos primarios de hepatocitos humanos. Las modificaciones de histonas se analizaron por Western Blot, utilizando anticuerpos específicos. Para determinar las actividades enzimáticas implicadas se ensayó el efecto de inhibidores específicos como el ZM443979 y ensayos de siRNA. La expresión de genes implicados en el control de la actividad inflamatoria, como NF-κB y COX-2, se determinó por RT-PCR cuantitativa.[Resultados] Hemos demostrado que el HCV a través de la proteína core inhibe la fosforilación del residuode Serina 10 de la histona H3 (H3Ser10ph), un marcador epigenético asociado a la regulación de la actividad transcripcional y la mitosis celular. Los ensayos con inhibidores de actividades enzimáticas implicadas en la fosforilación de histonas demostraron que el efecto sobre la H3Ser10 inducida por la proteína del core viral se inhibía en presencia de ZM443979, un inhibidor de la actividad Aurora quinasa B (AKB). Mediante ensayos de coinmunoprecipitación observamos una interacción directa entre la proteína del core viral y la AKB. La interacción con la proteína del core indujo una inhibición de la actividad quinasa de la AKB. Asociado a la inhibición de la actividad AKB se observó una inhibición de transcripción de NF-κB y COX-2, genes implicados en el control de la respuesta inflamatoria. La sobreexpresión de AKB revirtió este efecto y disminuyó la infectividad extracelular del virus, de manera opuesta la inhibición de la actividad de Aurora B aumentó la infectividad viral.[Conclusiones] La proteína del core del VHC interacciona con AKB inhibiendo su actividad quinasa y disminuyendo los niveles de fosforilación de la H3Ser10ph así como la expresión de NF-κB y COX-2, dos genes implicados en la regulación de la respuesta inflamatoria. Este mecanismo podría ser una nueva estrategia del VHC para asegurar su infectividad y persistencia en la célula huésped.N

Plasma Ribavirin Trough Concentrations at Week 4 Predict Hepatitis C Virus (HCV) Relapse in HIV-HCV-Coinfected Patients Treated for Chronic Hepatitis C▿

The influence of ribavirin trough concentrations (RBV Ctrough) on the risk of hepatitis C virus (HCV) relapse was retrospectively analyzed in 99 HIV-HCV-coinfected patients who achieved end-of-treatment response with pegylated alpha interferon plus weight-based RBV. The independent predictors (odds ratio [OR] [95% confidence interval (CI)]) of HCV relapse were RBV plasma Ctrough of <2.5 μg/ml (4.5 [1.3 to 15.5]), baseline serum HCV RNA (2.5 [1.2 to 5.1]), and HCV genotype 1 or 4 (13.3 [2.6 to 66.7]). Monitoring of RBV Ctrough may permit early adjustment of RBV dosage to avoid HCV relapse