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1.
PLoS One ; 15(8): e0237156, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32780756

RESUMO

Ischemic neuropathy is common in subjects with critical limb ischemia, frequently causing chronic neuropathic pain. However, neuropathic pain caused by ischemia is hard to control despite the restoration of an adequate blood flow. Here, we used a rat model of ischemic-reperfusion nerve injury (IRI) to investigate possible effects of hepatocyte growth factor (HGF) against ischemic neuropathy. Hemagglutinating virus of Japan (HVJ) liposomes containing plasmids encoded with HGF was delivered into the peripheral nervous system by retrograde axonal transport following its repeated injections into the tibialis anterior muscle in the right hindlimb. First HGF gene transfer was done immediately after IRI, and repeated at 1, 2 and 3 weeks later. Rats with IRI exhibited pronounced mechanical allodynia and thermal hyperalgesia, decreased blood flow and skin temperature, and lowered thresholds of plantar stimuli in the hind paw. These were all significantly improved by HGF gene transfer, as also were sciatic nerve conduction velocity and muscle action potential amplitudes. Histologically, HGF gene transfer resulted in a significant increase of endoneurial microvessels in sciatic and tibial nerves and promoted nerve regeneration which were confirmed by morphometric analysis. Neovascularization was observed in the contralateral side of peripheral nerves as well. In addition, IRI elevated mRNA levels of P2X3 and P2Y1 receptors, and transient receptor potential vanilloid receptor subtype 1 (TRPV1) in sciatic nerves, dorsal root ganglia and spinal cord, and these elevated levels were inhibited by HGF gene transfer. In conclusion, HGF gene transfer is a potent candidate for treatment of acute ischemic neuropathy caused by reperfusion injury, because of robust angiogenesis and enhanced nerve regeneration.


Assuntos
Terapia Genética/métodos , Fator de Crescimento de Hepatócito/genética , Neuralgia/terapia , Traumatismo por Reperfusão/terapia , Animais , Modelos Animais de Doenças , Gânglios Espinais/metabolismo , Técnicas de Transferência de Genes , Vetores Genéticos , Fator de Crescimento de Hepatócito/metabolismo , Humanos , Hiperalgesia/metabolismo , Lipossomos/metabolismo , Masculino , Ratos , Ratos Wistar , Nervo Isquiático/metabolismo , Vírus Sendai/genética , Resultado do Tratamento
2.
Virus Res ; 286: 198074, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32589897

RESUMO

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel human coronavirus causing the pandemic of severe pneumonia (Coronavirus Disease 2019, COVID-19). SARS-CoV-2 is highly pathogenic in human, having posed immeasurable public health challenges to the world. Innate immune response is critical for the host defense against viral infection and the dysregulation of the host innate immune responses probably aggravates SARS-CoV-2 infection, contributing to the high morbidity and lethality of COVID-19. It has been reported that some coronavirus proteins play an important role in modulating innate immunity of the host, but few studies have been conducted on SARS-CoV-2. In this study, we screened the viral proteins of SARS-CoV-2 and found that the viral ORF6, ORF8 and nucleocapsid proteins were potential inhibitors of type I interferon signaling pathway, a key component for antiviral response of host innate immune. All the three proteins showed strong inhibition on type I interferon (IFN-ß) and NF-κB-responsive promoter, further examination revealed that these proteins were able to inhibit the interferon-stimulated response element (ISRE) after infection with Sendai virus, while only ORF6 and ORF8 proteins were able to inhibit the ISRE after treatment with interferon beta. These findings would be helpful for the further study of the detailed signaling pathway and unveil the key molecular player that may be targeted.


Assuntos
Betacoronavirus/genética , Interações Hospedeiro-Patógeno/genética , Interferon beta/genética , NF-kappa B/genética , Proteínas do Nucleocapsídeo/genética , Proteínas Virais/genética , Betacoronavirus/imunologia , Regulação da Expressão Gênica , Genes Reporter , Células HEK293 , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunidade Inata , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/imunologia , Interferon beta/imunologia , Luciferases/genética , Luciferases/metabolismo , NF-kappa B/imunologia , Proteínas do Nucleocapsídeo/imunologia , Fosfoproteínas , Plasmídeos/química , Plasmídeos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Elementos de Resposta , Vírus Sendai/genética , Vírus Sendai/imunologia , Transdução de Sinais , Transfecção/métodos , Proteínas Virais/imunologia
3.
FEBS Lett ; 594(5): 864-877, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31705658

RESUMO

Respirovirus C protein blocks the type I interferon (IFN)-stimulated activation of the JAK-STAT pathway. It has been reported that C protein inhibits IFN-α-stimulated tyrosine phosphorylation of STATs, but the underlying mechanism is poorly understood. Here, we show that the C protein of Sendai virus (SeV), a member of the Respirovirus genus, binds to the IFN receptor subunit IFN-α/ß receptor subunit (IFNAR)2 and inhibits IFN-α-stimulated tyrosine phosphorylation of the upstream receptor-associated kinases, JAK1 and TYK2. Analysis of various SeV C mutant (Cm) proteins demonstrates the importance of the inhibitory effect on receptor-associated kinase phosphorylation for blockade of JAK-STAT signaling. Furthermore, this inhibitory effect and the IFNAR2 binding capacity are observed for all the respirovirus C proteins examined. Our results suggest that respirovirus C protein inhibits activation of the receptor-associated kinases JAK1 and TYK2 possibly through interaction with IFNAR2.


Assuntos
Receptor de Interferon alfa e beta/metabolismo , Vírus Sendai/metabolismo , Transdução de Sinais , Proteínas Virais/metabolismo , Linhagem Celular , Células HEK293 , Humanos , Janus Quinase 1/metabolismo , Mutação , Fosforilação , Fatores de Transcrição STAT/metabolismo , Vírus Sendai/genética , TYK2 Quinase/metabolismo , Proteínas Virais/genética
4.
J Mol Med (Berl) ; 97(12): 1685-1694, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31786669

RESUMO

In an earlier study, a novel Sendai virus-vectored anti-tuberculosis vaccine encoding Ag85A and Ag85B (SeV85AB) was constructed and shown to elicit antigen-specific T cell responses and protection against Mycobacterium tuberculosis (Mtb) infection in a murine model. In this study, we evaluate whether the immune responses induced by this novel vaccine might be elevated by a recombinant DNA vaccine expressing the same antigen in a heterologous prime-boost vaccination strategy. The results showed that both SeV85AB prime-DNA boost (SeV85AB-DNA) and DNA prime-SeV85AB boost (DNA-SeV85AB) vaccination strategies significantly enhanced the antigen-specific T cell responses induced by the separate vaccines. The SeV85AB-DNA immunization regimen induced higher levels of recall T cell responses after Mtb infection and conferred better immune protection compared with DNA-SeV85AB or a single immunization. Collectively, our study lends strong evidence that a DNA vaccine boost might be included in a novel SeV85AB immunization strategy designed to enhance the immune protection against Mtb. KEY MESSAGES: A heterologous prime-boost regimen with a novel recombinant SeV85AB and a DNA vaccine increase the T cell responses above those from a single vaccine. The heterologous prime-boost regimen provided protection against Mtb infection. The DNA vaccine might be included in a novel SeV85AB immunization strategy designed to enhance the immune protection against Mtb.


Assuntos
Aciltransferases/imunologia , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Mycobacterium tuberculosis/imunologia , Vacinas contra a Tuberculose/genética , Tuberculose/imunologia , Vacinas de DNA/imunologia , Aciltransferases/genética , Animais , Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Linfócitos T CD8-Positivos/imunologia , Feminino , Vetores Genéticos , Imunização Secundária , Interferon gama/metabolismo , Interleucina-2/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Mycobacterium tuberculosis/genética , Vírus Sendai/genética , Tuberculose/prevenção & controle , Vacinas contra a Tuberculose/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Vacinação
5.
Sci Rep ; 9(1): 16862, 2019 11 14.
Artigo em Inglês | MEDLINE | ID: mdl-31727944

RESUMO

Patients with acute myeloid leukemia frequently present translocations of MLL gene. Rearrangements of MLL protein (MLL-r) in complexes that contain the histone methyltransferase DOT1L are common, which elicit abnormal methylation of lysine 79 of histone H3 at MLL target genes. Phase 1 clinical studies with pinometostat (EPZ-5676), an inhibitor of DOT1L activity, demonstrated the therapeutic potential for targeting DOT1L in MLL-r leukemia patients. We previously reported that down-regulation of DOT1L increases influenza and vesicular stomatitis virus replication and decreases the antiviral response. Here we show that DOT1L inhibition also reduces Sendai virus-induced innate response and its overexpression decreases influenza virus multiplication, reinforcing the notion of DOT1L controlling viral replication. Accordingly, genes involved in the host innate response against pathogens (RUBICON, TRIM25, BCL3) are deregulated in human lung epithelial cells treated with pinometostat. Concomitantly, deregulation of some of these genes together with that of the MicroRNA let-7B, may account for the beneficial effects of pinometostat treatment in patients with MLL-r involving DOT1L. These results support a possible increased vulnerability to infection in MLL-r leukemia patients undergoing pinometostat treatment. Close follow up of infection should be considered in pinometostat therapy to reduce some severe side effects during the treatment.


Assuntos
Antineoplásicos/efeitos adversos , Benzimidazóis/efeitos adversos , Inibidores Enzimáticos/efeitos adversos , Regulação Leucêmica da Expressão Gênica , Histona-Lisina N-Metiltransferase/genética , Vírus da Influenza A Subtipo H1N1/genética , Infecções Oportunistas/induzido quimicamente , Células A549 , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/imunologia , Proteína 3 do Linfoma de Células B/genética , Proteína 3 do Linfoma de Células B/imunologia , Suscetibilidade a Doenças , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Histona-Lisina N-Metiltransferase/imunologia , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Vírus da Influenza A Subtipo H1N1/crescimento & desenvolvimento , Vírus da Influenza A Subtipo H1N1/metabolismo , Influenza Humana/induzido quimicamente , Influenza Humana/genética , Influenza Humana/imunologia , Influenza Humana/virologia , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/imunologia , Leucemia Mieloide Aguda/patologia , MicroRNAs/genética , MicroRNAs/imunologia , Infecções Oportunistas/genética , Infecções Oportunistas/imunologia , Infecções Oportunistas/virologia , Vírus Sendai/genética , Vírus Sendai/crescimento & desenvolvimento , Vírus Sendai/metabolismo , Transdução de Sinais , Fatores de Transcrição/genética , Fatores de Transcrição/imunologia , Proteínas com Motivo Tripartido/genética , Proteínas com Motivo Tripartido/imunologia , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/imunologia , Replicação Viral
6.
Cell Rep ; 29(7): 1909-1922.e5, 2019 11 12.
Artigo em Inglês | MEDLINE | ID: mdl-31722206

RESUMO

Reprogramming somatic cells to induced pluripotent stem cells (iPSCs) is accompanied by dramatic changes in epigenetic programs, including silencing of endogenous and exogenous retroviruses. Here, we utilized replication-defective and persistent Sendai virus (SeVdp)-based vectors to monitor retroviral silencing during reprogramming. We observed that retroviral silencing occurred at an early reprogramming stage without a requirement for KLF4 or the YY1-binding site in the retroviral genome. Insertional chromatin immunoprecipitation (iChIP) enabled us to isolate factors assembled on the silenced provirus, including components of inhibitor of histone acetyltransferase (INHAT), which includes the SET/TAF-I oncoprotein. Knockdown of SET/TAF-I in mouse embryonic fibroblasts (MEFs) diminished retroviral silencing during reprogramming, and overexpression of template activating factor-I α (TAF-Iα), a SET/TAF-I isoform predominant in embryonic stem cells (ESCs), reinforced retroviral silencing by an SeVdp-based vector that is otherwise defective in retroviral silencing. Our results indicate an important role for TAF-Iα in retroviral silencing during reprogramming.


Assuntos
Técnicas de Reprogramação Celular , Reprogramação Celular , Retrovirus Endógenos , Inativação Gênica , Células-Tronco Embrionárias Murinas , Animais , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Retrovirus Endógenos/genética , Retrovirus Endógenos/metabolismo , Chaperonas de Histonas/genética , Chaperonas de Histonas/metabolismo , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Células-Tronco Embrionárias Murinas/virologia , Vírus Sendai/genética , Vírus Sendai/metabolismo , Fator de Transcrição YY1/genética , Fator de Transcrição YY1/metabolismo
7.
Stem Cell Res ; 40: 101574, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31627126

RESUMO

The familial form of Alzheimer's disease (FAD), which is caused by mutations in PRESENILIN 1 (PSEN1) and amyloid precursor protein (APP) genes, represents less than 5% of all AD cases and has an early-onset. We report the generation and characterization of an iPSC line derived from a FAD patient carrying the PSEN1-G206D mutation. The iPSC line maintained the original genotype, a normal karyotype, was free from Sendai viral vectors and reprogramming factors (OCT4, SOX2, KLF4 and c-MYC), presented a typical morphology, expressed endogenous pluripotency markers, and could be differentiated into ectodermal, mesodermal and endodermal cells, confirming its pluripotency.


Assuntos
Doença de Alzheimer/genética , Linhagem Celular/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Presenilina-1/genética , Adulto , Doença de Alzheimer/metabolismo , Diferenciação Celular , Linhagem Celular/metabolismo , Células Cultivadas , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Masculino , Mutação de Sentido Incorreto , Presenilina-1/metabolismo , Vírus Sendai/genética , Vírus Sendai/fisiologia , Integração Viral
8.
Viruses ; 11(11)2019 10 24.
Artigo em Inglês | MEDLINE | ID: mdl-31652964

RESUMO

Pangolins are endangered animals in urgent need of protection. Identifying and cataloguing the viruses carried by pangolins is a logical approach to evaluate the range of potential pathogens and help with conservation. This study provides insight into viral communities of Malayan Pangolins (Manis javanica) as well as the molecular epidemiology of dominant pathogenic viruses between Malayan Pangolin and other hosts. A total of 62,508 de novo assembled contigs were constructed, and a BLAST search revealed 3600 ones (≥300 nt) were related to viral sequences, of which 68 contigs had a high level of sequence similarity to known viruses, while dominant viruses were the Sendai virus and Coronavirus. This is the first report on the viral diversity of pangolins, expanding our understanding of the virome in endangered species, and providing insight into the overall diversity of viruses that may be capable of directly or indirectly crossing over into other mammals.


Assuntos
Infecções por Coronavirus/veterinária , Coronavirus/isolamento & purificação , Mamíferos/virologia , Infecções por Respirovirus/veterinária , Vírus Sendai/isolamento & purificação , Animais , Coronavirus/classificação , Coronavirus/genética , Coronavirus/fisiologia , Infecções por Coronavirus/virologia , Espécies em Perigo de Extinção/estatística & dados numéricos , Metagenômica , Filogenia , Infecções por Respirovirus/virologia , Vírus Sendai/classificação , Vírus Sendai/genética , Vírus Sendai/fisiologia
9.
Methods Mol Biol ; 2048: 53-57, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31396928

RESUMO

The discovery and development of induced pluripotent stem cells (iPSCs) opened a novel venue for disease modeling, drug discovery, and personalized medicine. Additionally, iPSCs have been utilized for a wide variety of research and clinical applications without immunological and ethical concerns that encounter embryonic stem cells. Adoptive T cell immunotherapy is a form of cellular immunotherapy that involves transfusion of functional T cells. However, this approach requires T cell expansion and the process causes T cell exhaustion. As a result, highly expanded T cells have not proven to be particularly effective for treatments. This exhaustion issue could be overcome due to rejuvenation of T cells by reprogramming to pluripotency and redifferentiation to T cells. This is a potential therapeutic strategy for combating various types of cancer.


Assuntos
Reprogramação Celular/imunologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Cultura Primária de Células/métodos , Linfócitos T Citotóxicos/fisiologia , Reprogramação Celular/genética , Vetores Genéticos/genética , Humanos , Imunoterapia Adotiva/métodos , Neoplasias/imunologia , Neoplasias/terapia , Proteínas Recombinantes/genética , Vírus Sendai/genética , Fatores de Transcrição/genética , Transdução Genética/métodos
10.
Stem Cell Res ; 39: 101516, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31415975

RESUMO

We have generated and characterized seven human induced pluripotent stem cell (iPSC) lines derived from peripheral blood mononuclear cells (PBMCs) from a single family, including unaffected and affected individuals clinically diagnosed with Autism Spectrum Disorder (ASD). The reprogramming of the PBMCs was performed using non-integrative Sendai virus containing the reprogramming factors POU5F1 (OCT4), SOX2, KLF4 and MYC. All iPSC lines exhibited a normal karyotype and pluripotency was validated by immunofluorescence, flow cytometry and their ability to differentiate into the three embryonic germ layers. These iPSC lines are a valuable resource to study the molecular mechanisms underlying ASD.


Assuntos
Transtorno do Espectro Autista/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Leucócitos Mononucleares/citologia , Adulto , Reprogramação Celular/genética , Reprogramação Celular/fisiologia , Feminino , Citometria de Fluxo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Leucócitos Mononucleares/metabolismo , Masculino , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Vírus Sendai/genética , Adulto Jovem
11.
Stem Cell Res ; 39: 101527, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31408836

RESUMO

We have generated an induced pluripotent stem cell (iPSC) line KCLi003-A (iOP101) from epidermal keratinocytes of a female donor, heterozygous for the loss-of-function mutation p.R501X in the filaggrin gene (FLG), using non-integrating Sendai virus vectors. Derivation and expansion of iPSCs were performed under xeno-free culture conditions. Characterization and validation of KCLi003-A line included molecular karyotyping, mutation screening using restriction enzyme digestion, next generation sequencing (NGS), while pluripotency and differentiation potential were confirmed by expression of associated markers in vitro and by in vivo teratoma assay.


Assuntos
Células-Tronco Pluripotentes Induzidas/metabolismo , Proteínas de Filamentos Intermediários/genética , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Reprogramação Celular/genética , Reprogramação Celular/fisiologia , Imunofluorescência , Heterozigoto , Humanos , Repetições de Microssatélites/genética , Mycoplasma/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Vírus Sendai/genética
12.
Stem Cell Res ; 39: 101522, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31401456

RESUMO

Genetic polymorphism of apolipoprotein E (APOE) confers differential susceptibility to late-onset Alzheimer's disease (LOAD). The ε3 allele of APOE, the most common isoform, does not represent a risk factor for LOAD. In contrast, the ε4 allele is the strongest genetic risk factor for this disease. Here, we present the characterization of four iPSC lines generated from dermal fibroblasts of diagnosed sporadic AD patients using Sendai viral vectors encoding OCT4, SOX2, KLF4 and c-MYC. The iPSCs expressed endogenous pluripotency markers, could be differentiated into the three germ layers, maintained the original genotypes, and were free from Sendai vectors and reprogramming factors.


Assuntos
Corpos Embrioides/citologia , Apolipoproteínas E/genética , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Linhagem Celular , Técnicas de Genotipagem/métodos , Humanos , Imuno-Histoquímica , Cariotipagem , Repetições de Microssatélites/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Vírus Sendai/genética
13.
J Interferon Cytokine Res ; 39(11): 711-719, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31268382

RESUMO

Interferon (IFN), the first ever-described cytokine, has a potent activity against viruses. Soon since its discovery, quantification of IFN has been an important issue. Most of the traditional methods to measure IFN biological activity rely on indirect methods that quantify dyes retained by IFN-protected cells against a lytic virus, or by techniques that indirectly quantify viral replication by measuring the expression level of viral-encoded reporter proteins such as the green fluorescent protein (GFP). In both cases, the IFN units are determined by the quantification of an effective dose 50, defined as the IFN dose that prevents 50% cell death of 50% reduction of the maximal amount of GFP intensity. In this study we propose the use of an alternative approach to measure IFN activity by calculating the minimal IFN dose 50 as the amount of IFN able to completely protect 50% of the cells from infection measured by the total absence of virus-dependent GFP signal in a cell culture plate. This sensitive approach could be used to easily quantify the Z value to determine IFN bioassay robustness. We believe that this approximation could be interesting to be considered by the IFN community.


Assuntos
Bioensaio , Interferon Tipo I/análise , Animais , Células Cultivadas , Chlorocebus aethiops , Humanos , Proteínas Recombinantes/análise , Vírus Sendai/genética , Vírus Sendai/crescimento & desenvolvimento , Vírus Sendai/isolamento & purificação , Células Vero
14.
Stem Cell Res ; 39: 101493, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31326747

RESUMO

Two clones of human induced pluripotent stem cells (iPSCs) were generated from dermal fibroblasts isolated from a one-year-old Thai patient with X-linked osteogenesis imperfecta. The patient harbored a mutation, p.N459S, in the MBTPS2 gene. The cells were reprogrammed using an integration-free Sendai virus containing KLF4, c-MYC, OCT4 and SOX2. Both of the established iPSC lines (MDCUi001-A and MDCUi001-B) maintained normal karyotype, expressed pluripotent markers and differentiated into all three germ layers.


Assuntos
Células-Tronco Pluripotentes Induzidas/metabolismo , Osteogênese Imperfeita/genética , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Linhagem Celular , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibroblastos/fisiologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Cariótipo , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Masculino , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Vírus Sendai/genética , Tailândia
15.
Stem Cell Res ; 39: 101498, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31326748

RESUMO

Huntington disease (HD) is an autosomal dominant, neurodegenerative disease caused by a CAG repeat expansion within the coding sequence of the HTT gene, resulting in a highly toxic protein with an expanded polyglutamine stretch that forms typical protein aggregates throughout the brain. We generated human induced pluripotent stem cells (hiPSCs) from two HD patients using non-integrating Sendai virus (SeV). The hiPSCs display a normal karyotype, express all pluripotency markers, have the same CAG repeat expansion as the original fibroblasts and are able to differentiate into the three germ layers in vitro.


Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Células Cultivadas , Feminino , Humanos , Doença de Huntington/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Cariótipo , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Vírus Sendai/genética
16.
Mol Cancer Ther ; 18(8): 1430-1438, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31171582

RESUMO

In clinical N0 (cN0) cases with head and neck squamous cell carcinoma (HNSCC), a treatment selection is still controversial: elective neck dissection or watchful waiting. We focused on sentinel lymph node (SLN)-targeted therapy using the urokinase-type plasminogen activator (uPA)-dependent oncolytic Sendai virus "BioKnife." The objectives of this study were to investigate BioKnife migration into SLNs and elucidate its antitumor effect on lymph node metastases (LNM). We established an orthotopic nude mouse model of HNSCC, with LNM being frequently induced. We inoculated HSC-3-M3, human highly metastatic tongue squamous cell carcinoma cells, in the tongue of the nude mice, and after 2 weeks, we injected BioKnife into the primary tumor. We tracked BioKnife migration into the SLNs by immunostaining, RT-PCR, and an in vivo imaging system. We also examined its antitumor effects and mechanisms through serial section analysis of lymph nodes. GFP reporter expression was clearly visible in the lymph nodes of virus groups, which corresponded to SLNs. Relative GFP mRNA was significantly increased in both the tongues and lymph nodes in the virus groups compared with that in the control group (P < 0.05). Serial section analysis showed that BioKnife infected cancer cells and exhibited significant antitumor effect against LNM compared with the control groups (P < 0.05). We detected apoptosis in LNM infected by BioKnife. BioKnife migrated into SLNs after its injection into the primary tumor and effectively suppressed LNM, suggesting that SLN-targeted therapy using BioKnife has great potential to provide a novel and promising alternative to elective neck dissection in cN0 patients with HNSCC.


Assuntos
Terapia Viral Oncolítica , Vírus Oncolíticos , Vírus Sendai , Linfonodo Sentinela/patologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/terapia , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Modelos Animais de Doenças , Humanos , Metástase Linfática , Camundongos , Micrometástase de Neoplasia , Vírus Oncolíticos/genética , Vírus Sendai/genética , Linfonodo Sentinela/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/diagnóstico por imagem , Ensaios Antitumorais Modelo de Xenoenxerto
17.
Stem Cell Res Ther ; 10(1): 185, 2019 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-31234949

RESUMO

BACKGROUND: Disease modeling with patient-derived induced pluripotent stem cells (iPSCs) is a powerful tool for elucidating the mechanisms underlying disease pathogenesis and developing safe and effective treatments. Patient peripheral blood (PB) cells are used for iPSC generation in many cases since they can be collected with minimum invasiveness. To derive iPSCs that lack immunoreceptor gene rearrangements, hematopoietic stem and progenitor cells (HSPCs) are often targeted as the reprogramming source. However, the current protocols generally require HSPC mobilization and/or ex vivo expansion owing to their sparsity at the steady state and low reprogramming efficiencies, making the overall procedure costly, laborious, and time-consuming. METHODS: We have established a highly efficient method for generating iPSCs from non-mobilized PB-derived CD34+ HSPCs. The source PB mononuclear cells were obtained from 1 healthy donor and 15 patients and were kept frozen until the scheduled iPSC generation. CD34+ HSPC enrichment was done using immunomagnetic beads, with no ex vivo expansion culture. To reprogram the CD34+-rich cells to pluripotency, the Sendai virus vector SeVdp-302L was used to transfer four transcription factors: KLF4, OCT4, SOX2, and c-MYC. In this iPSC generation series, the reprogramming efficiencies, success rates of iPSC line establishment, and progression time were recorded. After generating the iPSC frozen stocks, the cell recovery and their residual transgenes, karyotypes, T cell receptor gene rearrangement, pluripotency markers, and differentiation capability were examined. RESULTS: We succeeded in establishing 223 iPSC lines with high reprogramming efficiencies from 15 patients with 8 different disease types. Our method allowed the rapid appearance of primary colonies (~ 8 days), all of which were expandable under feeder-free conditions, enabling robust establishment steps with less workload. After thawing, the established iPSC lines were verified to be pluripotency marker-positive and of non-T cell origin. A majority of the iPSC lines were confirmed to be transgene-free, with normal karyotypes. Their trilineage differentiation capability was also verified in a defined in vitro assay. CONCLUSION: This robust and highly efficient method enables the rapid and cost-effective establishment of transgene-free iPSC lines from a small volume of PB, thus facilitating the biobanking of patient-derived iPSCs and their use for the modeling of various diseases.


Assuntos
Antígenos CD34/metabolismo , Reprogramação Celular/fisiologia , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Vírus Sendai/genética , Adolescente , Adulto , Idoso , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Linhagem Celular , Reprogramação Celular/genética , Feminino , Citometria de Fluxo , Humanos , Cariotipagem , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Masculino , Pessoa de Meia-Idade , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Adulto Jovem
18.
PLoS One ; 14(5): e0216944, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31100083

RESUMO

Most viruses are known to spontaneously generate defective viral genomes (DVG) due to errors during replication. These DVGs are subgenomic and contain deletions that render them unable to complete a full replication cycle in the absence of a co-infecting, non-defective helper virus. DVGs, especially of the copyback type, frequently observed with paramyxoviruses, have been recognized to be important triggers of the antiviral innate immune response. DVGs have therefore gained interest for their potential to alter the attenuation and immunogenicity of vaccines. To investigate this potential, accurate identification and quantification of DVGs is essential. Conventional methods, such as RT-PCR, are labor intensive and will only detect primer sequence-specific species. High throughput sequencing (HTS) is much better suited for this undertaking. Here, we present an HTS-based algorithm called DVG-profiler to identify and quantify all DVG sequences in an HTS data set generated from a virus preparation. DVG-profiler identifies DVG breakpoints relative to a reference genome and reports the directionality of each segment from within the same read. The specificity and sensitivity of the algorithm was assessed using both in silico data sets as well as HTS data obtained from parainfluenza virus 5, Sendai virus and mumps virus preparations. HTS data from the latter were also compared with conventional RT-PCR data and with data obtained using an alternative algorithm. The data presented here demonstrate the high specificity, sensitivity, and robustness of DVG-profiler. This algorithm was implemented within an open source cloud-based computing environment for analyzing HTS data. DVG-profiler might prove valuable not only in basic virus research but also in monitoring live attenuated vaccines for DVG content and to assure vaccine lot to lot consistency.


Assuntos
Algoritmos , Mapeamento Cromossômico/estatística & dados numéricos , Vírus Defeituosos/genética , Genoma Viral , Vírus da Caxumba/genética , Vírus da Parainfluenza 5/genética , Vírus Sendai/genética , Animais , Mapeamento Cromossômico/métodos , Primers do DNA/síntese química , Primers do DNA/metabolismo , Conjuntos de Dados como Assunto , Vírus Defeituosos/classificação , Sequenciamento de Nucleotídeos em Larga Escala/estatística & dados numéricos , Humanos , Tipagem Molecular , Vírus da Caxumba/classificação , Vírus da Parainfluenza 5/classificação , Reação em Cadeia da Polimerase em Tempo Real , Vírus Sendai/classificação , Sensibilidade e Especificidade
19.
Cell Reprogram ; 21(2): 78-88, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30969880

RESUMO

Induced pluripotent stem cells (iPSCs) remain a promising approach to target diseases with a loss of functional parenchyma. This technology comes with a number of concerns for clinical applications, including teratogenic potential and genomic instability. Here we focused on evaluating the safety of cross-species Sendai viral reprogramming, as well as investigating the transcriptional dynamics during reprogramming and differentiation. We established that Sendai viral vectors carrying human Oct4, Sox2, Klf4, and c-Myc (OSKM) could produce mouse iPSCs free of transduced viral materials. Gene expression analysis revealed an efficient silencing of the virally-introduced human pluripotency factors and upregulation of the endogenous pluripotency network over time. In addition, single cell gene expression analysis of proof-of-principle-derived cardiomyocytes revealed distinct expression patterns indicative of subspecialized cardiac cell lineages. Moreover, our results demonstrate the importance of monitoring genomic aberrations before any clinical or preclinical applications, as we detected a high prevalence of chromosomal instability. Taken together, we demonstrated the successful use of a clinically germane method to reprogram terminally differentiated mouse cells and their potential to generate specialized cardiac cell types. Additionally, our results suggest a plasticity of OSKM to reprogram more divergent species and provide a new application of an established reprogramming approach.


Assuntos
Reprogramação Celular , Vetores Genéticos/administração & dosagem , Células-Tronco Pluripotentes Induzidas/citologia , Miócitos Cardíacos/citologia , Vírus Sendai/genética , Animais , Diferenciação Celular , Linhagem da Célula , Vetores Genéticos/genética , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Fatores de Transcrição Kruppel-Like/genética , Camundongos , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Proteínas Proto-Oncogênicas c-myc/genética , Fatores de Transcrição SOXB1/genética , Vírus Sendai/metabolismo , Análise de Célula Única
20.
PLoS One ; 14(4): e0215490, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31022207

RESUMO

Induced pluripotent stem cell (iPSC)-technology is an important platform in medicine and disease modeling. Physiological degeneration and disease onset are common occurrences in the aging population. iPSCs could offer regenerative medical options for age-related degeneration and disease in the elderly. However, reprogramming somatic cells from the elderly is inefficient when successful at all. Perhaps due to their low rates of replication in culture, traditional transduction and reprogramming approaches with centenarian fibroblasts met with little success. A simple and reproducible reprogramming process is reported here which enhances interactions of the cells with the viral vectors that leads to improved iPSC generation. The improved methods efficiently generates fully reprogrammed iPSC lines from 105-107 years old subjects in feeder-free conditions using an episomal, Sendai-Virus (SeV) reprogramming vector expressing four reprogramming factors. In conclusion, dermal fibroblasts from human subjects older than 100 years can be efficiently and reproducibly reprogrammed to fully pluripotent cells with minor modifications to the standard reprogramming procedures. Efficient generation of iPSCs from the elderly may provide a source of cells for the regeneration of tissues and organs with autologous cells as well as cellular models for the study of aging, longevity and age-related diseases.


Assuntos
Técnicas de Reprogramação Celular/métodos , Reprogramação Celular , Fibroblastos/fisiologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Adulto , Fatores Etários , Idoso de 80 Anos ou mais , Células Cultivadas , Vetores Genéticos/genética , Humanos , Hidrodinâmica , Recém-Nascido , Cultura Primária de Células , Reprodutibilidade dos Testes , Vírus Sendai/genética , Pele/citologia , Envelhecimento da Pele/fisiologia , Transfecção/métodos , Transplante Autólogo/métodos
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