RESUMEN
Tyrosine hydroxylase (TH) is the rate-limiting enzyme in dopamine biosynthesis catalyzing the tetrahydrobiopterin (BH4 )-dependent hydroxylation of tyrosine to L-DOPA. Here, we analyzed 25 TH variants associated with various degrees of dopa-responsive dystonia and evaluate the effect of each variant on protein stability, activity and cellular localization. Furthermore, we investigated the physical interaction between TH and human wildtype (wt) GTP cyclohydrolase 1 (GTPCH) and the effect of variants on this interaction. Our in vitro results classify variants according to their resistance to proteinase K digestion into three groups (stable, intermediate, unstable). Based on their cellular localization, two groups of variants can be identified, variant group one with cytoplasmic distribution and variant group two forming aggregates. These aggregates do not correlate with loss of enzymatic activity but nevertheless might be a good target for molecular chaperones. Unfortunately, no obvious correlation between the half-life of a variant and its enzymatic activity or between solubility, stability and enzymatic activity of a given variant could be found. Excitingly, some variants disrupt the physical interaction between TH and human wildtype GTPCH, thereby interfering with enzymatic activity and offering new druggable targets for therapy. Taken together, our results highlight the importance of an in-depth molecular analysis of each variant in order to be able to classify groups of disease variants and to find specific therapies for each subgroup. Stand-alone in silico analyses predict less precise the effect of specific variants and should be combined with other in vitro analyses in cellular model systems.
RESUMEN
Cell entry of the pandemic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is mediated by its spike protein S. As a main antigenic determinant, S protein is in focus of various therapeutic strategies. Besides particle-cell fusion, S mediates fusion between infected and uninfected cells resulting in syncytia formation. Here, we present sensitive assay systems with a high dynamic range and high signal-to-noise ratios covering not only particle-cell and cell-cell fusion but also fusion from without (FFWO). In FFWO, S-containing viral particles induce syncytia independently of de novo synthesis of S. Neutralizing antibodies, as well as sera from convalescent patients, inhibited particle-cell fusion with high efficiency. Cell-cell fusion, in contrast, was only moderately inhibited despite requiring levels of S protein below the detection limit of flow cytometry and Western blot. The data indicate that syncytia formation as pathological consequence during coronavirus disease 2019 (COVID-19) can proceed at low levels of S protein and may not be effectively prevented by antibodies.
RESUMEN
SAMHD1 is a phosphohydrolase maintaining cellular dNTP homeostasis but also acts as a critical regulator in innate immune responses due to its antiviral activity and association with autoimmune disease, leading to aberrant activation of interferon. SAMHD1 expression is differentially regulated by interferon in certain primary cells, but the underlying mechanism is not understood. Here, we report a detailed characterization of the promotor region, the 5'- and 3'-untranslated region (UTR) of SAMHD1, and the mechanism responsible for the cell type-dependent up-regulation of SAMHD1 protein by interferon. We demonstrate that induction of SAMHD1 by type I and II interferons depends on 3'-UTR post-transcriptional regulation, whereas the promoter drives basal expression levels. We reveal novel functional target sites for the microRNAs miR-181a, miR-30a, and miR-155 in the SAMHD1 3'-UTR. Furthermore, we demonstrate that down-regulation of endogenous miR-181a and miR-30a levels inversely correlates with SAMHD1 protein up-regulation upon type I and II interferon stimulation in primary human monocytes. These miRNAs are not modulated by interferon in macrophages or dendritic cells, and consequently protein levels of SAMHD1 remain unchanged. These results suggest that SAMHD1 is a non-classical interferon-stimulated gene regulated through cell type-dependent down-regulation of miR-181a and miR-30a in innate sentinel cells.
Asunto(s)
Interferón Tipo I/farmacología , Interferón gamma/farmacología , MicroARNs/genética , Monocitos/metabolismo , Proteínas de Unión al GTP Monoméricas/metabolismo , Regiones no Traducidas 3'/genética , Secuencia de Bases , Células Cultivadas , Células Dendríticas/citología , Células Dendríticas/efectos de los fármacos , Células Dendríticas/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Humanos , Monocitos/citología , Monocitos/efectos de los fármacos , Proteínas de Unión al GTP Monoméricas/genética , Proteína 1 que Contiene Dominios SAM y HDRESUMEN
Deoxynucleotide triphosphates (dNTPs) are essential for efficient hepatitis B virus (HBV) replication. Here, we investigated the influence of the restriction factor SAMHD1, a dNTP hydrolase (dNTPase) and RNase, on HBV replication. We demonstrated that silencing of SAMHD1 in hepatic cells increased HBV replication, while overexpression had the opposite effect. SAMHD1 significantly affected the levels of extracellular viral DNA as well as intracellular reverse transcription products, without affecting HBV RNAs or cccDNA. SAMHD1 mutations that interfere with the dNTPase activity (D137N) or in the catalytic center of the histidine-aspartate (HD) domain (D311A), and a phospho-mimetic mutation (T592E), abrogated the inhibitory activity. In contrast, a mutation diminishing the potential RNase but not dNTPase activity (Q548A) and a mutation disabling phosphorylation (T592A) did not affect antiviral activity. Moreover, HBV restriction by SAMHD1 was rescued by addition of deoxynucleosides. Although HBV infection did not directly affect protein level or phosphorylation of SAMHD1, the virus upregulated intracellular dATPs. Interestingly, SAMHD1 was dephosphorylated, thus in a potentially antiviral-active state, in primary human hepatocytes. Furthermore, SAMHD1 was upregulated by type I and II interferons in hepatic cells. These results suggest that SAMHD1 is a relevant restriction factor for HBV and restricts reverse transcription through its dNTPase activity.
Asunto(s)
Virus de la Hepatitis B/fisiología , Hepatocitos , Mutación Missense , Proteína 1 que Contiene Dominios SAM y HD/metabolismo , Replicación Viral/fisiología , Sustitución de Aminoácidos , Células Hep G2 , Hepatocitos/enzimología , Hepatocitos/patología , Hepatocitos/virología , Humanos , Proteína 1 que Contiene Dominios SAM y HD/genéticaRESUMEN
In general, cell-based medicinal products do not represent a uniform class of medicinal products, but instead comprise medicinal products with diverse regulatory classification as advanced-therapy medicinal products (ATMP), medicinal products (MP), tissue preparations, or blood products. Due to the legal and scientific consequences of the development and approval of MPs, classification should be clarified as early as possible. This paper describes the legal situation in Germany and highlights specific criteria and concepts for classification, with a focus on, but not limited to, ATMPs and non-ATMPs. Depending on the stage of product development and the specific application submitted to a competent authority, legally binding classification is done by the German Länder Authorities, Paul-Ehrlich-Institut, or European Medicines Agency. On request by the applicants, the Committee for Advanced Therapies may issue scientific recommendations for classification.
Asunto(s)
Productos Biológicos/clasificación , Productos Biológicos/uso terapéutico , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Aprobación de Drogas/legislación & jurisprudencia , Programas Nacionales de Salud/legislación & jurisprudencia , Alemania , HumanosRESUMEN
With the release of Regulation 1394/2007, a new framework for gene and cell therapy medicinal products and tissue-engineered products was established in the European Union. For all three product classes, called advanced therapy medicinal products, a centralised marketing authorisation became mandatory. The European Medicines Agency (EMA) together with its Committee for Advanced Therapies, Committee for Human Medicinal Products and the network of national agencies is responsible for scientific evaluation of the marketing authorisation applications. For a new application, data and information relating to manufacturing processes and quality control of the active substance and the final product have to be submitted for evaluation together with data from non-clinical and clinical safety and efficacy studies. Technical requirements for ATMPs are defined in the legislation, and guidance for different products is available through several EMA/CAT guidelines. Due to the diversity of ATMPs, a tailored approach for regulating these products is considered necessary. Thus, a risk-based approach has been introduced for ATMPs allowing flexibility for the regulatory requirements. Since the regulatory framework for ATMPs was established, five products have been licenced in the European Union. However, the pipeline of new ATMPs is much bigger, as seen from the significant numbers of different products discussed by the CAT in scientific advice and classification procedures. In 2013, a public consultation on the ATMP Regulation was conducted by the European Commission, and the results were published in 2014. The report proposes several improvements for the current framework and established procedures for the regulation of ATMPs.
Asunto(s)
Tratamiento Basado en Trasplante de Células y Tejidos/ética , Control de Medicamentos y Narcóticos/legislación & jurisprudencia , Terapia Genética/legislación & jurisprudencia , Mercadotecnía/legislación & jurisprudencia , Investigación Biomédica Traslacional/legislación & jurisprudencia , Animales , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Ensayos Clínicos como Asunto , Evaluación Preclínica de Medicamentos , Drogas en Investigación/farmacocinética , Drogas en Investigación/farmacología , Europa (Continente) , Terapia Genética/ética , Humanos , Aplicación de Nuevas Drogas en Investigación/legislación & jurisprudencia , Seguridad del Paciente/legislación & jurisprudencia , Guías de Práctica Clínica como Asunto , Control de Calidad , Proyectos de Investigación , Investigación Biomédica Traslacional/éticaRESUMEN
On September 11, 2014, a workshop entitled 'Advanced Therapy Medicinal Products: How to Bring Cell-Based Medicinal Product Successfully to the Market' was held at the 47th annual meeting of the German Society for Transfusion Medicine and Immunohematology (DGTI), co-organised by the European Medicines Agency (EMA) and the DGTI in collaboration with the German Stem Cell Network (GSCN). The workshop brought together over 160 participants from academia, hospitals, small- or medium-sized enterprise developers and regulators. At the workshop, speakers from EMA, the Committee for Advanced Therapies (CAT), industry and academia addressed the regulatory aspects of development and authorisation of advanced therapy medicinal products (ATMPs), classification of ATMPs and considerations on cell-based therapies for cardiac repair. The open forum discussion session allowed for a direct interaction between ATMP developers and the speakers from EMA and CAT.
RESUMEN
During the past decade, a large number of cell-based medicinal products have been tested in clinical trials for the treatment of various diseases and tissue defects. However, licensed products and those approaching marketing authorization are still few. One major area of challenge is the manufacturing and quality development of these complex products, for which significant manipulation of cells might be required. While the paradigms of quality, safety and efficacy must apply also to these innovative products, their demonstration may be demanding. Demonstration of comparability between production processes and batches may be difficult for cell-based medicinal products. Thus, the development should be built around a well-controlled manufacturing process and a qualified product to guarantee reproducible data from nonclinical and clinical studies.
Asunto(s)
Comercio , Trasplante de Células Madre/economía , Trasplante de Células Madre/legislación & jurisprudencia , Células Madre/citología , Ensayos Clínicos como Asunto , Unión Europea , Humanos , Control Social FormalRESUMEN
Gene therapy is a rapidly evolving field that needs an integrated approach, as acknowledged in the concept article on the revision of the guideline on gene transfer medicinal products. The first gene therapy application for marketing authorization was approved in the International Conference on Harmonisation (ICH) region in 2012, the product being Alipogene tiparvovec. The regulatory process for this product has been commented on extensively, highlighting the challenges posed by such a novel technology. Here, as current or previous members of the Committee for Advanced Therapies, we share our perspectives and views on gene therapy as a treatment modality based on current common understanding and regulatory experience of gene therapy products in the European Union to date. It is our view that a tailored approach is needed for a given gene therapy product in order to achieve successful marketing authorization.
Asunto(s)
Seguridad de Productos para el Consumidor/legislación & jurisprudencia , Terapia Genética/legislación & jurisprudencia , Comercialización de los Servicios de Salud/legislación & jurisprudencia , Unión Europea , Vectores Genéticos/efectos adversos , Vectores Genéticos/uso terapéutico , HumanosRESUMEN
LINE-1 (L1) retrotransposons are mobile genetic elements whose extensive proliferation resulted in the generation of ≈ 34% of the human genome. They have been shown to be a cause of single-gene diseases. Moreover, L1-encoded endonuclease can elicit double-strand breaks that may lead to genomic instability. Mammalian cells adopted strategies restricting mobility and deleterious consequences of uncontrolled retrotransposition. The human APOBEC3 protein family of polynucleotide cytidine deaminases contributes to intracellular defense against retroelements. APOBEC3 members inhibit L1 retrotransposition by 35-99%. However, genomic L1 retrotransposition events that occurred in the presence of L1-restricting APOBEC3 proteins are devoid of detectable G-to-A hypermutations, suggesting one or multiple deaminase-independent L1 restricting mechanisms. We set out to uncover the mechanism of APOBEC3C (A3C)-mediated L1 inhibition and found that it is deaminase independent, requires an intact dimerization site and the RNA-binding pocket mutation R122A abolishes L1 restriction by A3C. Density gradient centrifugation of L1 ribonucleoprotein particles, subcellular co-localization of L1-ORF1p and A3C and co-immunoprecipitation experiments indicate that an RNA-dependent physical interaction between L1 ORF1p and A3C dimers is essential for L1 restriction. Furthermore, we demonstrate that the amount of L1 complementary DNA synthesized by L1 reverse transcriptase is reduced by ≈ 50% if overexpressed A3C is present.
Asunto(s)
Citidina Desaminasa/metabolismo , Elementos de Nucleótido Esparcido Largo , Proteínas/metabolismo , ADN Polimerasa Dirigida por ARN/metabolismo , Proteínas Portadoras/análisis , Citidina Desaminasa/química , Citidina Desaminasa/genética , Gránulos Citoplasmáticos/química , Gránulos Citoplasmáticos/enzimología , ADN Helicasas , Células HeLa , Humanos , Mutación , Proteínas de Unión a Poli-ADP-Ribosa , Multimerización de Proteína , Proteínas/análisis , Proteínas/química , ARN Helicasas , Proteínas con Motivos de Reconocimiento de ARNRESUMEN
In the past decade, the therapeutic value of mesenchymal stromal cells (MSCs) has been studied in various indications, thereby taking advantage of their immunosuppressive properties. Easy procurement from bone marrow, adipose tissue or other sources and conventional in vitro expansion culture have made their clinical use attractive. Bridging the gap between current scientific knowledge and regulatory prospects on the transformation potential and possible tumorigenicity of MSCs, the Cell Products Working Party and the Committee for Advanced Therapies organized a meeting with leading European experts in the field of MSCs. This meeting elucidated the risk of potential tumorigenicity related to MSC-based therapies from two angles: the scientific perspective and the regulatory point of view. The conclusions of this meeting, including the current regulatory thinking on quality, nonclinical and clinical aspects for MSCs, are presented in this review, leading to a clearer way forward for the development of such products.
Asunto(s)
Carcinogénesis , Proliferación Celular , Trasplante de Células Madre Mesenquimatosas/efectos adversos , Células Madre Mesenquimatosas/citología , Tejido Adiposo/citología , Células de la Médula Ósea/citología , Técnicas de Cultivo de Célula , Diferenciación Celular/genética , Humanos , Células Madre Mesenquimatosas/metabolismoRESUMEN
Increasing scientific knowledge and technical innovations in the areas of cell biology, biotechnology and medicine resulted in the development of promising therapeutic approaches for the prevention and treatment of human diseases. Advanced therapy medicinal products (ATMPs) reflect a complex and innovative class of biopharmaceuticals as these products are highly research-driven, characterised by innovative manufacturing processes and heterogeneous with regard to their origin, type and complexity. This class of ATMP integrates gene therapy medicinal products, somatic cell therapy medicinal products and tissue engineering products and are often individualized and patient-specific products. Multiple challenges arise from the nature of ATMPs, which are often developed by micro, small and medium sized enterprises, university and academia, for whom regulatory experiences are limited and regulatory requirements are challenging. Regulatory guidance such as the reflection paper on classification of ATMPs and guidelines highlighting product-specific issues support academic research groups and pharmaceutical companies to foster the development of safe and effective ATMPs. This review provides an overview on the European regulatory aspects of ATMPs and highlights specific regulatory tools such as the ATMP classification procedure, a discussion on the hospital exemption for selected ATMPs as well as borderline issues towards transplants/transfusion products.
RESUMEN
Unlike activated CD4(+) T cells, resting CD4(+) T cells are highly resistant to productive HIV-1 infection. Early after HIV-1 entry, a major block limits reverse transcription of incoming viral genomes. Here we show that the deoxynucleoside triphosphate triphosphohydrolase SAMHD1 prevents reverse transcription of HIV-1 RNA in resting CD4(+) T cells. SAMHD1 is abundantly expressed in resting CD4(+) T cells circulating in peripheral blood and residing in lymphoid organs. The early restriction to infection in unstimulated CD4(+) T cells is overcome by HIV-1 or HIV-2 virions into which viral Vpx is artificially or naturally packaged, respectively, or by addition of exogenous deoxynucleosides. Vpx-mediated proteasomal degradation of SAMHD1 and elevation of intracellular deoxynucleotide pools precede successful infection by Vpx-carrying HIV. Resting CD4(+) T cells from healthy donors following SAMHD1 silencing or from a patient with Aicardi-Goutières syndrome homozygous for a nonsense mutation in SAMHD1 were permissive for HIV-1 infection. Thus, SAMHD1 imposes an effective restriction to HIV-1 infection in the large pool of noncycling CD4(+) T cells in vivo. Bypassing SAMHD1 was insufficient for the release of viral progeny, implicating other barriers at later stages of HIV replication. Together, these findings may unveil new ways to interfere with the immune evasion and T cell immunopathology of pandemic HIV-1.
Asunto(s)
Linfocitos T CD4-Positivos , VIH-1 , Proteínas de Unión al GTP Monoméricas , Enfermedades Autoinmunes del Sistema Nervioso/genética , Enfermedades Autoinmunes del Sistema Nervioso/virología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Infecciones por VIH , VIH-1/genética , VIH-1/metabolismo , VIH-2/genética , VIH-2/metabolismo , Proteínas del Virus de la Inmunodeficiencia Humana/genética , Proteínas del Virus de la Inmunodeficiencia Humana/metabolismo , Humanos , Proteínas de Unión al GTP Monoméricas/genética , Proteínas de Unión al GTP Monoméricas/metabolismo , Malformaciones del Sistema Nervioso/genética , Malformaciones del Sistema Nervioso/virología , Transcripción Reversa/genética , Proteína 1 que Contiene Dominios SAM y HD , Proteínas Reguladoras y Accesorias Virales/genética , Proteínas Reguladoras y Accesorias Virales/metabolismo , Virión/genética , Virión/crecimiento & desarrollo , Replicación ViralRESUMEN
The human immunodeficiency virus type 1 accessory protein Vif is important for viral infectivity because it counteracts the antiviral protein APOBEC3G (A3G). ³²P metabolic labelling of stimulated cells revealed in vivo phosphorylation of the control protein, whereas no serine/threonine phosphorylation was detected for Vif or the A3G protein. These data were confirmed by in vitro kinase assays using active recombinant kinase. Mitogen-activated protein kinase/extracellular signal-regulated kinase 2 efficiently phosphorylated its target ELK, but failed to phosphorylate Vif. Putative serine/threonine phosphorylation point mutations in Vif (T96, S144, S165, T188) using single-round infection assays demonstrated that these mutations did not alter Vif activity, with the exception of Vif.T96E. Interestingly, T96E and not T96A was functionally impaired, indicating that this residue is critical for Vif-A3G physical interaction and activity. Our data suggest that Vif and A3G are not serine/threonine phosphorylated in human cells and phosphorylation is not linked to their functional activities.
Asunto(s)
Citidina Desaminasa/metabolismo , VIH-1/clasificación , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/metabolismo , Desaminasa APOBEC-3G , Secuencia de Aminoácidos , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/genética , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico , Citidina Desaminasa/genética , Regulación de la Expresión Génica , Células HEK293 , VIH-1/genética , Humanos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos , Fosforilación , Mutación Puntual , Serina/metabolismo , Treonina/metabolismo , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
Myeloid blood cells are largely resistant to infection with human immunodeficiency virus type 1 (HIV-1). Recently, it was reported that Vpx from HIV-2/SIVsm facilitates infection of these cells by counteracting the host restriction factor SAMHD1. Here, we independently confirmed that Vpx interacts with SAMHD1 and targets it for ubiquitin-mediated degradation. We found that Vpx-mediated SAMHD1 degradation rendered primary monocytes highly susceptible to HIV-1 infection; Vpx with a T17A mutation, defective for SAMHD1 binding and degradation, did not show this activity. Several single nucleotide polymorphisms in the SAMHD1 gene have been associated with Aicardi-Goutières syndrome (AGS), a very rare and severe autoimmune disease. Primary peripheral blood mononuclear cells (PBMC) from AGS patients homozygous for a nonsense mutation in SAMHD1 (R164X) lacked endogenous SAMHD1 expression and support HIV-1 replication in the absence of exogenous activation. Our results indicate that within PBMC from AGS patients, CD14+ cells were the subpopulation susceptible to HIV-1 infection, whereas cells from healthy donors did not support infection. The monocytic lineage of the infected SAMHD1 -/- cells, in conjunction with mostly undetectable levels of cytokines, chemokines and type I interferon measured prior to infection, indicate that aberrant cellular activation is not the cause for the observed phenotype. Taken together, we propose that SAMHD1 protects primary CD14+ monocytes from HIV-1 infection confirming SAMHD1 as a potent lentiviral restriction factor.
Asunto(s)
Enfermedades Autoinmunes del Sistema Nervioso/genética , Predisposición Genética a la Enfermedad/genética , Infecciones por VIH/genética , Proteínas de Unión al GTP Monoméricas/deficiencia , Proteínas de Unión al GTP Monoméricas/genética , Células Mieloides/virología , Malformaciones del Sistema Nervioso/genética , Enfermedades Autoinmunes del Sistema Nervioso/metabolismo , Enfermedades Autoinmunes del Sistema Nervioso/virología , Separación Celular , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , VIH-1/metabolismo , Humanos , Immunoblotting , Inmunoprecipitación , Receptores de Lipopolisacáridos/metabolismo , Microscopía Confocal , Mutación Missense , Células Mieloides/metabolismo , Malformaciones del Sistema Nervioso/metabolismo , Malformaciones del Sistema Nervioso/virología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína 1 que Contiene Dominios SAM y HD , Espectrometría de Masas en Tándem , TransfecciónRESUMEN
Human CD34(+) hematopoietic stem cells (HSCs) exhibit the potential to differentiate into a variety of specialized blood cells. The distinct intracellular mechanisms that control cell fate and lineage commitment of these multipotent cells are not well defined. In this study, we investigate and modulate the signaling processes during HSC differentiation toward myeloid dendritic cells (mDCs). DC differentiation induced by the cytokines Granulocyte macrophage colony-stimulating factor (GM-CSF) and Interleukin-4 (IL-4) led to activation of the Extracellular-signal-regulated kinase (ERK), protein kinase C (PKC), and Janus kinase (JAK)/Signal Transducer and Activator of Transcription (STAT) but not the SAPK/c-Jun NH(2) -terminal kinase and p38 mitogen-activated protein kinase signaling pathways. From the activated signaling pathways the PKC isoform δ was found to phosphorylate the transcription factor PU.1, which is described as one of the key factors for myeloid HSC differentiation. On molecular level, PKCδ regulated PU.1 activity by affecting its transactivation activity, whereas its DNA binding activity remained unaffected. This was accompanied by PKCδ-induced phosphorylation of the PU.1 transactivation domain. Furthermore, treatment with PKC- and ERK1/2-specific signaling inhibitors impaired both HSC differentiation toward mDCs as well as phosphorylation-mediated transactivation activity of PU.1. Taken together, these results provide new insights into the molecular mechanisms promoting the differentiation process of HSCs toward mDCs and introduce the PKC isoform δ as critical mediator.
Asunto(s)
Células Dendríticas/metabolismo , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Proteína Quinasa C-delta/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Transactivadores/metabolismo , Animales , Antígenos CD34/inmunología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Línea Celular , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Células Madre Hematopoyéticas/inmunología , Humanos , Interleucina-4/metabolismo , Quinasas Janus/metabolismo , MAP Quinasa Quinasa 4 , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Fosforilación , Factor de Transcripción STAT1/metabolismo , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
BACKGROUND: The non-pathogenic course of SIV infection in its natural host is characterized by robust viral replication in the absence of chronic immune activation and T cell proliferation. In contrast, acutely lethal enteropathic SIVsmm strain PBj induces a strong immune activation and causes a severe acute and lethal disease in pig-tailed macaques after cross-species transmission. One important pathogenicity factor of the PBj virus is the PBj-Nef protein, which contains a conserved diacidic motif and, unusually, an immunoreceptor tyrosine-based activation motif (ITAM). RESULTS: Mutation of the diacidic motif in the Nef protein of the SIVsmmPBj abolishes the acute phenotype of this virus. In vitro, wild-type and mutant PBj (PBj-Nef202/203GG) viruses replicated to similar levels in macaque PBMCs, but PBj-Nef202/203GG no longer triggers ERK mitogen-activated protein (MAP) kinase pathway including an alteration of a Nef-associated Raf-1/ERK-2 multiprotein signaling complex. Moreover, stimulation of IL-2 and down-modulation of CD4 and CD28 were impaired in the mutant virus. Pig-tailed macaques infected with PBj-Nef202/203GG did not show enteropathic complications and lethality as observed with wild-type PBj virus, despite efficient replication of both viruses in vivo. Furthermore, PBj-Nef202/203GG infected animals revealed reduced T-cell activation in periphery lymphoid organs and no detectable induction of IL-2 and IL-6. CONCLUSIONS: In sum, we report here that mutation of the diacidic motif in the PBj-Nef protein abolishes disease progression in pig-tailed macaques despite efficient replication. These data suggest that alterations in the ability of a lentivirus to promote T cell activation and proliferation can have a dramatic impact on its pathogenic potential.
Asunto(s)
Colon/patología , Productos del Gen nef/química , Activación de Linfocitos , Mutación , Virus de la Inmunodeficiencia de los Simios/patogenicidad , Linfocitos T/inmunología , Secuencias de Aminoácidos , Animales , Células Cultivadas , Colon/virología , Productos del Gen nef/genética , Productos del Gen nef/metabolismo , Humanos , Linfopenia/virología , Macaca nemestrina , Enfermedades de los Monos/inmunología , Enfermedades de los Monos/patología , Enfermedades de los Monos/virología , Fenotipo , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/fisiopatología , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Virus de la Inmunodeficiencia de los Simios/genética , Virus de la Inmunodeficiencia de los Simios/inmunología , Virus de la Inmunodeficiencia de los Simios/metabolismo , Viremia/virología , Replicación ViralRESUMEN
Advanced therapy medicinal products (ATMPs), which include gene therapy medicinal products, somatic cell therapy medicinal products and tissue-engineered products, are at the cutting edge of innovation and offer a major hope for various diseases for which there are limited or no therapeutic options. They have therefore been subject to considerable interest and debate. Following the European regulation on ATMPs, a consolidated regulatory framework for these innovative medicines has recently been established. Central to this framework is the Committee for Advanced Therapies (CAT) at the European Medicines Agency (EMA), comprising a multidisciplinary scientific expert committee, representing all EU member states and European Free Trade Association countries, as well as patient and medical associations. In this article, the CAT discusses some of the typical issues raised by developers of ATMPs, and highlights the opportunities for such companies and research groups to approach the EMA and the CAT as a regulatory advisor during development.
Asunto(s)
Terapia Genética/legislación & jurisprudencia , Regulación Gubernamental , Trasplante de Células Madre/legislación & jurisprudencia , Ingeniería de Tejidos/legislación & jurisprudencia , Unión Europea , Terapia Genética/métodos , Humanos , Trasplante de Células Madre/métodosRESUMEN
The accessory protein Vpx is encoded by lentiviruses of the human immunodeficiency virus type 2 (HIV-2) and the simian immunodeficiency SIVsm/SIVmac lineage. It is packaged into virions and is indispensable in early steps of monocyte infection. HIV-1, which does not encode Vpx, is not able to infect human monocytes, but Vpx enables infection with HIV-1. The underlying mechanism is not completely understood. In this work, we focus on Vpx-mediated intracellular postentry events as counteraction of host cell proteins. We found that Vpx binds to apolipoprotein B mRNA-editing catalytic polypeptide 3 family member A (APOBEC3A; A3A), a member of the family of cytidine deaminases, present in monocytes. This interaction led to a reduction of the steady-state protein level of A3A. A single-point mutation in Vpx (H82A) abrogated binding to A3A and single-round infection of monocytes by HIV-1. Taken together, our data indicate that lentiviral Vpx counteracts A3A in human monocytes.