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1.
Eur J Immunol ; 51(6): 1412-1422, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33576494

RESUMO

Heterologous polyclonal antibodies might represent an alternative to the use of convalescent plasma or monoclonal antibodies (mAbs) in coronavirus disease (COVID-19) by targeting multiple antigen epitopes. However, heterologous antibodies trigger human natural xenogeneic antibody responses particularly directed against animal-type carbohydrates, mainly the N-glycolyl form of the neuraminic acid (Neu5Gc) and the α1,3-galactose, potentially leading to serum sickness or allergy. Here, we immunized cytidine monophosphate-N-acetylneuraminic acid hydroxylase and α1,3-galactosyl-transferase (GGTA1) double KO pigs with the Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike receptor binding domain to produce glyco-humanized polyclonal neutralizing antibodies lacking Neu5Gc and α1,3-galactose epitopes. Animals rapidly developed a hyperimmune response with anti-SARS-CoV-2 end-titers binding dilutions over one to a million and end-titers neutralizing dilutions of 1:10 000. The IgG fraction purified and formulated following clinical Good Manufacturing Practices, named XAV-19, neutralized spike/angiotensin converting enzyme-2 interaction at a concentration <1 µg/mL, and inhibited infection of human cells by SARS-CoV-2 in cytopathic assays. We also found that pig GH-pAb Fc domains fail to interact with human Fc receptors, thereby avoiding macrophage-dependent exacerbated inflammatory responses and a possible antibody-dependent enhancement. These data and the accumulating safety advantages of using GH-pAbs in humans warrant clinical assessment of XAV-19 against COVID-19.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , COVID-19/imunologia , COVID-19/terapia , SARS-CoV-2/imunologia , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/imunologia , Anticorpos Neutralizantes/genética , Anticorpos Neutralizantes/farmacologia , Anticorpos Antivirais/genética , Anticorpos Antivirais/farmacologia , COVID-19/genética , Galactosiltransferases/deficiência , Galactosiltransferases/imunologia , Células HEK293 , Humanos , Imunização Passiva , SARS-CoV-2/genética , Ácidos Siálicos/genética , Ácidos Siálicos/imunologia , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Suínos , Soroterapia para COVID-19
2.
New Phytol ; 226(1): 126-141, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31580482

RESUMO

As Arctic soils warm, thawed permafrost releases nitrogen (N) that could stimulate plant productivity and thus offset soil carbon losses from tundra ecosystems. Although mycorrhizal fungi could facilitate plant access to permafrost-derived N, their exploration capacity beyond host plant root systems into deep, cold active layer soils adjacent to the permafrost table is unknown. We characterized root-associated fungi (RAF) that colonized ericoid (ERM) and ectomycorrhizal (ECM) shrub roots and occurred below the maximum rooting depth in permafrost thaw-front soil in tussock and shrub tundra communities. We explored the relationships between root and thaw front fungal composition and plant uptake of a 15 N tracer applied at the permafrost boundary. We show that ERM and ECM shrubs associate with RAF at the thaw front providing evidence for potential mycelial connectivity between roots and the permafrost boundary. Among shrubs and tundra communities, RAF connectivity to the thaw boundary was ubiquitous. The occurrence of particular RAF in both roots and thaw front soil was positively correlated with 15 N recovered in shrub biomass Taxon-specific RAF associations could be a mechanism for the vertical redistribution of deep, permafrost-derived nutrients, which may alleviate N limitation and stimulate productivity in warming tundra.


Assuntos
Pergelissolo , Tundra , Regiões Árticas , Ecossistema , Nitrogênio/metabolismo , Solo
3.
Neurobiol Dis ; 124: 263-275, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30471417

RESUMO

Amyotrophic Lateral Sclerosis (ALS) is a neural disorder gradually leading to paralysis of the whole body. Alterations in superoxide dismutase SOD1 gene have been linked with several variants of familial ALS. Here, we investigated a transgenic (Tg) cloned swine model expressing the human pathological hSOD1G93A allele. As in patients, these Tg pigs transmitted the disease to the progeny with an autosomal dominant trait and showed ALS onset from about 27 months of age. Post mortem analysis revealed motor neuron (MN) degeneration, gliosis and hSOD1 protein aggregates in brainstem and spinal cord. Severe skeletal muscle pathology including necrosis and inflammation was observed at the end stage, as well. Remarkably, as in human patients, these Tg pigs showed a quite long presymptomatic phase in which gradually increasing amounts of TDP-43 were detected in peripheral blood mononuclear cells. Thus, this transgenic swine model opens the unique opportunity to investigate ALS biomarkers even before disease onset other than testing novel drugs and possible medical devices.


Assuntos
Esclerose Lateral Amiotrófica/patologia , Neurônios Motores/patologia , Doenças Musculares/genética , Degeneração Neural/genética , Superóxido Dismutase-1/genética , Proteinopatias TDP-43/genética , Esclerose Lateral Amiotrófica/genética , Animais , Animais Geneticamente Modificados , Modelos Animais de Doenças , Humanos , Doenças Musculares/patologia , Degeneração Neural/patologia , Suínos , Proteinopatias TDP-43/patologia
4.
Xenotransplantation ; 26(5): e12524, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31115108

RESUMO

Two well-characterized carbohydrate epitopes are absent in humans but present in other mammals. These are galactose-α1,3-galactose (αGal) and N-glycolylneuraminic acid (Neu5Gc) which are introduced by the activities of two enzymes including α(1,3) galactosyltransferase (encoded by the GGTA1 gene) and CMP-Neu5Gc hydroxylase (encoded by the CMAH gene) that are inactive in humans but present in cattle. Hence, bovine-derived products are antigenic in humans who receive bioprosthetic heart valves (BHVs) or those that suffer from red meat syndrome. Using programmable nucleases, we disrupted (knockout, KO) GGTA1 and CMAH genes encoding for the enzymes that catalyse the synthesis of αGal and Neu5Gc, respectively, in both male and female bovine fibroblasts. The KO in clonally selected fibroblasts was detected by polymerase chain reaction (PCR) and confirmed by Sanger sequencing. Selected fibroblasts colonies were used for somatic cell nuclear transfer (SCNT) to produce cloned embryos that were implanted in surrogate recipient heifers. Fifty-three embryos were implanted in 33 recipients heifers; 3 pregnancies were carried to term and delivered 3 live calves. Primary cell cultures were established from the 3 calves and following molecular analyses confirmed the genetic deletions. FACS analysis showed the double-KO phenotype for both antigens confirming the mutated genotypes. Availability of such cattle double-KO model lacking both αGal and Neu5Gc offers a unique opportunity to study the functionality of BHV manufactured with tissues of potentially lower immunogenicity, as well as a possible new clinical approaches to help patients with red meat allergy syndrome due to the presence of these xenoantigens in the diet.


Assuntos
Animais Geneticamente Modificados , Antígenos Heterófilos/metabolismo , Monofosfato de Citidina/análogos & derivados , Galactose/metabolismo , Galactosiltransferases/genética , Técnicas de Inativação de Genes , Oxigenases de Função Mista/genética , Ácidos Neuramínicos/metabolismo , Animais , Antígenos Heterófilos/imunologia , Bioprótese , Bovinos , Monofosfato de Citidina/imunologia , Monofosfato de Citidina/metabolismo , Feminino , Fibroblastos/imunologia , Hipersensibilidade Alimentar/imunologia , Galactose/imunologia , Galactosiltransferases/deficiência , Próteses Valvulares Cardíacas , Humanos , Masculino , Oxigenases de Função Mista/deficiência , Ácidos Neuramínicos/imunologia , Transplante Heterólogo
5.
PLoS Genet ; 11(2): e1004951, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25659124

RESUMO

Many recurrent chromosome translocations in cancer result in the generation of fusion genes that are directly implicated in the tumorigenic process. Precise modeling of the effects of cancer fusion genes in mice has been inaccurate, as constructs of fusion genes often completely or partially lack the correct regulatory sequences. The reciprocal t(2;13)(q36.1;q14.1) in human alveolar rhabdomyosarcoma (A-RMS) creates a pathognomonic PAX3-FOXO1 fusion gene. In vivo mimicking of this translocation in mice is complicated by the fact that Pax3 and Foxo1 are in opposite orientation on their respective chromosomes, precluding formation of a functional Pax3-Foxo1 fusion via a simple translocation. To circumvent this problem, we irreversibly inverted the orientation of a 4.9 Mb syntenic fragment on chromosome 3, encompassing Foxo1, by using Cre-mediated recombination of two pairs of unrelated oppositely oriented LoxP sites situated at the borders of the syntenic region. We tested if spatial proximity of the Pax3 and Foxo1 loci in myoblasts of mice homozygous for the inversion facilitated Pax3-Foxo1 fusion gene formation upon induction of targeted CRISPR-Cas9 nuclease-induced DNA double strand breaks in Pax3 and Foxo1. Fluorescent in situ hybridization indicated that fore limb myoblasts show a higher frequency of Pax3/Foxo1 co-localization than hind limb myoblasts. Indeed, more fusion genes were generated in fore limb myoblasts via a reciprocal t(1;3), which expressed correctly spliced Pax3-Foxo1 mRNA encoding Pax3-Foxo1 fusion protein. We conclude that locus proximity facilitates chromosome translocation upon induction of DNA double strand breaks. Given that the Pax3-Foxo1 fusion gene will contain all the regulatory sequences necessary for precise regulation of its expression, we propose that CRISPR-Cas9 provides a novel means to faithfully model human diseases caused by chromosome translocation in mice.


Assuntos
Proteínas de Fusão Oncogênica/genética , Fatores de Transcrição Box Pareados/genética , Rabdomiossarcoma Alveolar/genética , Translocação Genética/genética , Animais , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Humanos , Hibridização in Situ Fluorescente , Camundongos , Mioblastos/metabolismo , Mioblastos/patologia , RNA Mensageiro/biossíntese , Rabdomiossarcoma Alveolar/metabolismo , Rabdomiossarcoma Alveolar/patologia
6.
Xenotransplantation ; 21(5): 431-43, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25040113

RESUMO

BACKGROUND: Human corneal allografting is an established procedure to cure corneal blindness. However, a shortage of human donor corneas as well as compounding economic, cultural, and organizational reasons in many countries limit its widespread use. Artificial corneas as well as porcine corneal xenografts have been considered as possible alternatives. To date, all preclinical studies using de-cellularized pig corneas have shown encouraging graft survival results; however, relatively few studies have been conducted in pig to non-human primate (NHP) models, and particularly using genetically engineered donors. METHODS: In this study, we assessed the potential benefit of using either hCTLA4-Ig transgenic or α1,3-Galactosyl Transferase (GT) Knock-Out (KO) plus transgenic hCD39/hCD55/hCD59/fucosyl-transferase pig lines in an anterior lamellar keratoplasty pig to NHP model. RESULTS: Corneas from transgenic animals expressing hCTLA4-Ig under the transcriptional control of a neuron-specific enolase promoter showed transgene expression in corneal keratocytes of the stroma and expression was maintained after transplantation. Although a first acute rejection episode occurred in all animals during the second week post-keratoplasty, the median final rejection time was 70 days in the hCTLA4-Ig group vs. 21 days in the wild-type (WT) control group. In contrast, no benefit for corneal xenograft survival from the GTKO/transgenic pig line was found. At rejection, cell infiltration in hCTLA4Ig transgenic grafts was mainly composed of macrophages with fewer CD3+ CD4+ and CD79+ cells than in other types of grafts. Anti-donor xenoantibodies increased dramatically between days 9 and 14 post-surgery in all animals. CONCLUSIONS: Local expression of the hCTLA4-Ig transgene dampens rejection of xenogeneic corneal grafts in this pig-to-NHP lamellar keratoplasty model. The hCTLA4-Ig transgene seems to target T-cell responses without impacting humoral responses, the control of which would presumably require additional peripheral immunosuppression.


Assuntos
Ceratócitos da Córnea/metabolismo , Transplante de Córnea/métodos , Rejeição de Enxerto/prevenção & controle , Imunoconjugados/metabolismo , Transgenes , Transplante Heterólogo/métodos , Abatacepte , Animais , Animais Geneticamente Modificados , Biomarcadores/metabolismo , Ceratócitos da Córnea/imunologia , Rejeição de Enxerto/genética , Rejeição de Enxerto/imunologia , Sobrevivência de Enxerto/genética , Sobrevivência de Enxerto/imunologia , Imunoconjugados/genética , Macaca fascicularis , Masculino , Modelos Animais , Sus scrofa/genética
7.
Genomics ; 101(1): 24-9, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22982297

RESUMO

The possibility to genotype embryos prior to implantation would have advantages for increasing the speed of selection of cattle. Reliable genotyping requires more DNA than can be obtained from biopsies of embryos, if they are to remain viable. Multiple displacement amplification (MDA) is a whole genome amplification technique used to increase the amount of DNA from biopsies for analysis. Reduced genome coverage resulting in Allele Drop Out (ADO) at heterozygous loci or missing genotypes are drawbacks of MDA. The present article describes the correlation between the input DNA quantity or embryo biopsy size and MDA success. Missing genotypes and ADO drastically increased when fewer than 30-40 cells or the genomic equivalents were used. However, embryo viability was found to be reduced if biopsied with more than 10 cells. Therefore, in vitro cell culture was investigated as a means to increase the number of cells available and the genotyping reliability.


Assuntos
Bovinos/genética , Técnicas de Genotipagem , Técnicas de Amplificação de Ácido Nucleico , Alelos , Animais , Biópsia , Bovinos/embriologia , Clonagem de Organismos , Embrião de Mamíferos/química , Embrião de Mamíferos/patologia , Genótipo , Análise de Sequência de DNA
8.
Biol Reprod ; 89(3): 68, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23926281

RESUMO

In the pig, the efficiency of in vitro embryo production and somatic cell nuclear transfer (SCNT) procedures remains limited. It has been suggested that prematuration treatments (pre-IVM) based on the prolongation of a patent, bidirectional crosstalk between the oocyte and the cumulus cells through gap junction mediate communication (GJC), with the maintenance of a proper level of cAMP, could improve the developmental capability of oocytes. The aim of this study was to assess: 1) dose-dependent effects of cilostamide on nuclear maturation kinetics, 2) the relationship between treatments on GJC functionality and large-scale chromatin configuration changes, and 3) the impact of treatments on developmental competence acquisition after parthenogenetic activation (PA) and SCNT. Accordingly, cumulus-oocyte complexes were collected from 3- to 6-mm antral follicles and cultured for 24 h in defined culture medium with or without 1 µM cilostamide. GJC functionality was assessed by Lucifer yellow microinjection, while chromatin configuration was evaluated by fluorescence microscopy after nuclear staining. Cilostamide administration sustained functional coupling for up to 24 h of culture and delayed meiotic resumption, as only 25.6% of cilostamide-treated oocytes reached the pro-metaphase I stage compared to the control (69.7%; P < 0.05). Moreover, progressive chromatin condensation was delayed before meiotic resumption based upon G2/M biomarker phosphoprotein epitope acquisition using immunolocalization. Importantly, cilostamide treatment under these conditions improved oocyte developmental competence, as reflected in higher blastocyst quality after both parthenogenetic activation and SCNT.


Assuntos
Comunicação Celular/efeitos dos fármacos , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Junções Comunicantes/efeitos dos fármacos , Técnicas de Transferência Nuclear/veterinária , Oócitos/efeitos dos fármacos , Partenogênese/efeitos dos fármacos , Quinolonas/farmacologia , Suínos/fisiologia , Animais , Células Cultivadas , Células do Cúmulo/efeitos dos fármacos , Células do Cúmulo/fisiologia , Relação Dose-Resposta a Droga , Feminino , Meiose/efeitos dos fármacos , Oócitos/fisiologia , Oogênese/efeitos dos fármacos , Partenogênese/fisiologia
9.
Xenotransplantation ; 20(4): 252-61, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23865597

RESUMO

BACKGROUND: Glutaraldehyde fixation does not guarantee complete tissue biocompatibility in current clinical bioprosthetic heart valves (BHVs). Particularly, circulating anti-αGal human antibodies increase significantly from just 10 days after a BHV implantation. The inactivation of such epitope should be mandatory to meet the requirements for a perspectively safe clinical application; nevertheless, its quantitative assessment in commercially available BHVs has never been carried out. METHODS: In this investigation, seven different models of BHVs were tested. The number of epitopes was determined with reference to a standard αGal source by an ELISA test. The presence of xenoantigen was subsequently confirmed by immunofluorescence analysis. Porcine tissue, knockout for the αGal epitopes, was used as negative control. RESULTS: Epic™ valve was the only model among those tested, in which the αGal antigen appeared to be completely shielded. Composite Trifecta™ valve exhibited conflicting results: cusps of bovine pericardial tissue were devoid of reactive αGal epitopes, while the stent cover strip of porcine pericardium still maintained 30% of active antigens originally present in native tissue. All other tested BHVs express an αGal amount not significantly different from that exhibited by porcine Mosaic(®) valve (5.2 ± 0.6 × 10(10) each 10 mg of tissue). CONCLUSIONS: For the first time, the quantitative evaluation of the αGal epitope in heart valve bioprostheses, already in clinical practice for about 40 yrs, was finally determined. Such quantification might provide indications of biocompatibility relevant for the selection of bioprosthetic devices and an increase in the confidence of the patient. It might become a major quality control tool in the production and redirection of future investigation in the quest for αGal-free long-lasting substitutes.


Assuntos
Epitopos/imunologia , Galactosiltransferases/imunologia , Glutaral/farmacologia , Próteses Valvulares Cardíacas , Valvas Cardíacas/efeitos dos fármacos , Valvas Cardíacas/imunologia , Transplante Heterólogo/métodos , Animais , Anticorpos Anti-Idiotípicos/imunologia , Antígenos/imunologia , Bovinos , Ensaio de Imunoadsorção Enzimática/métodos , Epitopos/genética , Galactosiltransferases/genética , Técnicas de Inativação de Genes , Rejeição de Enxerto/imunologia , Humanos , Masculino , Teste de Materiais , Modelos Animais , Suínos
10.
Cancers (Basel) ; 15(21)2023 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-37958436

RESUMO

Melanoma brain metastasis (MBM) is significantly associated with poor prognosis and is diagnosed in 80% of patients at autopsy. Circulating tumor cells (CTCs) are "seeds" of metastasis and the smallest functional units of cancer. Our multilevel approach has previously identified a CTC RPL/RPS gene signature directly linked to MBM onset. We hypothesized that targeting ribogenesis prevents MBM/metastasis in CTC-derived xenografts. We treated parallel cohorts of MBM mice with FDA-approved protein translation inhibitor omacetaxine with or without CDK4/CDK6 inhibitor palbociclib, and monitored metastatic development and cell proliferation. Necropsies and IVIS imaging showed decreased MBM/extracranial metastasis in drug-treated mice, and RNA-Seq on mouse-blood-derived CTCs revealed downregulation of four RPL/RPS genes. However, mitochondrial stress tests and RT-qPCR showed that omacetaxine and palbociclib inversely affected glycolytic metabolism, demonstrating that dual targeting of cell translation/proliferation is critical to suppress plasticity in metastasis-competent CTCs. Equally relevant, we provide the first-ever functional metabolic characterization of patient-derived circulating neoplastic cells/CTCs.

11.
Cancer Res Commun ; 3(2): 309-324, 2023 02.
Artigo em Inglês | MEDLINE | ID: mdl-36860657

RESUMO

The importance of the immune microenvironment in ovarian cancer progression, metastasis, and response to therapies has become increasingly clear, especially with the new emphasis on immunotherapies. To leverage the power of patient-derived xenograft (PDX) models within a humanized immune microenvironment, three ovarian cancer PDXs were grown in humanized NBSGW (huNBSGW) mice engrafted with human CD34+ cord blood-derived hematopoietic stem cells. Analysis of cytokine levels in the ascites fluid and identification of infiltrating immune cells in the tumors demonstrated that these humanized PDX (huPDX) established an immune tumor microenvironment similar to what has been reported for patients with ovarian cancer. The lack of human myeloid cell differentiation has been a major setback for humanized mouse models, but our analysis shows that PDX engraftment increases the human myeloid population in the peripheral blood. Analysis of cytokines within the ascites fluid of huPDX revealed high levels of human M-CSF, a key myeloid differentiation factor as well as other elevated cytokines that have previously been identified in ovarian cancer patient ascites fluid including those involved in immune cell differentiation and recruitment. Human tumor-associated macrophages and tumor-infiltrating lymphocytes were detected within the tumors of humanized mice, demonstrating immune cell recruitment to tumors. Comparison of the three huPDX revealed certain differences in cytokine signatures and in the extent of immune cell recruitment. Our studies show that huNBSGW PDX models reconstitute important aspects of the ovarian cancer immune tumor microenvironment, which may recommend these models for preclinical therapeutic trials. Significance: huPDX models are ideal preclinical models for testing novel therapies. They reflect the genetic heterogeneity of the patient population, enhance human myeloid differentiation, and recruit immune cells to the tumor microenvironment.


Assuntos
Neoplasias Ovarianas , Cavidade Peritoneal , Humanos , Camundongos , Animais , Feminino , Xenoenxertos , Ascite , Neoplasias Ovarianas/terapia , Citocinas , Microambiente Tumoral
12.
Cancer Res Commun ; 2(11): 1436-1448, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36407834

RESUMO

Melanoma brain metastasis (MBM) is linked to poor prognosis and low overall survival. We hypothesized that melanoma circulating tumor cells (CTCs) possess a gene signature significantly expressed and associated with MBM. Employing a multi-pronged approach, we provide first-time evidence identifying a common CTC gene signature for ribosomal protein large/small subunits (RPL/RPS) which associate with MBM onset and progression. Experimental strategies involved capturing, transcriptional profiling and interrogating CTCs, either directly isolated from blood of melanoma patients at distinct stages of MBM progression or from CTC-driven MBM in experimental animals. Second, we developed the first Magnetic Resonance Imaging (MRI) CTC-derived MBM xenograft model (MRI-MBM CDX) to discriminate MBM spatial and temporal growth, recreating MBM clinical presentation and progression. Third, we performed the comprehensive transcriptional profiling of MRI-MBM CDXs, along with longitudinal monitoring of CTCs from CDXs possessing/not possessing MBM. Our findings suggest that enhanced ribosomal protein content/ribogenesis may contribute to MBM onset. Since ribosome modifications drive tumor progression and metastatic development by remodeling CTC translational events, overexpression of the CTC RPL/RPS gene signature could be implicated in MBM development. Collectively, this study provides important insights for relevance of the CTC RPL/RPS gene signature in MBM, and identify potential targets for therapeutic intervention to improve patient care for melanoma patients diagnosed with or at high-risk of developing MBM.


Assuntos
Neoplasias Encefálicas , Melanoma , Células Neoplásicas Circulantes , Animais , Humanos , Melanoma/genética , Células Neoplásicas Circulantes/metabolismo , Neoplasias Encefálicas/genética , Proteínas Ribossômicas/genética
13.
Carcinogenesis ; 32(4): 452-61, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21177767

RESUMO

Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma in children with an annual incidence of five new cases per million. Alveolar rhabdomyosarcoma (ARMS) is characterized by the t(2;13) or t(1;13) chromosomal translocations, which generate the PAX3-FOXO1 or PAX7-FOXO1 fusion genes, respectively. The oncogenic activity of PAX3-FOXO1 has been demonstrated in vitro and in vivo, yet expression of the fusion protein alone in primary myoblasts or a mouse model is insufficient for tumorigenic transformation. To identify genes cooperating with PAX3-FOXO1 in ARMS tumorigenesis, we generated a retroviral complementary DNA (cDNA) expression library from the Rh30 ARMS cell line. Arf-/- myoblasts expressing PAX3-FOXO1 and the retroviral cDNA library rapidly formed tumors after subcutaneous injection into NOD-SCID mice. Tumors formed by Arf-/-/PAX3-FOXO1/MarX-library myoblasts contained an unknown cDNA, encoding the C-terminus of the Homo sapiens hypothetical protein, FLJ10404, herein named IRIZIO. Expression of full length IRIZIO cDNA also cooperated with PAX3-FOXO1 in the transformation of Arf-/- myoblasts. Given that IRIZIO is expressed at increased levels in RMS, it might contribute to rhabdomyosarcomagenesis in humans.


Assuntos
Transformação Celular Neoplásica , Proteínas de Neoplasias/metabolismo , Proteínas de Fusão Oncogênica/fisiologia , Oncogenes , Fatores de Transcrição Box Pareados/fisiologia , Rabdomiossarcoma Alveolar/etiologia , Animais , Linhagem Celular , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Camundongos SCID , Mioblastos/patologia , Proteínas de Neoplasias/genética , RNA Mensageiro/análise , Proteína do Retinoblastoma/fisiologia , Rabdomiossarcoma Alveolar/genética , Proteína Supressora de Tumor p53/fisiologia
14.
Reproduction ; 141(4): 453-65, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21239525

RESUMO

The most successful development of interspecies somatic cell nuclear transfer (iSCNT) embryos has been achieved in closely related species. The analyses of embryonic gene activity in iSCNT embryos of different species combinations have revealed the existence of significant aberrations in expression of housekeeping genes and genes dependent on the major embryonic genome activation (EGA). However, there are many studies with successful blastocyst (BL) development of iSCNT embryos derived from donor cells and oocytes of animal species with distant taxonomical relations (inter-family/inter-class) that should indicate proper EGA at least in terms of RNA polymerase I activation, nucleoli formation, and activation of genes engaged in morula and BL formation. We investigated the ability of bovine, porcine, and rabbit oocytes to activate embryonic nucleoli formation in the nuclei of somatic cells of different mammalian species. In iSCNT embryos, nucleoli precursor bodies originate from the oocyte, while most proteins engaged in the formation of mature nucleoli should be transcribed from genes de novo in the donor nucleus at the time of EGA. Thus, the success of nucleoli formation depends on species compatibility of many components of this complex process. We demonstrate that the time and cell stage of nucleoli formation are under the control of recipient ooplasm. Oocytes of the studied species possess different abilities to support nucleoli formation. Formation of nucleoli, which is a complex but small part of the whole process of EGA, is essential but not absolutely sufficient for the development of iSCNT embryos to the morula and BL stages.


Assuntos
Nucléolo Celular/metabolismo , Quimerismo/embriologia , Técnicas de Transferência Nuclear , Oócitos/metabolismo , Oócitos/ultraestrutura , Animais , Bovinos , Clonagem de Organismos/métodos , Embrião de Mamíferos , Feminino , Fertilização in vitro , Oócitos/citologia , Fosfoproteínas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Coelhos , Especificidade da Espécie , Coloração e Rotulagem , Suínos , Doadores de Tecidos , Nucleolina
15.
Reproduction ; 140(2): 273-85, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20530093

RESUMO

The best results of inter-species somatic cell nuclear transfer (iSCNT) in mammals were obtained using closely related species that can hybridise naturally. However, in the last years, many reports describing blastocyst development following iSCNT between species with distant taxonomical relations (inter-classes, inter-order and inter-family) have been published. This indicates that embryonic genome activation (EGA) in xeno-cytoplasm is possible, albeit very rarely. Using a bovine-pig (inter-family) iSCNT model, we studied the basic characteristics of EGA: expression and activity of RNA polymerase II (RNA Pol II), formation of nucleoli (as an indicator of RNA polymerase I (RNA Pol I) activity), expression of the key pluripotency gene NANOG and alteration of mitochondrial mass. In control embryos (obtained by IVF or iSCNT), EGA was characterised by RNA Pol II accumulation and massive production of poly-adenylated transcripts (detected with oligo dT probes) in blastomere nuclei, and formation of nucleoli as a result of RNA Pol I activity. Conversely, iSCNT embryos were characterised by the absence of accumulation and low activity of RNA Pol II and inability to form active mature nucleoli. Moreover, in iSCNT embryos, NANOG was not expressed, and mitochondria mass was significantly lower than in intra-species embryos. Finally, the complete developmental block at the 16-25-cell stage for pig-bovine iSCNT embryos and at the four-cell stage for bovine-pig iSCNT embryos strongly suggests that EGA is not taking place in iSCNT embryos. Thus, our experiments clearly demonstrate poor nucleus-cytoplasm compatibility between these animal species.


Assuntos
Bovinos/fisiologia , Embrião de Mamíferos/fisiologia , Desenvolvimento Embrionário/fisiologia , Técnicas de Transferência Nuclear/veterinária , Suínos/fisiologia , Animais , Bovinos/genética , Núcleo Celular/fisiologia , Colágeno Tipo VI/genética , Colágeno Tipo VI/fisiologia , Citoplasma/fisiologia , DNA/química , DNA/genética , Feminino , Masculino , Mitocôndrias/fisiologia , Reação em Cadeia da Polimerase/veterinária , Gravidez , RNA Polimerase I/genética , RNA Polimerase I/fisiologia , RNA Polimerase II/genética , RNA Polimerase II/fisiologia , Suínos/genética
16.
Xenotransplantation ; 17(6): 397-410, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-21158942

RESUMO

BACKGROUND: Techniques for genetic engineering of swine are providing genetically modified animals of importance for the field of xenotransplantation, animal models for human diseases and for a variety of research applications. Many of these modifications have been directed toward avoiding naturally existing cellular and antibody responses to species-specific antigens. METHODS: A number of techniques are today available to engineering the genome of mammals, these range from the well established less efficient method of DNA microinjection into the zygote, the use of viral vectors, to the more recent use of somatic cell nuclear transfer. The use of enzymatic engineering that are being developed now will refine the precision of the genetic modification combined with the use of new vectors like transposons. RESULTS: The use of somatic cell nuclear transfer is currently the most efficient way to generate genetically modified pigs. The development of enzymatic engineering with zinc-finger nucleases, recombinases and transposons will revolutionize the field. Nevertheless, genetic engineering in large domesticated animals will remain a challenging task. CONCLUSIONS: Recent improvements in several fields of cell and molecular biology offer new promises and opportunities toward an easier, cost-effective and efficient generation of transgenic pigs.


Assuntos
Animais Geneticamente Modificados , Engenharia Genética/métodos , Microinjeções , Animais , Técnicas de Transferência de Genes , Humanos , Técnicas de Transferência Nuclear , Suínos
17.
Nature ; 424(6949): 635, 2003 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-12904778

RESUMO

Several animal species, including sheep, mice, cattle, goats, rabbits, cats, pigs and, more recently, mules have been reproduced by somatic cell cloning, with the offspring being a genetic copy of the animal donor of the nuclear material used for transfer into an enucleated oocyte. Here we use this technology to clone an adult horse and show that it is possible to establish a viable, full-term pregnancy in which the surrogate mother is also the nuclear donor. The cloned offspring is therefore genetically identical to the mare who carried it, challenging the idea that maternal immunological recognition of fetal antigens influences the well-being of the fetus and the outcome of the pregnancy.


Assuntos
Clonagem de Organismos , Cavalos/imunologia , Cavalos/fisiologia , Gravidez/imunologia , Gêmeos , Animais , Núcleo Celular/genética , Núcleo Celular/fisiologia , Feminino , Cavalos/genética , Masculino , Modelos Imunológicos , Gravidez/genética , Gêmeos/genética
18.
bioRxiv ; 2020 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-34013271

RESUMO

Perfusion of convalescent plasma (CP) has demonstrated a potential to improve the pneumonia induced by SARS-CoV-2, but procurement and standardization of CP are barriers to its wide usage. Many monoclonal antibodies (mAbs) have been developed but appear insufficient to neutralize SARS-CoV-2 unless two or three of them are being combined. Therefore, heterologous polyclonal antibodies of animal origin, that have been used for decades to fight against infectious agents might represent a highly efficient alternative to the use of CP or mAbs in COVID-19 by targeting multiple antigen epitopes. However, conventional heterologous polyclonal antibodies trigger human natural xenogeneic antibody responses particularly directed against animal-type carbohydrate epitopes, mainly the N-glycolyl form of the neuraminic acid (Neu5Gc) and the Gal α1,3-galactose (αGal), ultimately forming immune complexes and potentially leading to serum sickness or allergy. To circumvent these drawbacks, we engineered animals lacking the genes coding for the cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) and α1,3-galactosyl-transferase (GGTA1) enzymes to produce glyco-humanized polyclonal antibodies (GH-pAb) lacking Neu5Gc and α-Gal epitopes. We found that pig IgG Fc domains fail to interact with human Fc receptors and thereby should confer the safety advantage to avoiding macrophage dependent exacerbated inflammatory responses, a drawback possibly associated with antibody responses against SARS-CoV-2 or to avoiding a possible antibody-dependent enhancement (ADE). Therefore, we immunized CMAH/GGTA1 double knockout (DKO) pigs with the SARS-CoV-2 spike receptor-binding domain (RBD) to elicit neutralizing antibodies. Animals rapidly developed a hyperimmune response with anti-SARS-CoV-2 end-titers binding dilutions over one to a million and end-titers neutralizing dilutions of 1:10,000. The IgG fraction purified and formulated following clinical Good Manufacturing Practices, named XAV-19, neutralized Spike/angiotensin converting enzyme-2 (ACE-2) interaction at a concentration < 1µg/mL and inhibited infection of human cells by SARS-CoV-2 in cytopathic assays. These data and the accumulating safety advantages of using glyco-humanized swine antibodies in humans warranted clinical assessment of XAV-19 to fight against COVID-19.

19.
Trends Biotechnol ; 26(9): 469-74, 2008 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-18675481

RESUMO

The recent vote in the British Parliament allows scientists in principle to create hybrid embryos by transferring human somatic cell nuclei into animal oocytes. This vote opens a fascinating new area of research with the central aim of generating interspecific lines of embryonic stem cells (ESCs) that could potentially be used to understand development, differentiation, gene expression and genomic compatibility. It will also promote human cell therapies, as well as the pharmaceutical industry's search for new drug targets. If this approach is to be successful, many biological questions need to be answered and, in addition, some moral and ethical aspects must be taken into account.


Assuntos
Embrião de Mamíferos/citologia , Células-Tronco Embrionárias/citologia , Técnicas de Transferência Nuclear/tendências , Criação de Embriões para Pesquisa/ética , Animais , Linhagem Celular , Reprogramação Celular/fisiologia , Clonagem de Organismos/ética , Destinação do Embrião/ética , Embrião de Mamíferos/fisiologia , Humanos , Células-Tronco Multipotentes/citologia , Células-Tronco Multipotentes/metabolismo , Técnicas de Transferência Nuclear/ética , Técnicas de Transferência Nuclear/legislação & jurisprudência , Oócitos/citologia , Criação de Embriões para Pesquisa/legislação & jurisprudência , Criação de Embriões para Pesquisa/métodos , Células-Tronco Totipotentes/citologia , Células-Tronco Totipotentes/metabolismo
20.
Mol Reprod Dev ; 75(5): 731-43, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-18058811

RESUMO

Histone modification genes in bovine embryos: The mRNA expression pattern of histone-related genes was determined in bovine oocytes and embryos. We compared immature and in vitro-matured oocytes, either before or after enucleation and activation, in vitro produced embryos (zygotes, 8-16 cell stages, blastocysts), embryos cloned with female or male donor cells; parthenogenetic embryos, and in vivo-derived blastocysts to detect deviations from the normal expression pattern. A sensitive semi-quantitative endpoint RT-PCR assay was used to reveal differences in histone deacetylation [histone deacetylase 2 (HDAC2)]; histone acetylation [histone acetyltransferase 1 (HAT1)]; histone methylation [histone methyltransferases (SUV39H1, G9A)]; heterochromatin formation [heterochromatin protein 1 (HP1)]; and chromatin-mediated transcription regulation [zygote arrest 1 (ZAR1)]. With the exception of ZAR1, these mRNAs were present throughout preimplantation development. The relative abundance of mRNAs for histone methyltransferases (SUV39H1 and G9A) and for heterochromatin-associated protein (HP1) differed significantly before and after activation of the bovine embryonic genome. The similarity of HAT1 gene expression in 8-16 cell embryos and blastocysts suggests that histone acetylation is primarily affected by in vitro culture only prior to embryonic genome activation. HDAC2 gene mRNA expression was not affected by in vitro culture and/or cloning before and after activation of the embryonic genome. The donor cell line affected mRNA expression patterns of genes involved in reprogramming cloned embryos suggesting epigenetic dysregulation. Results show that both in vitro production and somatic cloning alter the mRNA expression of histone modifying genes in bovine embryos.


Assuntos
Blastocisto/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Histonas/metabolismo , Proteínas Nucleares/biossíntese , Processamento de Proteína Pós-Traducional/fisiologia , RNA Mensageiro/biossíntese , Acetilação , Animais , Blastocisto/citologia , Bovinos , Clonagem de Organismos/métodos , Técnicas de Cultura Embrionária/métodos , Epigênese Genética/fisiologia , Feminino , Histonas/genética , Masculino , Proteínas Nucleares/genética , Partenogênese/fisiologia , RNA Mensageiro/genética , Especificidade da Espécie
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