RESUMO
BACKGROUND: Epstein barr virus (EBV) infection of B cells is now understood to be one of the triggering events for the development of Multiple Sclerosis (MS), a progressive immune-mediated disease of the central nervous system. EBV infection is also linked to expression of human endogenous retroviruses (HERVs) of the HERV-W group, a further risk factor for the development of MS. Ocrelizumab is a high-potency disease-modifying treatment (DMT) for MS, which depletes B cells by targeting CD20. OBJECTIVES: We studied the effects of ocrelizumab on gene expression in peripheral blood mononuclear cells (PBMC) from paired samples from 20 patients taken prior to and 6 months after beginning ocrelizumab therapy. We hypothesised that EBV and HERV-W loads would be lower in post-treatment samples. METHODS: Samples were collected in Paxgene tubes, subject to RNA extraction and Illumina paired end short read mRNA sequencing with mapping of sequence reads to the human genome using Salmon and differential gene expression compared with DeSeq2. Mapping was also performed separately to the HERV-D database of HERV sequences and the EBV reference sequence. RESULTS: Patient samples were more strongly clustered by individual rather than disease type (relapsing/remitting or primary progressive), treatment (pre and post), age, or sex. Fourteen genes, all clearly linked to B cell function were significantly down regulated in the post treatment samples. Interestingly only one pre-treatment sample had detectable EBV RNA and there were no significant differences in HERV expression (of any group) between pre- and post-treatment samples. CONCLUSIONS: While EBV and HERV expression are clearly linked to triggering MS pathogenesis, it does not appear that high level expression of these viruses is a part of the ongoing disease process or that changes in virus load are associated with ocrelizumab treatment.
Assuntos
Anticorpos Monoclonais Humanizados , Linfócitos B , Retrovirus Endógenos , Leucócitos Mononucleares , Humanos , Retrovirus Endógenos/efeitos dos fármacos , Feminino , Masculino , Adulto , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Linfócitos B/efeitos dos fármacos , Anticorpos Monoclonais Humanizados/farmacologia , Pessoa de Meia-Idade , Fatores Imunológicos/farmacologia , RNA Viral , Esclerose Múltipla/tratamento farmacológico , Esclerose Múltipla/virologia , Esclerose Múltipla/imunologia , Herpesvirus Humano 4 , Expressão Gênica/efeitos dos fármacosRESUMO
In this work, we are reporting that "Shock and Kill", a therapeutic approach designed to eliminate latent HIV from cell reservoirs, is extrapolatable to cancer therapy. This is based on the observation that malignant cells express a spectrum of human endogenous retroviral elements (HERVs) which can be transcriptionally boosted by HDAC inhibitors. The endoretroviral gene HERV-V2 codes for an envelope protein, which resembles syncytins. It is significantly overexpressed upon exposure to HDAC inhibitors and can be effectively targeted by simultaneous application of TLR7/8 agonists, triggering intrinsic apoptosis. We demonstrated that this synergistic cytotoxic effect was accompanied by the functional disruption of the TLR7/8-NFκB, Akt/PKB, and Ras-MEK-ERK signalling pathways. CRISPR/Cas9 ablation of TLR7 and HERV-V1/V2 curtailed apoptosis significantly, proving the pivotal role of these elements in driving cell death. The effectiveness of this new approach was confirmed in ovarian tumour xenograft studies, revealing a promising avenue for future cancer therapies.
Assuntos
Adjuvantes Imunológicos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Retrovirus Endógenos/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Neoplasias Ovarianas/tratamento farmacológico , Receptor 7 Toll-Like/agonistas , Ativação Viral/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Depsipeptídeos/farmacologia , Retrovirus Endógenos/genética , Retrovirus Endógenos/metabolismo , Feminino , Humanos , Imiquimode/farmacologia , Imunidade Inata/efeitos dos fármacos , Camundongos Nus , Neoplasias Ovarianas/imunologia , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/virologia , Pteridinas/farmacologia , Transdução de Sinais , Receptor 7 Toll-Like/genética , Receptor 7 Toll-Like/metabolismo , Células Tumorais Cultivadas , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Vorinostat/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Endogenous retroviruses (ERVs) play a role in a variety of biological processes, including embryogenesis and cancer. DNA methyltransferase inhibitors (DNMTi)-induced ERV expression triggers interferon responses in ovarian cancer cells via the viral sensing machinery. Baseline expression of ERVs also occurs in cancer cells, though this process is poorly understood and previously unexplored in epithelial ovarian cancer (EOC). Here, the prognostic and immunomodulatory consequences of baseline ERV expression was assessed in EOC. METHODS: ERV expression was assessed using EOC transcriptional data from The Cancer Genome Atlas (TCGA) and from an independent cohort (Hammersmith Hospital, HH), as well as from untreated or DNMTi-treated EOC cell lines. Least absolute shrinkage and selection operator (LASSO) logistic regression defined an ERV expression score to predict patient prognosis. Immunohistochemistry (IHC) was conducted on the HH cohort. Combination of DNMTi treatment with γδ T cells was tested in vitro, using EOC cell lines and patient-derived tumor cells. RESULTS: ERV expression was found to define clinically relevant subsets of EOC patients. An ERV prognostic score was successfully generated in TCGA and validated in the independent cohort. In EOC patients from this cohort, a high ERV score was associated with better survival (log-rank p=0.0009) and correlated with infiltration of CD8+PD1+T cells (r=0.46, p=0.0001). In the TCGA dataset, a higher ERV score was found in BRCA1/2 mutant tumors, compared to wild type (p=0.015), while a lower ERV score was found in CCNE1 amplified tumors, compared to wild type (p=0.019). In vitro, baseline ERV expression dictates the level of ERV induction in response to DNMTi. Manipulation of an ERV expression threshold by DNMTi resulted in improved EOC cell killing by cytotoxic immune cells. CONCLUSIONS: These findings uncover the potential for baseline ERV expression to robustly inform EOC patient prognosis, influence tumor immune infiltration and affect antitumor immunity.
Assuntos
Proteína BRCA1/genética , Proteína BRCA2/genética , Carcinoma Epitelial do Ovário/genética , Ciclina E/genética , Retrovirus Endógenos , Perfilação da Expressão Gênica/métodos , Proteínas Oncogênicas/genética , Neoplasias Ovarianas/genética , Carcinoma Epitelial do Ovário/tratamento farmacológico , Carcinoma Epitelial do Ovário/imunologia , Linhagem Celular Tumoral , Decitabina/farmacologia , Decitabina/uso terapêutico , Retrovirus Endógenos/efeitos dos fármacos , Feminino , Amplificação de Genes , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Linfócitos Intraepiteliais/efeitos dos fármacos , Linfócitos Intraepiteliais/imunologia , Mutação , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/imunologia , Prognóstico , Análise de Sequência de RNA , Análise de SobrevidaRESUMO
BACKGROUND: Gene therapy has held promises for treating specific genetic diseases. However, the key to clinical application depends on effective gene delivery. METHODS: Using a large animal model, we developed two pharmaceutical formulations for gene delivery in the pigs' vagina, which were made up of poly (ß-amino ester) (PBAE)-plasmid polyplex nanoparticles (NPs) based two gel materials, modified montmorillonite (mMMT) and hectorite (HTT). FINDINGS: By conducting flow cytometry of the cervical cells, we found that PBAE-GFP-NPs-mMMT gel was more efficient than PBAE-GFP-NPs-HTT gel in delivering exogenous DNA intravaginally. Next, we designed specific CRISPR/SpCas9 sgRNAs targeting porcine endogenous retroviruses (PERVs) and evaluated the genome editing efficacy in vivo. We discovered that PERV copy number in vaginal epithelium could be significantly reduced by the local delivery of the PBAE-SpCas9/sgRNA NPs-mMMT gel. Comparable genome editing results were also obtained by high-fidelity version of SpCas9, SpCas9-HF1 and eSpCas9, in the mMMT gel. Further, we confirmed that the expression of topically delivered SpCas9 was limited to the vagina/cervix and did not diffuse to nearby organs, which was relatively safe with low toxicity. INTERPRETATION: Our data suggested that the PBAE-NPs mMMT vaginal gel is an effective preparation for local gene therapy, yielding insights into novel therapeutic approaches to sexually transmitted disease in the genital tract. FUNDING: This work was supported by the National Science and Technology Major Project of the Ministry of science and technology of China (No. 2018ZX10301402); the National Natural Science Foundation of China (81761148025, 81871473 and 81402158); Guangzhou Science and Technology Programme (No. 201704020093); National Ten Thousand Plan-Young Top Talents of China, Fundamental Research Funds for the Central Universities (17ykzd15 and 19ykyjs07); Three Big Constructions-Supercomputing Application Cultivation Projects sponsored by National Supercomputer Center In Guangzhou; the National Research FFoundation (NRF) South Africa under BRICS Multilateral Joint Call for Proposals; grant 17-54-80078 from the Russian Foundation for Basic Research.
Assuntos
Colo do Útero/citologia , Retrovirus Endógenos/genética , Dosagem de Genes/efeitos dos fármacos , Polímeros/química , RNA Guia de Cinetoplastídeos/administração & dosagem , Administração Intravaginal , Animais , Bentonita/química , Sistemas CRISPR-Cas , Células Cultivadas , Colo do Útero/química , Retrovirus Endógenos/efeitos dos fármacos , Feminino , Edição de Genes , Terapia Genética , Camundongos , Modelos Animais , Nanopartículas , Plasmídeos/administração & dosagem , Plasmídeos/genética , Silicatos/química , Suínos , Cremes, Espumas e Géis VaginaisRESUMO
BACKGROUND: Human endogenous retroviruses (HERVs) are genetic elements in the human genome, which resulted from ancient retroviral germline infections. HERVs have strong transcriptional promoters and enhancers that affect a cell's transcriptome. They also encode proteins that can exert effects in human cells. This review examines how our increased understanding of HERVs have led to their potential use as biomarkers and immunologic targets. MATERIAL AND METHODS: PubMed/Medline, Embase, Web of Science, and Cochrane databases were used in a systematic search to identify all articles studying the potential impact of HERVs on surgical diseases. The search included studies that involved clinical patient samples in diseases including cancer, inflammatory conditions, and autoimmune disease. Articles focused on conditions not routinely managed by surgeons were excluded. RESULTS: Eighty six articles met inclusion and quality criteria for this review and were included. Breast cancer and melanoma have robust evidence regarding the use of HERVs as potential tumor markers and immunologic targets. Reported evidence of the activity of HERVs in colorectal cancer, pancreatic cancer, hepatocellular cancer, prostate and ovarian cancer, germ cell tumors as well as idiopathic pulmonary hypertension, and the inflammatory response in burns was also reviewed. CONCLUSIONS: Increasingly convincing evidence indicates that HERVs may play a role in solid organ malignancy and present important biomarkers or immunologic targets in multiple cancers. Innovative investigation of HERVs is a valuable focus of translational research and can deepen our understanding of cellular physiology and the effects of endogenous retroviruses on human biology. As strategies for treatment continue to focus on genome-based interventions, understanding the impact of endogenous retroviruses on human disease will be critical.
Assuntos
Biomarcadores Tumorais/genética , Retrovirus Endógenos/genética , Regulação Neoplásica da Expressão Gênica/imunologia , Neoplasias/genética , Pesquisa Translacional Biomédica , Antineoplásicos Imunológicos/farmacologia , Antineoplásicos Imunológicos/uso terapêutico , Biomarcadores Tumorais/antagonistas & inibidores , Retrovirus Endógenos/efeitos dos fármacos , Retrovirus Endógenos/imunologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genoma Humano , Humanos , Neoplasias/diagnóstico , Neoplasias/tratamento farmacológico , Transcriptoma/efeitos dos fármacos , Transcriptoma/genética , Transcriptoma/imunologiaRESUMO
Mesenchymal tumor subpopulations secrete pro-tumorigenic cytokines and promote treatment resistance1-4. This phenomenon has been implicated in chemorefractory small cell lung cancer and resistance to targeted therapies5-8, but remains incompletely defined. Here, we identify a subclass of endogenous retroviruses (ERVs) that engages innate immune signaling in these cells. Stimulated 3 prime antisense retroviral coding sequences (SPARCS) are oriented inversely in 3' untranslated regions of specific genes enriched for regulation by STAT1 and EZH2. Derepression of these loci results in double-stranded RNA generation following IFN-γ exposure due to bi-directional transcription from the STAT1-activated gene promoter and the 5' long terminal repeat of the antisense ERV. Engagement of MAVS and STING activates downstream TBK1, IRF3, and STAT1 signaling, sustaining a positive feedback loop. SPARCS induction in human tumors is tightly associated with major histocompatibility complex class 1 expression, mesenchymal markers, and downregulation of chromatin modifying enzymes, including EZH2. Analysis of cell lines with high inducible SPARCS expression reveals strong association with an AXL/MET-positive mesenchymal cell state. While SPARCS-high tumors are immune infiltrated, they also exhibit multiple features of an immune-suppressed microenviroment. Together, these data unveil a subclass of ERVs whose derepression triggers pathologic innate immune signaling in cancer, with important implications for cancer immunotherapy.
Assuntos
Retrovirus Endógenos/metabolismo , Imunidade Inata/efeitos dos fármacos , Interferons/farmacologia , Neoplasias/imunologia , Neoplasias/virologia , Animais , Linhagem Celular Tumoral , Retrovirus Endógenos/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos Nus , Neoplasias/genética , RNA Antissenso/genéticaRESUMO
We describe a newly identified therapeutic target for type 1 diabetes (T1D): an envelope protein of endogenous retroviral origin, human endogenous retrovirus W envelope (HERV-W-Env). HERV-W-Env was found to be detected in the blood of ~60% of patients with T1D and is expressed in acinar pancreatic cells of 75% of patients with T1D at post mortem examination. Preclinical experiments showed that this protein displays direct cytotoxicity on human ß-islet cells. In vivo HERV-W-Env impairs the insulin and glucose metabolism in transgenic mice expressing HERV-W-Env. GNbAC1, an IgG4 monoclonal antibody, has been developed to specifically target HERV-W-Env and to neutralize the effect of HERV-W-Env in vitro and in vivo. GNbAC1 is currently in clinical development for multiple sclerosis and > 300 subjects have been administered with GNbAC1 so far. GNbAC1 is now being tested in T1D in the RAINBOW-T1D study, which is a randomized placebo-controlled study with the objective of showing the safety and pharmacodynamic response of GNbAC1 in patients who have had T1D with a maximum of 4 years' duration. GNbAC1 is being tested vs placebo at the dose of 6 mg/kg in 60 patients during six repeated administrations for 6 months; a 6-month open-label extension will follow. The primary endpoint is to assess safety, and secondary endpoints are the pharmacodynamic responses to GNbAC1. GNbAC1 targeting HERV-W-Env is currently in clinical development in T1D, with the first safety and pharmacodynamic study. If the study results are positive, this may open the door to the development of an innovative non-immunomodulatory disease-modifying treatment for T1D.
Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Diabetes Mellitus Tipo 1/tratamento farmacológico , Retrovirus Endógenos/efeitos dos fármacos , Produtos do Gene env/efeitos dos fármacos , Fatores Imunológicos/farmacologia , Diabetes Mellitus Tipo 1/virologia , Retrovirus Endógenos/imunologia , Produtos do Gene env/sangue , Produtos do Gene env/imunologia , HumanosRESUMO
Inhibitors of DNA methyltransferases (DNMTis) or histone deacetylases (HDACis) are epigenetic drugs which are investigated since decades. Several have been approved and are applied in the treatment of hematopoietic and lymphatic malignancies, although their mode of action has not been fully understood. Two recent findings improved mechanistic insights: i) activation of human endogenous retroviral elements (HERVs) with concomitant synthesis of double-stranded RNAs (dsRNAs), and ii) massive activation of promoters from long terminal repeats (LTRs) which originated from past HERV invasions. These dsRNAs activate an antiviral response pathway followed by apoptosis. LTR promoter activation leads to synthesis of non-annotated transcripts potentially encoding novel or cryptic proteins. Here, we discuss the current knowledge of the molecular effects exerted by epigenetic drugs with a focus on DNMTis and HDACis. We highlight the role in LTR activation and provide novel data from both in vitro and in vivo epigenetic drug treatment.
Assuntos
Antineoplásicos/uso terapêutico , DNA (Citosina-5-)-Metiltransferases/genética , Retrovirus Endógenos/efeitos dos fármacos , Inibidores Enzimáticos/uso terapêutico , Epigênese Genética , Neoplasias Hematológicas/tratamento farmacológico , Inibidores de Histona Desacetilases/uso terapêutico , Histona Desacetilases/genética , Apoptose/efeitos dos fármacos , Apoptose/genética , DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , DNA (Citosina-5-)-Metiltransferases/metabolismo , Retrovirus Endógenos/genética , Retrovirus Endógenos/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias Hematológicas/enzimologia , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/patologia , Histona Desacetilases/metabolismo , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Interações Hospedeiro-Patógeno/genética , Humanos , Regiões Promotoras Genéticas , RNA de Cadeia Dupla , Sequências Repetidas Terminais , Ativação Viral/efeitos dos fármacosRESUMO
The expression of human endogenous retroviruses (HERVs) has been associated with Multiple Sclerosis (MS). The MS-related retrovirus (MSRV/HERV-W) has the potential to activate inflammatory immunity, which could promote both susceptibility and progression toward MS. A connection between HERVs and MS is also supported by the observation that people infected with the human immunodeficiency virus (HIV) may have a lower risk of developing MS than the HIV non-infected, healthy population. This may be due to suppression of HERV expression by antiretroviral therapies (ART) used to treat HIV infection. In this pilot study, we compared RNA expression of the envelope gene of MSRV/HERV-W, as well as Toll-like receptors (TLR) 2 and 4, in a small cohort of HIV+ patients with MS patients and healthy controls (HC). An increased expression of MSRV/HERV-Wenv and TLR2 RNA was detected in blood of MS patients compared with HIV patients and HC, while TLR4 was increased in both MS and HIV patients. There was, however, no difference in MSRV/HERV-Wenv, TLR2 and TLR4 expression between ART-treated and -untreated HIV patients. The viral protein Env was expressed mainly by B cells and monocytes, but not by T cells and EBV infection could induce the expression of MSRV/HERV-Wenv in Lymphoblastoid cell lines (LCLs). LCLs were therefore used as an in vitro system to test the efficacy of ART in inhibiting the expression of MSRV/HERV-Wenv. Efavirenz (a non-nucleoside reverse transcriptase inhibitor) alone or different combined drugs could reduce MSRV/HERV-Wenv expression in vitro. Further, experiments are needed to clarify the potential role of ART in protection from MS.
Assuntos
Retrovirus Endógenos/efeitos dos fármacos , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Infecções por HIV/tratamento farmacológico , Esclerose Múltipla/tratamento farmacológico , Inibidores da Transcriptase Reversa/administração & dosagem , Adulto , Alcinos , Benzoxazinas/administração & dosagem , Estudos de Coortes , Ciclopropanos , Combinação de Medicamentos , Retrovirus Endógenos/genética , Retrovirus Endógenos/imunologia , Feminino , Regulação Viral da Expressão Gênica/imunologia , Produtos do Gene env/sangue , Produtos do Gene env/imunologia , Produtos do Gene env/isolamento & purificação , Infecções por HIV/complicações , Infecções por HIV/imunologia , Infecções por HIV/virologia , Humanos , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla/complicações , Esclerose Múltipla/imunologia , Esclerose Múltipla/virologia , Projetos Piloto , RNA Viral/isolamento & purificação , Receptor 2 Toll-Like/sangue , Receptor 2 Toll-Like/imunologia , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/sangue , Receptor 4 Toll-Like/imunologia , Receptor 4 Toll-Like/metabolismo , Resultado do Tratamento , Adulto JovemRESUMO
BACKGROUND: Inhaled particles have been shown to produce systemic changes in DNA methylation. Global hypomethylation has been associated to viral sequence reactivation, possibly linked to the activation of pro-inflammatory pathways occurring after exposure. This observation provides a rationale to investigate viral sequence (both exogenous and endogenous) methylation in association to metal-rich particulate matter exposure. To verify this hypothesis, we chose the Wp promoter of the Epstein-Barr Virus (EBV-Wp) and the promoter of the human-endogenous-retrovirus w (HERV-w), respectively as a paradigm of an exogenous and an endogenous retroviral sequence, to be investigated by bisulfite PCR Pyrosequencing. We enrolled 63 male workers in an electric furnace steel plant, exposed to high level of metal-rich particulate matter. RESULTS: Comparing samples obtained in the first day of a work week (time 0-baseline, after 2 days off work) and the samples obtained after 3 days of work (time 1-post exposure), the mean methylation of EBV-Wp was significantly higher at baseline compared to post-exposure (meanbaseline = 56.7%5mC; meanpost-exposure = 47.9%5mC; p-value = 0.009), whereas the mean methylation of HERV-w did not significantly differ. Individual exposure to inhalable particles and metals was estimated based on measures in all working areas and time spent by the study subjects in each area. In a regression model adjusted for age, body mass index and smoking, PM and metal components had a positive association with EBV-Wp methylation (i.e. PM10: ß = 5.99, p-value < 0.038; nickel: ß = 17.82, p-value = 0.02; arsenic: ß = 13.59, p-value < 0.015). CONCLUSIONS: The difference observed comparing baseline and post-exposure samples may be suggestive of a rapid change in EBV methylation induced by air particles, while correlation between EBV methylation and PM/metal exposure may represent a more stable adaptive mechanism. Future studies investigating a larger panel of viral sequences could better elucidate possible mechanisms and their role in pro-inflammatory pathways leading to systemic health effects.
Assuntos
Poluentes Atmosféricos/toxicidade , Metilação de DNA/efeitos dos fármacos , Metalurgia , Metais/toxicidade , Exposição Ocupacional , Material Particulado/toxicidade , Adulto , DNA Viral/metabolismo , Retrovirus Endógenos/efeitos dos fármacos , Retrovirus Endógenos/metabolismo , Herpesvirus Humano 4/efeitos dos fármacos , Herpesvirus Humano 4/metabolismo , Humanos , Itália , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , AçoRESUMO
Human endogenous retroviruses (HERVs), remnants of ancestral viral genomic insertions, are known to represent 8% of the human genome and are associated with several pathologies. In particular, the envelope protein of HERV-W family (HERV-W-Env) has been involved in multiple sclerosis pathogenesis. Investigations to detect HERV-W-Env in a few other autoimmune diseases were negative, except in type-1 diabetes (T1D). In patients suffering from T1D, HERV-W-Env protein was detected in 70% of sera, and its corresponding RNA was detected in 57% of peripheral blood mononuclear cells. While studies on human Langerhans islets evidenced the inhibition of insulin secretion by HERV-W-Env, this endogenous protein was found to be expressed by acinar cells in 75% of human T1D pancreata. An extensive immunohistological analysis further revealed a significant correlation between HERV-W-Env expression and macrophage infiltrates in the exocrine part of human pancreata. Such findings were corroborated by in vivo studies on transgenic mice expressing HERV-W-env gene, which displayed hyperglycemia and decreased levels of insulin, along with immune cell infiltrates in their pancreas. Altogether, these results strongly suggest an involvement of HERV-W-Env in T1D pathogenesis. They also provide potentially novel therapeutic perspectives, since unveiling a pathogenic target in T1D.
Assuntos
Diabetes Mellitus Tipo 1/tratamento farmacológico , Diabetes Mellitus Tipo 1/virologia , Retrovirus Endógenos/efeitos dos fármacos , Proteínas do Envelope Viral/fisiologia , Animais , Antivirais/uso terapêutico , Estudos de Coortes , Diabetes Mellitus Tipo 1/complicações , Retrovirus Endógenos/genética , Retrovirus Endógenos/patogenicidade , Feminino , Humanos , Hiperglicemia/complicações , Insulina/metabolismo , Antagonistas da Insulina/farmacologia , Ilhotas Pancreáticas/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , RNA Viral/sangue , Proteínas do Envelope Viral/efeitos dos fármacosRESUMO
Human endogenous retroviruses (HERVs) make up 8% of the human genome. The HERV type K (HERV-K) HML-2 (HK2) family contains proviruses that are the most recent entrants into the human germ line and are transcriptionally active. In HIV-1 infection and cancer, HK2 genes produce retroviral particles that appear to be infectious, yet the replication capacity of these viruses and potential pathogenicity has been difficult to ascertain. In this report, we screened the efficacy of commercially available reverse transcriptase inhibitors (RTIs) at inhibiting the enzymatic activity of HK2 RT and HK2 genomic replication. Interestingly, only one provirus, K103, was found to encode a functional RT among those examined. Several nucleoside analogue RTIs (NRTIs) blocked K103 RT activity and consistently inhibited the replication of HK2 genomes. The NRTIs zidovudine (AZT), stavudine (d4T), didanosine (ddI), and lamivudine (3TC), and the nucleotide RTI inhibitor tenofovir (TDF), show efficacy in blocking K103 RT. HIV-1-specific nonnucleoside RTIs (NNRTIs), protease inhibitors (PIs), and integrase inhibitors (IIs) did not affect HK2, except for the NNRTI etravirine (ETV). The inhibition of HK2 infectivity by NRTIs appears to take place at either the reverse transcription step of the viral genome prior to HK2 viral particle formation and/or in the infected cells. Inhibition of HK2 by these drugs will be useful in suppressing HK2 infectivity if these viruses prove to be pathogenic in cancer, neurological disorders, or other diseases associated with HK2. The present studies also elucidate a key aspect of the life cycle of HK2, specifically addressing how they do, and/or did, replicate.IMPORTANCE Endogenous retroviruses are relics of ancestral virus infections in the human genome. The most recent of these infections was caused by HK2. While HK2 often remains silent in the genome, this group of viruses is activated in HIV-1-infected and cancer cells. Recent evidence suggests that these viruses are infectious, and the potential exists for HK2 to contribute to disease. We show that HK2, and specifically the enzyme that mediates virus replication, can be inhibited by a panel of drugs that are commercially available. We show that several drugs block HK2 with different efficacies. The inhibition of HK2 replication by antiretroviral drugs appears to occur in the virus itself as well as after infection of cells. Therefore, these drugs might prove to be an effective treatment by suppressing HK2 infectivity in diseases where these viruses have been implicated, such as cancer and neurological syndromes.
Assuntos
Retrovirus Endógenos/efeitos dos fármacos , Retrovirus Endógenos/genética , Genoma Viral/efeitos dos fármacos , Inibidores da Transcriptase Reversa/farmacologia , Transcrição Reversa/efeitos dos fármacos , Fármacos Anti-HIV/farmacologia , Linhagem Celular Tumoral , Retrovirus Endógenos/enzimologia , Retrovirus Endógenos/patogenicidade , Humanos , Inibidores de Integrase/farmacologia , Lamivudina/farmacologia , Inibidores de Proteases/farmacologia , Estavudina/farmacologia , Replicação Viral/efeitos dos fármacos , Replicação Viral/genética , Zidovudina/farmacologiaRESUMO
Porcine endogenous retroviruses (PERVs) are integrated in the genome of all pigs; they are released as infectious particles, and under certain conditions they can infect human cells. Therefore, they represent a risk when pigs are used as sources of cells, tissues, or organs for xenotransplantation. Xenotransplantation is under development due to the increasing shortage of human transplants. Whereas most porcine microorganisms which may be able to induce a disease (zoonosis) in the transplant recipient can be eliminated, this is not possible in the case of PERVs. Antiretroviral drugs which had been developed for the treatment of human immunodeficiency virus-1 (HIV-1) infections have been tested in vitro for their efficacy in inhibiting PERV replication. Inhibitors of the viral reverse transcriptase and of the integrase have been found effective. The most effective inhibitor of the reverse transcriptase was azidothymidine (AZT); the integrase inhibitors were the most potent inhibitors of PERV. Although in the past PERV transmission has not been observed after experimental or clinical xenotransplantation of pig cells or organs, and although PERVs may one day be inactivated in pigs by genome editing using CRISPR/Cas, knowing which antiretroviral drugs can effectively restrict PERV infection will still be important.
Assuntos
Antirretrovirais/uso terapêutico , Retrovirus Endógenos/efeitos dos fármacos , Infecções por Retroviridae/tratamento farmacológico , Transplante Heterólogo , Animais , Xenoenxertos , Humanos , Inibidores de Integrase/uso terapêutico , Infecções por Retroviridae/virologia , Inibidores da Transcriptase Reversa/uso terapêutico , Sus scrofa , Zidovudina/uso terapêutico , Zoonoses/tratamento farmacológico , Zoonoses/prevenção & controle , Zoonoses/transmissão , Zoonoses/virologiaRESUMO
BACKGROUND: While antiretroviral therapies have improved life expectancy and reduced viral loads in HIV-1-positive individuals, the cessation of treatment results in a rebound of viral replication. This suggests that a reservoir of latently-infected cells remains within these patients, the identity of which is ill-defined and therefore difficult to target therapeutically. Current strategies are aimed at using drugs such as histone deacetylase (HDAC) inhibitors to induce the expression of latent HIV-1 proviruses in order to activate and ultimately eradicate this reservoir of infected cells. One concern with the use of HDAC inhibitors is that they could up-regulate human endogenous retroviruses (HERVs), as well as HIV-1, with potentially pathophysiological consequences. RESULTS: In this study, we analysed the transcription of HERV genes in HIV-1 latency T cell (J-LAT 8.4) and monocyte (U1) models following treatment with the HDAC inhibitors, vorinostat, panobinostat and romidepsin. We examined the expression of HERV-K (HML-2) env and pol, as well as the co-opted genes HERV-W env (syncytin-1), HERV-FRD env (syncytin-2), in these cell lines. Finally, we investigated HERV expression in primary human T cells. CONCLUSIONS: We show that HDAC inhibitors did not substantially increase the transcription of the analysed HERV env or pol genes, suggesting that histone acetylation is not crucial for controlling HERV expression in these experimental models and in ex vivo primary human T cells. Importantly, this indicates that unwanted HERV expression does not appear to be a barrier to the use of HDAC inhibitors in HIV-1 cure strategies.
Assuntos
Retrovirus Endógenos/efeitos dos fármacos , Retrovirus Endógenos/fisiologia , HIV-1/efeitos dos fármacos , Inibidores de Histona Desacetilases/metabolismo , Provírus/efeitos dos fármacos , Provírus/fisiologia , Ativação Viral/efeitos dos fármacos , Linhagem Celular , Produtos do Gene env/análise , Produtos do Gene env/genética , Produtos do Gene pol/análise , Produtos do Gene pol/genética , Humanos , Monócitos/efeitos dos fármacos , Monócitos/virologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/virologia , Transcrição GênicaRESUMO
We studied HERV expression in cell lines after hypoxia, mitogenic stimulation, and demethylation, to better understand if hypoxia may play a role in ERV activation also within the nervous system, as represented by neuroblastoma cell lines. The level of RNA of four human ERV groups (HERVs) (HERVE, I/T, H, and W), and three housekeeping genes, of different cell lines including A549, COS-1, Namalwa, RD-L and Vero-E6, as well as human neuroblastoma cell lines SH-SY5Y, SK-N-DZ, and SK-N-AS were studied using reverse transcription and real-time quantitative PCR (QPCR). During the course of recovery from hypoxia a pronounced and selective activation of RNA expression of HERVW-like sequences, but not of HERVE, I/T, H, and three housekeeping genes, was found in the neuroblastoma cell lines, most pronounced in SK-N-DZ. In the SK-N-DZ cell line, we also tested the expression of HERVs after chemical treatments. HERVW-like sequences were selectively upregulated by 5-azacytidine, a demethylating agent. Some HERVW loci seem especially responsive to hypoxia and demethylation. HERV expression in neuroblastoma cells is selectively and profoundly influenced by some physiological and chemical stimuli.
Assuntos
Retrovirus Endógenos/genética , Neuroblastoma/genética , Neuroblastoma/virologia , RNA Viral/genética , Azacitidina/farmacologia , Hipóxia Celular , Linhagem Celular Tumoral , Proliferação de Células/genética , Retrovirus Endógenos/classificação , Retrovirus Endógenos/efeitos dos fármacos , Retrovirus Endógenos/fisiologia , Inibidores Enzimáticos/farmacologia , Humanos , Oxigênio/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Transcrição Reversa , Regulação para Cima , Ativação Viral/efeitos dos fármacos , Ativação Viral/genéticaRESUMO
Human endogenous retroviruses (HERV) represent about 8 % of the human genome. Some of these genetic elements are expressed in pathological circumstances. A HERV protein, the multiple sclerosis-associated retrovirus (MSRV) envelope protein (MSRV-Env), is expressed in the blood and active brain lesions of multiple sclerosis (MS) patients. It possesses pro-inflammatory and myelinotoxic properties. The patterns of expression and pathogenic properties of MSRV-Env make it a relevant drug target for MS therapeutics-in particular for preventing neurodegeneration, a key component of progressive forms of MS. An immunoglobulin G4 monoclonal antibody (mAb), called GNbAC1, has been developed to neutralize this pathogenic target. After showing neutralizing effects in vitro and in mouse models of MS, GNbAC1 is now in phase II clinical development. MSRV-related biomarkers such as MSRV-Env and MSRV polymerase (MSRV-Pol) gene transcripts are overexpressed in the blood and cerebrospinal fluid of patients with MS. These biomarkers may have prognostic value for long-term MS evolution, and their transcription levels in blood decline during treatments with GNbAC1, which has also been reported in patients administered reference MS drugs such as natalizumab or interferon-ß. GNbAC1 as a new MSRV-Env-antagonist mAb could be a specific and causal treatment for MS, with a particular application for progressive forms of the disease. For possible use in companion diagnostic tests, MSRV-associated biomarkers could open the door to a personalized therapeutic approach for MS.
Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Retrovirus Endógenos/efeitos dos fármacos , Produtos do Gene env/antagonistas & inibidores , Esclerose Múltipla/tratamento farmacológico , Animais , Anticorpos Monoclonais Humanizados/uso terapêutico , Ensaios Clínicos Fase II como Assunto , Modelos Animais de Doenças , Retrovirus Endógenos/genética , Retrovirus Endógenos/metabolismo , Produtos do Gene env/sangue , Produtos do Gene env/líquido cefalorraquidiano , Produtos do Gene env/genética , Humanos , Camundongos , Esclerose Múltipla/virologia , Medicina de Precisão , RNA Viral/sangue , RNA Viral/líquido cefalorraquidiano , RNA Viral/efeitos dos fármacosRESUMO
GNbAC1 is a humanized monoclonal antibody targeting MSRV-Env, an endogenous retroviral protein, which is expressed in multiple sclerosis (MS) lesions, is pro-inflammatory and inhibits oligodendrocyte precursor cell differentiation. This paper describes the open-label extension up to 12months of a trial testing GNbAC1 in 10 MS patients at 2 and 6mg/kg. The primary objective was to assess GNbAC1 safety, and other objectives were pharmacokinetic and pharmacodynamic assessments. During the extended study, no safety issues occurred in the 8 remaining patients. No anti-GNbAC1 antibodies were detected. GNbAC1 appears well tolerated.
Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Retrovirus Endógenos/efeitos dos fármacos , Esclerose Múltipla/tratamento farmacológico , Proteínas do Envelope Viral/antagonistas & inibidores , Idoso , Anticorpos Monoclonais Humanizados/farmacologia , Estudos de Coortes , Retrovirus Endógenos/imunologia , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla/imunologia , Proteínas do Envelope Viral/imunologiaRESUMO
BACKGROUND: Several viruses were reported as co-factors triggering the pathogenesis of multiple sclerosis (MS), including the endogenous retroviruses of the HERV-W family, that were also proposed as biomarkers of disease progression and therapy outcome. OBJECTIVE: The objective of this article is to clarify whether in MS patients treatment with natalizumab has effects on MSRV/syncytin-1/HERV-W expression and the possible relationship with disease outcome. METHODS: Peripheral blood mononuclear cells were collected from 22 patients with relapsing-remitting disease, at entry and after three, six and 12 months of treatment with natalizumab. The cell subpopulations and the expression of MSRVenv/syncytin-1/HERV-Wenv were analyzed by flow cytometry and by discriminatory env-specific RT-PCR assays. RESULTS: By flow cytometry the relative amounts of T, NK and monocyte subpopulations were shown to remain fairly constant. A relative increase of B lymphocytes was observed at three to six months (p = 0.033). The MSRVenv and syncitin-1 transcripts were reduced at six to 12 months of therapy (p = 0.0001). Accordingly, at month 12, the plasma-membrane levels of the HERV-Wenv protein were reduced (p = 0.0001). B cells, NK and monocytes but not T cells expressed the HERV-Wenv protein. None of the patients relapsed during therapy. CONCLUSION: Effective therapy with natalizumab downregulates MSRV/syncytin-1/HERV-W expression.
Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Retrovirus Endógenos/efeitos dos fármacos , Produtos do Gene env/análise , Esclerose Múltipla Recidivante-Remitente/tratamento farmacológico , Esclerose Múltipla Recidivante-Remitente/virologia , Proteínas da Gravidez/análise , Adulto , Estudos de Coortes , Feminino , Citometria de Fluxo , Humanos , Leucócitos Mononucleares/virologia , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Natalizumab , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Adulto JovemRESUMO
Caffeine and aspirin have been suggested to be involved in neurologic diseases, such as schizophrenia, and previous data have revealed that abnormal expression of HERV-W elements may be an important factor in the etiopathogenesis of those diseases. In this article, we reported that caffeine and aspirin contributed to the expression of HERV-W env and gag in Human SH-SY5Y neuroblastoma cells. Semi-quantitative RT-PCR and quantitative Real-time PCR were used to detect the mRNA of HERV-W env and gag in cells exposed to caffeine or aspirin. Western blotting was used to detect the protein of HERV-W env. Luciferase activity assay was employed to detect the activity of HERV-W env promoter. It was found that both caffeine and aspirin could increase the expression of HERV-W env and gag in human SH-SY5Y neuroblastoma cells. Caffeine could activate the HERV-W env promoter, while aspirin could not. With previous studies we can conjecture that HERVs might play a bridging role between environmental factors, such as drugs and neurologic diseases.
Assuntos
Aspirina/metabolismo , Cafeína/metabolismo , Retrovirus Endógenos/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacos , Western Blotting , Linhagem Celular Tumoral , Perfilação da Expressão Gênica , Produtos do Gene env/biossíntese , Produtos do Gene env/genética , Produtos do Gene gag/biossíntese , Produtos do Gene gag/genética , Genes Reporter , Humanos , Luciferases/análise , Luciferases/genética , Neurônios/efeitos dos fármacos , Neurônios/virologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo RealRESUMO
BACKGROUND: Proposed co-factors triggering the pathogenesis of multiple sclerosis (MS) are the Epstein Barr virus (EBV), and the potentially neuropathogenic MSRV (MS-associated retrovirus) and syncytin-1, of the W family of human endogenous retroviruses. METHODOLOGY/PRINCIPAL FINDINGS: In search of links, the expression of HERV-W/MSRV/syncytin-1, with/without exposure to EBV or to EBV glycoprotein350 (EBVgp350), was studied on peripheral blood mononuclear cells (PBMC) from healthy volunteers and MS patients, and on astrocytes, by discriminatory env-specific RT-PCR assays, and by flow cytometry. Basal expression of HERV-W/MSRV/syncytin-1 occurs in astrocytes and in monocytes, NK, and B, but not in T cells. This uneven expression is amplified in untreated MS patients, and dramatically reduced during therapy. In astrocytes, EBVgp350 stimulates the expression of HERV-W/MSRV/syncytin-1, with requirement of the NF-κB pathway. In EBVgp350-treated PBMC, MSRVenv and syncytin-1 transcription is activated in B cells and monocytes, but not in T cells, nor in the highly expressing NK cells. The latter cells, but not the T cells, are activated by proinflammatory cytokines. CONCLUSIONS/SIGNIFICANCE: In vitro EBV activates the potentially immunopathogenic and neuropathogenic HERV-W/MSRV/syncytin-1, in cells deriving from blood and brain. In vivo, pathogenic outcomes would depend on abnormal situations, as in late EBV primary infection, that is often symptomatic, or/and in the presence of particular host genetic backgrounds. In the blood, HERV-Wenv activation might induce immunopathogenic phenomena linked to its superantigenic properties. In the brain, toxic mechanisms against oligodendrocytes could be established, inducing inflammation, demyelination and axonal damage. Local stimulation by proinflammatory cytokines and other factors might activate further HERV-Ws, contributing to the neuropathogenity. In MS pathogenesis, a possible model could include EBV as initial trigger of future MS, years later, and HERV-W/MSRV/syncytin-1 as actual contributor to MS pathogenicity, in striking parallelism with disease behaviour.