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1.
J Wildl Dis ; 58(3): 537-549, 2022 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-35704504

RESUMO

Growing populations of Wild Turkeys (Meleagris gallopavo) may result in increased disease transmission among wildlife and spillover to poultry. Lymphoproliferative disease virus (LPDV) is an avian retrovirus that is widespread in Wild Turkeys of eastern North America, and infections may influence mortality and parasite co-infections. We aimed to identify individual and spatial risk factors of LPDV in Maine's Wild Turkeys. We also surveyed for co-infections between LPDV and reticuloendotheliosis virus (REV), Mycoplasma gallisepticum, and Salmonella pullorum to estimate trends in prevalence and examine covariance with LPDV. From 2017 to 2020, we sampled tissues from hunter-harvested (n=72) and live-captured (n=627) Wild Turkeys, in spring and winter, respectively, for molecular detection of LPDV and REV. In a subset of captured individuals (n=235), we estimated seroprevalence of the bacteria M. gallisepticum and S. pullorum using a plate agglutination test. Infection rates for LPDV and REV were 59% and 16% respectively, with a co-infection rate of 10%. Seroprevalence for M. gallisepticum and S. pullorum were 74% and 3.4%, with LPDV co-infection rates of 51% and 2.6%, respectively. Infection with LPDV and seroprevalence of M. gallisepticum and S. pullorum decreased, whereas REV infection increased, between 2018 and 2020. Females (64%), adults (72%), and individuals sampled in spring (76%) had higher risks of LPDV infection than males (47%), juveniles (39%), and individuals sampled in winter (57%). Furthermore, LPDV infection increased with percent forested cover (ß=0.014±0.007) and decreased with percent agriculture cover for juveniles (ß=-0.061±0.018) sampled in winter. These data enhance our understanding of individual and spatial predictors of LPDV infection in Wild Turkeys and aid in assessing the associated risk to Wild Turkey populations and poultry operations.


Assuntos
Alpharetrovirus , Doenças das Aves , Coinfecção , Vírus da Reticuloendoteliose , Viroses , Animais , Animais Selvagens , Doenças das Aves/epidemiologia , Coinfecção/epidemiologia , Coinfecção/veterinária , Feminino , Masculino , Aves Domésticas , Estudos Soroepidemiológicos , Perus , Viroses/veterinária
2.
Front Immunol ; 12: 751138, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34804035

RESUMO

Immune cell therapeutics are increasingly applied in oncology. Especially chimeric antigen receptor (CAR) T cells are successfully used to treat several B cell malignancies. Efforts to engineer CAR T cells for improved activity against solid tumors include co-delivery of pro-inflammatory cytokines in addition to CARs, via either constitutive cytokine expression or inducible cytokine expression triggered by CAR recognition of its target antigen-so-called "T cells redirected for universal cytokine-mediated killing" (TRUCKs) or fourth-generation CARs. Here, we tested the hypothesis that TRUCK principles could be expanded to improve anticancer functions of NK cells. A comparison of the functionality of inducible promoters responsive to NFAT or NFκB in NK cells showed that, in contrast to T cells, the inclusion of NFκB-responsive elements within the inducible promoter construct was essential for CAR-inducible expression of the transgene. We demonstrated that GD2CAR-specific activation induced a tight NFκB-promoter-driven cytokine release in NK-92 and primary NK cells together with an enhanced cytotoxic capacity against GD2+ target cells, also shown by increased secretion of cytolytic cytokines. The data demonstrate biologically relevant differences between T and NK cells that are important when clinically translating the TRUCK concept to NK cells for the treatment of solid malignancies.


Assuntos
Imunoterapia Adotiva , Células Matadoras Naturais/imunologia , NF-kappa B/genética , Alpharetrovirus/genética , Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/terapia , Linhagem Celular , Movimento Celular , Técnicas de Cocultura , Citocinas/imunologia , Vetores Genéticos , Glioblastoma/imunologia , Glioblastoma/terapia , Humanos , NF-kappa B/imunologia , Fatores de Transcrição NFATC/genética , Fatores de Transcrição NFATC/imunologia
3.
Viruses ; 13(7)2021 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-34372571

RESUMO

Anti-cancer activity can be improved by engineering immune cells to express chimeric antigen receptors (CARs) that recognize tumor-associated antigens. Retroviral vector gene transfer strategies allow stable and durable transgene expression. Here, we used alpharetroviral vectors to modify NK-92 cells, a natural killer cell line, with a third-generation CAR designed to target the IL-3 receptor subunit alpha (CD123), which is strongly expressed on the surface of acute myeloid leukemia (AML) cells. Alpharetroviral vectors also contained a transgene cassette to allow constitutive expression of human IL-15 for increased NK cell persistence in vivo. The anti-AML activity of CAR-NK-92 cells was tested via in vitro cytotoxicity assays with the CD123+ AML cell line KG-1a and in vivo in a patient-derived xenotransplantation CD123+ AML model. Unmodified NK-92 cells or NK-92 cells modified with a truncated version of the CAR that lacked the signaling domain served as controls. Alpharetroviral vector-modified NK-92 cells stably expressed the transgenes and secreted IL-15. Anti-CD123-CAR-NK-92 cells exhibited enhanced anti-AML activity in vitro and in vivo as compared to control NK-92 cells. Our data (1) shows the importance of IL-15 expression for in vivo persistence of NK-92 cells, (2) supports continued investigation of anti-CD123-CAR-NK cells to target AML, and (3) points towards potential strategies to further improve CAR-NK anti-AML activity.


Assuntos
Imunoterapia Adotiva/métodos , Subunidade alfa de Receptor de Interleucina-3/imunologia , Leucemia Mieloide Aguda/tratamento farmacológico , Idoso , Alpharetrovirus/genética , Animais , Linhagem Celular Tumoral , Feminino , Terapia Genética , Vetores Genéticos/genética , Humanos , Subunidade alfa de Receptor de Interleucina-3/genética , Subunidade alfa de Receptor de Interleucina-3/metabolismo , Células Matadoras Naturais/imunologia , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/imunologia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Cultura Primária de Células , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/imunologia , Transdução Genética , Transgenes , Ensaios Antitumorais Modelo de Xenoenxerto
4.
Viruses ; 13(1)2021 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-33477490

RESUMO

The assembly of a hexameric lattice of retroviral immature particles requires the involvement of cell factors such as proteins and small molecules. A small, negatively charged polyanionic molecule, myo-inositol hexaphosphate (IP6), was identified to stimulate the assembly of immature particles of HIV-1 and other lentiviruses. Interestingly, cryo-electron tomography analysis of the immature particles of two lentiviruses, HIV-1 and equine infectious anemia virus (EIAV), revealed that the IP6 binding site is similar. Based on this amino acid conservation of the IP6 interacting site, it is presumed that the assembly of immature particles of all lentiviruses is stimulated by IP6. Although this specific region for IP6 binding may be unique for lentiviruses, it is plausible that other retroviral species also recruit some small polyanion to facilitate the assembly of their immature particles. To study whether the assembly of retroviruses other than lentiviruses can be stimulated by polyanionic molecules, we measured the effect of various polyanions on the assembly of immature virus-like particles of Rous sarcoma virus (RSV), a member of alpharetroviruses, Mason-Pfizer monkey virus (M-PMV) representative of betaretroviruses, and murine leukemia virus (MLV), a member of gammaretroviruses. RSV, M-PMV and MLV immature virus-like particles were assembled in vitro from truncated Gag molecules and the effect of selected polyanions, myo-inostol hexaphosphate, myo-inositol, glucose-1,6-bisphosphate, myo-inositol hexasulphate, and mellitic acid, on the particles assembly was quantified. Our results suggest that the assembly of immature particles of RSV and MLV was indeed stimulated by the presence of myo-inostol hexaphosphate and myo-inositol, respectively. In contrast, no effect on the assembly of M-PMV as a betaretrovirus member was observed.


Assuntos
Membrana Celular/química , Membrana Celular/metabolismo , Interações Hospedeiro-Patógeno , Polieletrólitos/química , Retroviridae/fisiologia , Montagem de Vírus , Alpharetrovirus/fisiologia , Animais , Betaretrovirus/fisiologia , Células Cultivadas , Gammaretrovirus/fisiologia , Produtos do Gene gag/química , Produtos do Gene gag/metabolismo , Polieletrólitos/metabolismo , Retroviridae/ultraestrutura , Vírion
5.
Kidney Int ; 97(3): 528-537, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31932071

RESUMO

Prior studies reported that haploinsufficiency of the transcription factor ETS-1 is renoprotective in Dahl salt-sensitive rats, but the mechanism is unclear. Here, we tested whether ETS-1 is involved in hypertension-induced renal microvascular pathology and autoregulatory impairment. Hypertension was induced in salt-sensitive rats and salt-sensitive rats that are heterozygous with 1 wild-type or reference allele of Ets1 (SSEts1+/-) by feeding a diet containing 4% sodium chloride for 1 week. Increases in blood pressure did not differ. However, phosphorylated ETS-1 increased in afferent arterioles of hypertensive salt-sensitive rats, but not in hypertensive SSEts1+/- rats. Afferent arterioles of hypertensive salt-sensitive rats showed increased monocyte chemotactic protein-1 expression and infiltration of CD68 positive monocytes/macrophages. Isolated kidney microvessels showed increased mRNA expression of vascular cell adhesion molecule, intercellular adhesion molecule, P-selectin, fibronectin, transforming growth factor-ß, and collagen I in hypertensive salt-sensitive rats compared with hypertensive SSEts1+/- rats. Using the in vitro blood-perfused juxtamedullary nephron preparation, pressure-mediated afferent arteriolar responses were significantly blunted in hypertensive salt-sensitive rats compared to hypertensive SSEts1+/- rats. Over a 65-170 mm Hg pressure range tested baseline arteriolar diameters averaged 15.1 µm and remained between 107% and 89% of baseline diameter in hypertensive salt-sensitive rats vs. 114% and 73% in hypertensive SSEts1+/- rats (significantly different). Thus, ETS-1 participates in renal arteriolar pathology and autoregulation and thereby is involved in hypertension-mediated kidney injury in salt-sensitive rats.


Assuntos
Alpharetrovirus , Hipertensão , Proteína Proto-Oncogênica c-ets-1/genética , Animais , Pressão Sanguínea , Hipertensão/genética , Rim , Oncogenes , Ratos , Ratos Endogâmicos Dahl
6.
Hum Gene Ther Methods ; 30(3): 102-120, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30997855

RESUMO

In cellular immunotherapies, natural killer (NK) cells often demonstrate potent antitumor effects in high-risk cancer patients. But Good Manufacturing Practice (GMP)-compliant manufacturing of clinical-grade NK cells in high numbers for patient treatment is still a challenge. Therefore, new protocols for isolation and expansion of NK cells are required. In order to attack resistant tumor entities, NK cell killing can be improved by genetic engineering using alpharetroviral vectors that encode for chimeric antigen receptors (CARs). The aim of this work was to demonstrate GMP-grade manufacturing of NK cells using the CliniMACS® Prodigy device (Prodigy) with implemented applicable quality controls. Additionally, the study aimed to define the best time point to transduce expanding NK cells with alpharetroviral CAR vectors. Manufacturing and clinical-scale expansion of primary human NK cells were performed with the Prodigy starting with 8-15.0 × 109 leukocytes (including 1.1-2.3 × 109 NK cells) collected by small-scale lymphapheresis (n = 3). Positive fraction after immunoselection, in-process controls (IPCs), and end product were quantified by flow cytometric no-wash, single-platform assessment, and gating strategy using positive (CD56/CD16/CD45), negative (CD14/CD19/CD3), and dead cell (7-aminoactinomycine [7-AAD]) discriminators. The three runs on the fully integrated manufacturing platform included immunomagnetic separation (CD3 depletion/CD56 enrichment) followed by NK cell expansion over 14 days. This process led to high NK cell purities (median 99.1%) and adequate NK cell viabilities (median 86.9%) and achieved a median CD3+ cell depletion of log -3.6 after CD3 depletion and log -3.7 after immunomagnetic CD3 depletion and consecutive CD56 selection. Subsequent cultivation of separated NK cells in the CentriCult® chamber of Prodigy resulted in approximately 4.2-8.5-fold NK cell expansion rates by adding of NK MACS® basal medium containing NK MACS® supplement, interleukin (IL)-2/IL-15 and initial IL-21. NK cells expanded for 14 days revealed higher expression of natural cytotoxicity receptors (NKp30, NKp44, NKp46, and NKG2D) and degranulation/apoptotic markers and stronger cytolytic properties against K562 compared to non-activated NK cells before automated cultivation. Moreover, expanded NK cells had robust growth and killing activities even after cryopreservation. As a crucial result, it was possible to determine the appropriate time period for optimal CAR transduction of cultivated NK cells between days 8 and 14, with the highest anti-CD123 CAR expression levels on day 14. The anti-CD123 CAR NK cells showed retargeted killing and degranulation properties against CD123-expressing KG1a target cells, while basal cytotoxicity of non-transduced NK cells was determined using the CD123-negative cell line K562. Time-lapse imaging to monitor redirected effector-to-target contacts between anti-CD123 CAR NK and KG1a showed long-term effector-target interaction. In conclusion, the integration of the clinical-scale expansion procedure in the automated and closed Prodigy system, including IPC samples and quality controls and optimal time frames for NK cell transduction with CAR vectors, was established on 48-well plates and resulted in a standardized GMP-compliant overall process.


Assuntos
Alpharetrovirus/genética , Engenharia Celular , Células Matadoras Naturais , Receptores de Antígenos Quiméricos/genética , Linhagem Celular , Sobrevivência Celular , Citocinas/metabolismo , Vetores Genéticos , Humanos , Controle de Qualidade , Transdução Genética
7.
Hum Gene Ther ; 30(4): 381-401, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30734584

RESUMO

The introduction of chimeric antigen receptors (CARs) to augment the anticancer activity of immune cells represents one of the major clinical advances in recent years. This work demonstrates that sorted CAR natural killer (NK) cells have improved antileukemia activity compared to control NK cells that lack a functional CAR. However, in terms of viability, effectiveness, risk of side effects, and clinical practicality and applicability, an important question is whether gene-modified NK cell lines represent better CAR effector cells than primary human donor CAR-NK (CAR-dNK) cells. Comparison of the functional activities of sorted CAR-NK cells generated using the NK-92 cell line with those generated from primary human dNK cells demonstrated that CAR-NK-92 cells had stronger cytotoxic activity against leukemia cells compared to CAR-dNK cells. CAR-NK-92 and CAR-dNK cells had similar CD107a surface expression upon co-incubation with leukemia cells. However, CAR-NK-92 cells secreted higher granzyme A and interleukin-17A levels, while CAR-dNK cells secreted more tumor necrosis factor alpha, interferon gamma, and granulysin. In addition, CAR-NK-92 cells revealed a significantly higher potential for adverse side effects against nonmalignant cells. In short, this work shows the feasibility for further development of CAR-NK strategies to treat leukemia.


Assuntos
Imunoterapia Adotiva , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Leucemia Mieloide Aguda/etiologia , Leucemia Mieloide Aguda/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos Quiméricos/metabolismo , Alpharetrovirus/genética , Animais , Biomarcadores , Biomarcadores Tumorais , Comunicação Celular/imunologia , Degranulação Celular/imunologia , Linhagem Celular Tumoral , Citocinas/genética , Citocinas/metabolismo , Citotoxicidade Imunológica , Modelos Animais de Doenças , Expressão Gênica , Vetores Genéticos/genética , Humanos , Imunofenotipagem , Imunoterapia Adotiva/métodos , Leucemia Mieloide Aguda/patologia , Leucemia Mieloide Aguda/terapia , Masculino , Camundongos , Pessoa de Meia-Idade , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos Quiméricos/genética , Especificidade do Receptor de Antígeno de Linfócitos T , Transgenes
8.
J Wildl Dis ; 55(1): 113-122, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30124393

RESUMO

The successful reintroduction of Wild Turkeys ( Meleagris gallopavo) to Ontario, Canada, has led to established populations in southern portions of the province and currently allows for biannual hunting seasons. These populations geographically overlap Domestic Turkey farms, an important sector of the provincial agri-food industry. Potential pathogen transmission between Wild Turkeys and Domestic Turkeys ( Meleagris gallopavo) is a concern, because they are susceptible to infection with many of the same pathogens and have direct and indirect contact in outdoor or open farm settings and contaminated environmental substrates. However, data concerning potential poultry pathogens in Wild Turkeys in Canada are scarce. Thus, we assessed the prevalence and geographic distribution of geographically relevant viruses in Ontario Wild Turkeys. Oropharyngeal and cloacal swabs were tested for avian influenza viruses (AIV) by real-time reverse transcriptase (RT)-PCR ( n=207), pooled tissues for lymphoproliferative disease virus (LPDV; n=183) and reticuloendotheliosis virus ( n=119) by PCR, and gross skin lesions by real-time RT-PCR for avian poxvirus ( n=8). We sequenced a fragment of the gag polyprotein (p31) gene of LPDV on a subset ( n=10) of LPDV-positive samples for phylogenetic analysis and tested additional upland game bird species ( n=39) and domestic fowl for LPDV ( n=17). To the best of our knowledge, we document the first detection of LPDV in Wild Turkeys in Canada, with a prevalence of 65% (119/183). Phylogenetic analysis revealed that LPDV sequences from Ontario were genetically similar to other North American strains and did not group into separate clades. Reticuloendotheliosis virus was detected in 4% (5/119) of LPDV-positive Wild Turkeys. Grossly evident skin lesions from five Wild Turkeys tested positive for poxvirus, and all turkeys tested negative for AIV. This study provides evidence of LPDV circulation in Canada and provides a baseline for comparison with future Wild Turkey pathogen surveillance and monitoring in Ontario and elsewhere.


Assuntos
Alpharetrovirus/isolamento & purificação , Doenças das Aves/virologia , Infecções por Retroviridae/veterinária , Infecções Tumorais por Vírus/veterinária , Perus/virologia , Envelhecimento , Animais , Animais Selvagens , Doenças das Aves/epidemiologia , Ontário/epidemiologia , Vigilância da População , Vírus da Reticuloendoteliose Aviária , Infecções por Retroviridae/epidemiologia , Infecções por Retroviridae/virologia , Infecções Tumorais por Vírus/epidemiologia , Infecções Tumorais por Vírus/virologia
9.
Avian Dis ; 63(3): 506-510, 2019 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-31967435

RESUMO

This study describes the first recognized clinical case of lymphoproliferative disease virus (LPDV) in Canada and extends the range of LPDV in Canada through its detection in Manitoba and Quebec. We assessed the prevalence of LPDV in eastern wild turkeys (Meleagris gallopavo silvestris) with the use of whole, clotted blood from live birds in Manitoba (n = 65) and tissue samples collected postmortem in Quebec (n = 4). We tested for LPDV proviral DNA through PCR amplification and sequencing of a portion of the gag (p31) gene. Samples were also tested for reticuloendotheliosis virus (REV) by PCR. Twenty-four birds (34.8%) were positive for LPDV, including all diagnostic cases. One bird (1.4%) from Quebec had gross and microscopic lesions consistent with LPDV. Two turkeys (2.9%) were REV positive, one (1.4%) of which was co-infected with LPDV. Phylogenetic analysis of LPDV strains from Quebec and Manitoba grouped with previously sequenced samples from Ontario and publicly available sequences from a North American lineage. This study contributes valuable information toward ongoing surveillance and monitoring of LPDV in North America.


Virus de la enfermedad linfoproliferativa en pavos silvestres (Meleagris gallopavo) de Manitoba y Quebec, en Canadá. Este estudio describe el primer caso clínico reconocido del virus de la enfermedad linfoproliferativa (LPDV) en Canadá y extiende el rango de detección de este virus a través de su detección en Manitoba y Quebec. Se evaluó la prevalencia del virus de la enfermedad linfoproliferativa en pavos silvestres (Meleagris gallopavo silvestris) de la parte oriental, mediante el uso de sangre coagulada de aves vivas en Manitoba (n = 65) y de muestras de tejidos recolectadas postmortem en Quebec (n = 4). Se analizó el ADN proviral del virus de la enfermedad linfoproliferativa del pavo a través de la amplificación por PCR y la secuenciación de una parte del gene gag (p31). Las muestras también se analizaron para detectar el virus de la reticuloendoteliosis (REV) mediante PCR. Veinticuatro aves (34.8%) resultaron positivas para la presencia del virus de la enfermedad linfoproliferativa, incluyendo todos los casos diagnósticos. Un ave (1.4%) de Quebec tenía lesiones macroscópicas y microscópicas compatibles con este virus. Dos pavos (2.9%) fueron positivos a la presencia del virus de la reticuloendoteliosis, uno (1.4%) de los cuales se co-infectó con el virus de la enfermedad linfoproliferativa. El análisis filogenético de cepas del virus de la enfermedad linfoproliferativa de Quebec y Manitoba agrupó a estos virus con muestras previamente secuenciadas de Ontario y secuencias disponibles públicamente de un linaje de América del Norte. Este estudio aporta información valiosa para la vigilancia y el monitoreo continuos del virus de la enfermedad linfoproliferativa en América del Norte.


Assuntos
Alpharetrovirus/isolamento & purificação , Doenças das Aves/epidemiologia , Infecções por Retroviridae/veterinária , Infecções Tumorais por Vírus/veterinária , Perus , Animais , Doenças das Aves/virologia , Manitoba/epidemiologia , Prevalência , Quebeque/epidemiologia , Infecções por Retroviridae/epidemiologia , Infecções por Retroviridae/virologia , Infecções Tumorais por Vírus/epidemiologia , Infecções Tumorais por Vírus/virologia
10.
Front Immunol ; 10: 3123, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-32117200

RESUMO

Autologous chimeric antigen receptor-modified (CAR) T cells with specificity for CD19 showed potent antitumor efficacy in clinical trials against relapsed and refractory B-cell acute lymphoblastic leukemia (B-ALL). Contrary to T cells, natural killer (NK) cells kill their targets in a non-antigen-specific manner and do not carry the risk of inducing graft vs. host disease (GvHD), allowing application of donor-derived cells in an allogenic setting. Hence, unlike autologous CAR-T cells, therapeutic CD19-CAR-NK cells can be generated as an off-the-shelf product from healthy donors. Nevertheless, genetic engineering of peripheral blood (PB) derived NK cells remains challenging and optimized protocols are needed. In our study, we aimed to optimize the generation of CD19-CAR-NK cells by retroviral transduction to improve the high antileukemic capacity of NK cells. We compared two different retroviral vector platforms, the lentiviral and alpharetroviral, both in combination with two different transduction enhancers (Retronectin and Vectofusin-1). We further explored different NK cell isolation techniques (NK cell enrichment and CD3/CD19 depletion) to identify the most efficacious methods for genetic engineering of NK cells. Our results demonstrated that transduction of NK cells with RD114-TR pseudotyped retroviral vectors, in combination with Vectofusin-1 was the most efficient method to generate CD19-CAR-NK cells. Retronectin was potent in enhancing lentiviral/VSV-G gene delivery to NK cells but not alpharetroviral/RD114-TR. Furthermore, the Vectofusin-based transduction of NK cells with CD19-CARs delivered by alpharetroviral/RD114-TR and lentiviral/RD114-TR vectors outperformed lentiviral/VSV-G vectors. The final generated CD19-CAR-NK cells displayed superior cytotoxic activity against CD19-expressing target cells when compared to non-transduced NK cells achieving up to 90% specific killing activity. In summary, our findings present the use of RD114-TR pseudotyped retroviral particles in combination with Vectofusin-1 as a successful strategy to genetically modify PB-derived NK cells to achieve highly cytotoxic CD19-CAR-NK cells at high yield.


Assuntos
Alpharetrovirus/genética , Imunoterapia Adotiva/métodos , Células Matadoras Naturais/fisiologia , Lentivirus/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/imunologia , Receptores de Antígenos de Linfócitos T/genética , Antígenos CD19/imunologia , Antígenos CD19/metabolismo , Linhagem Celular Tumoral , Citotoxicidade Imunológica , Engenharia Genética , Vetores Genéticos , Humanos , Células Matadoras Naturais/transplante , Peptídeos/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Transdução Genética
11.
Cancer Gene Ther ; 26(3-4): 94-102, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30190513

RESUMO

We reported that inactivation of menin (the protein product of MEN1) increases activity of Dnmt1 and mediates DNA hypermethylation in the development of multiple endocrine neoplasia type 1 (MEN1) syndrome. We have developed a RCAS-TVA-based somatic gene transfer system that enables tissue-specific delivery of Dnmt1 to individual ß-cells of the pancreas in a RIP-TVA mouse model. In the present study, we mediated Dnmt1 expression in islet ß-cells in RIP-TVA mice by utilizing the RCAS-TVA system to test if the upregulation of Dnmt1 can promote ß-cell proliferation. In vitro, we demonstrated that upregulation of Dnmt1 increased ß-cell proliferation. In vivo, our results showed that the levels of serum insulin were increased in the RIP-TVA mice with RCASBP-Dnmt1 infection compared with wild-type control mice with RCASBP-Dnmt1 infection. Furthermore, we confirmed that mRNA and protein expression of Dnmt1 as well as Dnmt1 enzyme activity were upregulated in the RIP-TVA mice with RCASBP-Dnmt1 infection compared with wild-type control mice with RCASBP-Dnmt1 infection. Finally, we demonstrated that upregulation of Dnmt1 resulted in hyperplasia through ß-cell proliferation. We conclude that the upregulation of Dnmt1 promotes islet ß-cell proliferation and targeting Dnmt1 may be a promising therapy for patients suffering from pancreatic neuroendocrine tumors.


Assuntos
DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Ilhotas Pancreáticas/patologia , Neoplasia Endócrina Múltipla Tipo 1/patologia , Neoplasias Pancreáticas/patologia , Alpharetrovirus/genética , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Proliferação de Células/genética , Galinhas , DNA (Citosina-5-)-Metiltransferase 1/antagonistas & inibidores , DNA (Citosina-5-)-Metiltransferase 1/genética , Modelos Animais de Doenças , Fibroblastos , Técnicas de Transferência de Genes , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Humanos , Ilhotas Pancreáticas/metabolismo , Camundongos , Camundongos Transgênicos , Terapia de Alvo Molecular/métodos , Neoplasia Endócrina Múltipla Tipo 1/tratamento farmacológico , Neoplasia Endócrina Múltipla Tipo 1/genética , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Proteínas Proto-Oncogênicas/genética , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética
12.
Viruses ; 10(3)2018 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-29517993

RESUMO

Individual groups of retroviruses and retroviral vectors differ in their integration site preference and interaction with the host genome. Hence, immediately after infection genome-wide distribution of integrated proviruses is non-random. During long-term in vitro or persistent in vivo infection, the genomic position and chromatin environment of the provirus affects its transcriptional activity. Thus, a selection of long-term stably expressed proviruses and elimination of proviruses, which have been gradually silenced by epigenetic mechanisms, helps in the identification of genomic compartments permissive for proviral transcription. We compare here the extent and time course of provirus silencing in single cell clones of the K562 human myeloid lymphoblastoma cell line that have been infected with retroviral reporter vectors derived from avian sarcoma/leukosis virus (ASLV), human immunodeficiency virus type 1 (HIV) and murine leukaemia virus (MLV). While MLV proviruses remain transcriptionally active, ASLV proviruses are prone to rapid silencing. The HIV provirus displays gradual silencing only after an extended time period in culture. The analysis of integration sites of long-term stably expressed proviruses shows a strong bias for some genomic features-especially integration close to the transcription start sites of active transcription units. Furthermore, complex analysis of histone modifications enriched at the site of integration points to the accumulation of proviruses of all three groups in gene regulatory segments, particularly close to the enhancer loci. We conclude that the proximity to active regulatory chromatin segments correlates with stable provirus expression in various retroviral species.


Assuntos
Alpharetrovirus/genética , Cromatina/genética , Vetores Genéticos/genética , HIV-1/genética , Vírus da Leucemia Murina/genética , Provírus/genética , Sequências Reguladoras de Ácido Nucleico , Ativação Transcricional , Animais , Linhagem Celular , Elementos Facilitadores Genéticos , Epigênese Genética , Regulação Viral da Expressão Gênica , Inativação Gênica , Marcação de Genes , Humanos , Camundongos , Plasmídeos/genética , Estabilidade de RNA , Sítio de Iniciação de Transcrição , Integração Viral
13.
Curr Protoc Mol Biol ; 121: 23.17.1-23.17.7, 2018 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-29337371

RESUMO

The RCAS (replication-competent avian sarcoma leukosis virus long-terminal repeat with splice acceptor)-TVA (tumor virus A) gene delivery system has been successfully used in modeling human cancers. Based on this, we have recently developed a novel RCI-Oncogene (RCAS-Cre-IRES-Oncogene) gene delivery system that can be used to efficiently manipulate gene expression in spontaneous tumors in vivo. We used this system for tumor gene knockout (TuKO) and demonstrated a crucial role of FGFR1 in driving mammary tumor metastasis. This versatile tumor gene modification system can also be adapted into different configurations to address different questions in appropriate mutant mouse hosts. Here we describe a protocol using the TuKO approach to knock out a gene of interest in tumors in appropriate hosts. © 2018 by John Wiley & Sons, Inc.


Assuntos
Alpharetrovirus/genética , Técnicas de Inativação de Genes/métodos , Técnicas de Transferência de Genes , Neoplasias Mamárias Animais/genética , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Animais , Feminino , Humanos , Camundongos , Oncogenes
14.
Nucleic Acids Res ; 45(22): 12752-12765, 2017 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-29244184

RESUMO

Most retroviruses preferentially integrate into certain genomic locations and, as a result, their genome-wide integration patterns are non-random. We investigate the epigenetic landscape of integrated retroviral vectors and correlate it with the long-term stability of proviral transcription. Retroviral vectors derived from the avian sarcoma/leukosis virus expressing the GFP reporter were used to transduce the human myeloid lymphoblastoma cell line K562. Because of efficient silencing of avian retrovirus in mammalian cells, only ∼3% of established clones displayed stable proviral expression. We analyzed the vector integration sites in non-selected cells and in clones selected for the GFP expression. This selection led to overrepresentation of proviruses integrated in active transcription units, with particular accumulation in promoter-proximal areas. In parallel, we investigated the integration of vectors equipped with an anti-silencing CpG island core sequence. Such modification increased the frequency of stably expressing proviruses by one order. The modified vectors are also overrepresented in active transcription units, but stably expressed in distal parts of transcriptional units further away from promoters with marked accumulation in enhancers. These results suggest that integrated retroviruses subject to gradual epigenetic silencing during long-term cultivation. Among most genomic compartments, however, active promoters and enhancers protect the adjacent retroviruses from transcriptional silencing.


Assuntos
Alpharetrovirus/genética , Elementos Facilitadores Genéticos/genética , Regiões Promotoras Genéticas/genética , Transcrição Genética , Animais , Linhagem Celular , Ilhas de CpG/genética , Epigênese Genética , Inativação Gênica , Vetores Genéticos/genética , Humanos , Células K562 , Provírus/genética , Integração Viral/genética
15.
Biomaterials ; 97: 97-109, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27162078

RESUMO

Primary human T lymphocytes represent an important cell population for adoptive immunotherapies, including chimeric-antigen and T-cell receptor applications, as they have the capability to eliminate non-self, virus-infected and tumor cells. Given the increasing numbers of clinical immunotherapy applications, the development of an optimal vector platform for genetic T lymphocyte engineering, which allows cost-effective high-quality vector productions, remains a critical goal. Alpharetroviral self-inactivating vectors (ARV) have several advantages compared to other vector platforms, including a more random genomic integration pattern and reduced likelihood for inducing aberrant splicing of integrated proviruses. We developed an ARV platform for the transduction of primary human T lymphocytes. We demonstrated functional transgene transfer using the clinically relevant herpes-simplex-virus thymidine kinase variant TK.007. Proof-of-concept of alpharetroviral-mediated T-lymphocyte engineering was shown in vitro and in a humanized transplantation model in vivo. Furthermore, we established a stable, human alpharetroviral packaging cell line in which we deleted the entry receptor (SLC1A5) for RD114/TR-pseudotyped ARVs to prevent superinfection and enhance genomic integrity of the packaging cell line and viral particles. We showed that superinfection can be entirely prevented, while maintaining high recombinant virus titers. Taken together, this resulted in an improved production platform representing an economic strategy for translating the promising features of ARVs for therapeutic T-lymphocyte engineering.


Assuntos
Alpharetrovirus/metabolismo , Técnicas Genéticas , Vetores Genéticos/metabolismo , Linfócitos T/metabolismo , Montagem de Vírus , Sequência de Bases , Sistemas CRISPR-Cas/genética , Células Clonais , Genes Reporter , Células HEK293 , Humanos , Células Jurkat , Reprodutibilidade dos Testes , Linfócitos T/imunologia , Transdução Genética , Transgenes
16.
Mol Ther ; 24(7): 1216-26, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27138041

RESUMO

Retroviral engineering of hematopoietic stem cell-derived precursor T-cells (preTs) opens the possibility of targeted T-cell transfer across human leukocyte antigen (HLA)-barriers. Alpharetroviral vectors exhibit a more neutral integration pattern thereby reducing the risk of insertional mutagenesis. Cord blood-derived CD34+ cells were transduced and differentiated into preTs in vitro. Two promoters, elongation-factor-1-short-form, and a myeloproliferative sarcoma virus variant in combination with two commonly used envelopes were comparatively assessed choosing enhanced green fluorescent protein or a third-generation chimeric antigen receptor (CAR) against CD123 as gene of interest. Furthermore, the inducible suicide gene iCaspase 9 has been validated. Combining the sarcoma virus-derived promoter with a modified feline endogenous retrovirus envelope glycoprotein yielded in superior transgene expression and transduction rates. Fresh and previously frozen CD34+ cells showed similar transduction and expansion rates. Transgene-positive cells did neither show proliferative impairment nor alteration in their lymphoid differentiation profile. The sarcoma virus-derived promoter only could express sufficient levels of iCaspase 9 to mediate dimerizer-induced apoptosis. Finally, the CD123 CAR was efficiently expressed in CD34+ cells and proved to be functional when expressed on differentiated T-cells. Therefore, the transduction of CD34+ cells with alpharetroviral vectors represents a feasible and potentially safer approach for stem cell-based immunotherapies for cancer.


Assuntos
Alpharetrovirus/genética , Sangue Fetal/citologia , Engenharia Genética , Vetores Genéticos/genética , Células Precursoras de Linfócitos T/citologia , Células Precursoras de Linfócitos T/metabolismo , Antígenos CD34/metabolismo , Apoptose , Proteínas da Membrana Bacteriana Externa , Biomarcadores , Diferenciação Celular , Expressão Gênica , Técnicas de Transferência de Genes , Genes Reporter , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Subunidade alfa de Receptor de Interleucina-3/imunologia , Fenótipo , Regiões Promotoras Genéticas , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/citologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Transdução Genética , Transgenes
17.
J Mol Med (Berl) ; 94(1): 83-93, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26300042

RESUMO

UNLABELLED: Natural killer (NK) cells play an important role in tumor immunotherapy with their unique capability of killing transformed cells without the need for prior sensitization and without major histocompatibility complex (MHC)/peptide restriction. However, tumor cells can escape NK cell cytotoxicity by various tumor immune escape mechanisms. To overcome these escape mechanisms, NK cells can be modified to express chimeric antigen receptors (CARs), enhancing their tumor-specific cytotoxicity. To determine the most efficacious method to modify human NK cells, we compared different retroviral vector systems, retroviral pseudotypes, and transduction protocols. Using optimized transduction conditions, the highest transduction levels (up to 60%) were achieved with alpharetroviral vectors. Alpharetroviral-modified primary human NK cells exhibited no alteration in receptor expression and had similar degranulation activity as untransduced NK cells, thus demonstrating that alpharetroviral modification did not negatively affect NK cell cytotoxicity. Transduction of NK cells with an alpharetroviral vector containing a CD19 CAR expression cassette selectively enhanced NK cell cytotoxicity towards CD19-expressing leukemia cells, achieving nearly complete elimination of leukemia cells after 48 h. Taken together, alpharetroviral vectors are promising tools for NK cell-mediated cancer immunotherapy applications. KEY MESSAGES: Efficient modification of human NK cells using alpharetroviral vectors. Anti-CD19-CAR-NK cells exhibited improved cytotoxicity towards CD19(+) leukemia cells. Alpharetroviral vectors are promising tools for immunotherapy applications using NK cells.


Assuntos
Alpharetrovirus/genética , Antígenos CD19/genética , Citotoxicidade Imunológica/genética , Vetores Genéticos/genética , Células Matadoras Naturais/imunologia , Leucemia/terapia , Receptores de Antígenos/genética , Linhagem Celular Tumoral , Citotoxicidade Imunológica/imunologia , Terapia Genética/métodos , Proteínas de Fluorescência Verde/genética , Humanos , Imunoterapia Adotiva/métodos , Células Matadoras Naturais/citologia , Leucemia/imunologia , Receptores de Antígenos/biossíntese , Receptores de Antígenos/imunologia , Transdução Genética/métodos , Evasão Tumoral/imunologia
18.
Genet Mol Res ; 14(4): 14379-86, 2015 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-26600497

RESUMO

Endogenous retroviruses are regarded as ideal genetic markers for evolutionary analyses. Birds were some of the initial vertebrates found to contain endogenous retroviruses. However, few studies have investigated the presence and distribution of endogenous retroviruses in goose. In this study, we detected the avian sarcoma and leukosis virus gag gene in the genomic DNA of 8 Chinese native breeds using polymerase chain reaction method. The results indicated that a 1.2-kb avian sarcoma and leukosis virus gag sequence was integrated into all 8 goose breeds. The mean genetic pairwise distance was 0.918% among the investigated geese. To the best of our knowledge, this is the first report demonstrating the presence of the endogenous retroviruses in the domestic goose genome. The genetic structure should be further examined in the domestic goose.


Assuntos
Alpharetrovirus/genética , Anseriformes/genética , Evolução Molecular , Produtos do Gene gag/genética , Animais , Anseriformes/virologia , Cruzamento , DNA Mitocondrial/genética , Produtos do Gene gag/isolamento & purificação , Genoma
19.
BMC Mol Biol ; 16: 20, 2015 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-26608344

RESUMO

BACKGROUND: Gene expression is an inherently stochastic process, owing to its dynamic molecular nature. Protein amount distributions, which can be acquired by cytometry using a reporter gene, can inform about the mechanisms of the underlying microscopic molecular system. RESULTS: By using different clones of chicken erythroid progenitor cells harboring different integration sites of a CMV-driven mCherry protein, we investigated the dynamical behavior of such distributions. We show that, on short term, clone distributions can be quickly regenerated from small population samples with a high accuracy. On longer term, on the contrary, we show variations manifested by correlated fluctuation in the Mean Fluorescence Intensity. In search for a possible cause of this correlation, we demonstrate that in response to small temperature variations cells are able to adjust their gene expression rate: a modest (2 °C) increase in external temperature induces a significant down regulation of mean expression values, with a reverse effect observed when the temperature is decreased. Using a two-state model of gene expression we further demonstrate that temperature acts by modifying the size of transcription bursts, while the burst frequency of the investigated promoter is less systematically affected. CONCLUSIONS: For the first time, we report that transcription burst size is a key parameter for gene expression that metazoan cells from homeotherm animals can modify in response to an external thermal stimulus.


Assuntos
Eritroblastos/metabolismo , Células Precursoras Eritroides/metabolismo , Regulação da Expressão Gênica/fisiologia , Expressão Gênica/genética , Temperatura , Alpharetrovirus/genética , Animais , Linhagem Celular Transformada , Galinhas , Citometria de Fluxo , Fluorescência , Regulação da Expressão Gênica/genética , Genes Reporter/genética , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , Processos Estocásticos , Transcrição Genética/genética
20.
PLoS One ; 10(4): e0122644, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25897755

RESUMO

Lymphoproliferative disease virus (LPDV) is a poorly understood, oncogenic avian retrovirus of domestic turkeys that has historically been restricted to Europe and Israel. However, a recent study reported LPDV in multiple wild turkey diagnostic cases from throughout the eastern United States of America (USA). To better understand the distribution of LPDV in the eastern USA, we surveyed 1,164 reportedly asymptomatic hunter-harvested wild turkeys from 17 states for the presence of LPDV proviral DNA by PCR. In total, 564/1,164 (47%) turkeys were positive for LPDV. Wild turkeys from each state had a relatively high prevalence of LPDV, although statewide prevalence varied from 26 to 83%. Phylogenetic analysis revealed two major clades of LPDV in the USA, although one was at a low frequency suggesting restricted transmission, as well as significant clustering by state of isolation. To determine the best tissue to target for diagnostic purposes, liver, spleen, and bone marrow were tested from a subset of 15 hunter-harvested wild turkeys and 20 wild turkey diagnostic cases. Overall, bone marrow provided the highest level of detection for both hunter-harvested turkeys and diagnostic cases. The sensitivity of LPDV detection between tissues was not significantly different for diagnostic cases, but was for hunter-harvested birds. These results indicate that LPDV infection is common and widespread in wild turkey populations throughout the eastern USA, even without overt signs of disease.


Assuntos
Alpharetrovirus/genética , Doenças das Aves/virologia , Transtornos Linfoproliferativos/veterinária , Provírus/genética , Infecções por Retroviridae/veterinária , Perus/virologia , Animais , Doenças das Aves/epidemiologia , Monitoramento Epidemiológico , Feminino , Genes Virais , Transtornos Linfoproliferativos/epidemiologia , Transtornos Linfoproliferativos/virologia , Masculino , Dados de Sequência Molecular , Filogenia , Prevalência , Infecções por Retroviridae/epidemiologia , Infecções por Retroviridae/virologia , Análise de Sequência de DNA , Estados Unidos
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