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1.
Sci Rep ; 13(1): 5162, 2023 03 30.
Artigo em Inglês | MEDLINE | ID: mdl-36997583

RESUMO

The induction of antiviral innate immunity by systemic immunization with live virus can be employed to positively impact the response to therapeutic vaccination. We previously demonstrated that systemic immunization with a non-replicating MVA encoding CD40 ligand (CD40L) enhances innate immune cell activation and function, and triggers potent antitumor CD8+ T cell responses in different murine tumor models. Antitumor efficacy was increased when combined with tumor targeting antibodies. Here we report the development of TAEK-VAC-HerBy (TVH), a first-in-class human tumor antibody enhanced killing (TAEK) vaccine based on the non-replicating MVA-BN viral vector. It encodes the membrane bound form of human CD40L, HER2 and the transcription factor Brachyury. TVH is designed for therapeutic use in HER2- or Brachyury-expressing cancer patients in combination with tumor targeting antibodies. To preclude possible oncogenic activities in infected cells and to prevent binding of vaccine-encoded HER2 by monoclonal antibodies trastuzumab and pertuzumab, genetic modifications of HER2 were introduced in the vaccine. Brachyury was genetically modified to prevent nuclear localization of the protein thereby inhibiting its transcriptional activity. CD40L encoded in TVH enhanced human leukocyte activation and cytokine secretion in vitro. Lastly, TVH intravenous administration to non-human primates was proven immunogenic and safe in a repeat-dose toxicity study. Nonclinical data presented here highlight TVH as a first-in-class immunotherapeutic vaccine platform currently under clinical investigation.


Assuntos
Vacinas Anticâncer , Neoplasias , Humanos , Camundongos , Animais , Ligante de CD40/genética , Neoplasias/tratamento farmacológico , Linfócitos T CD8-Positivos , Anticorpos Antineoplásicos , Vaccinia virus/genética
2.
Front Immunol ; 13: 857440, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35479095

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a worldwide pandemic. Here, we present non-human primate immunogenicity and protective efficacy data generated with the capsid virus-like particle (cVLP)-based vaccine ABNCoV2 that has previously demonstrated immunogenicity in mice. In rhesus macaques, a single vaccination with either 15 or 100 µg ABNCoV2 induced binding and neutralizing antibodies in a dose-dependent manner, at levels comparable to those measured in human convalescents. A second vaccine administration led to a >50-fold increase in neutralizing antibodies, with 2-log higher mean levels in the 100-µg ABNCoV2 group compared with convalescent samples. Upon SARS-CoV-2 challenge, a significant reduction in viral load was observed for both vaccine groups relative to the challenge control group, with no evidence of enhanced disease. Remarkably, neutralizing antibody titers against an original SARS-CoV-2 isolate and against variants of concern were comparable, indicating a potential for broad protection afforded by ABNCoV2, which is currently in clinical testing.


Assuntos
COVID-19 , Vacinas Virais , Animais , Anticorpos Neutralizantes , Anticorpos Antivirais , COVID-19/prevenção & controle , Vacinas contra COVID-19 , Capsídeo , Proteínas do Capsídeo , Humanos , Macaca mulatta , SARS-CoV-2
3.
J Clin Invest ; 117(11): 3540-50, 2007 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17965774

RESUMO

Changes in cytoplasmic Ca2+ levels regulate a variety of fundamental cellular functions in virtually all cells. In nonexcitable cells, a major pathway of Ca2+ entry involves receptor-mediated depletion of intracellular Ca2+ stores followed by the activation of store-operated calcium channels in the plasma membrane. We have established a mouse line expressing an activating EF hand motif mutant of stromal interaction molecule 1 (Stim1), an ER receptor recently identified as the Ca2+ sensor responsible for activation of Ca2+ release-activated (CRAC) channels in T cells, whose function in mammalian physiology is not well understood. Mice expressing mutant Stim1 had macrothrombocytopenia and an associated bleeding disorder. Basal intracellular Ca2+ levels were increased in platelets, which resulted in a preactivation state, a selective unresponsiveness to immunoreceptor tyrosine activation motif-coupled agonists, and increased platelet consumption. In contrast, basal Ca2+ levels, but not receptor-mediated responses, were affected in mutant T cells. These findings identify Stim1 as a central regulator of platelet function and suggest a cell type-specific activation or composition of the CRAC complex.


Assuntos
Cálcio/metabolismo , Motivos EF Hand/genética , Hemorragia , Glicoproteínas de Membrana/metabolismo , Mutação , Ativação Plaquetária , Trombocitopenia , Animais , Medula Óssea/patologia , Canais de Cálcio/metabolismo , Fibrose/patologia , Hemorragia/genética , Hemorragia/metabolismo , Megacariócitos/citologia , Megacariócitos/metabolismo , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos , Ativação Plaquetária/genética , Glicoproteínas da Membrana de Plaquetas/metabolismo , Transdução de Sinais/fisiologia , Esplenomegalia/metabolismo , Molécula 1 de Interação Estromal , Linfócitos T/citologia , Linfócitos T/metabolismo , Trombocitopenia/genética , Trombocitopenia/metabolismo
4.
Oncogene ; 24(4): 561-72, 2005 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-15580301

RESUMO

HOXB4 overexpression mediates increased self-renewal of haematopoietic stem cells (HSCs) ex vivo. Since HOXB4-expanded HSCs retain normal differentiation potential and there is no leukaemia development from transduced HSCs, HOXB4 represents a promising tool for human HSC therapy. However, the increased proliferation capacity of HOXB4 overexpressing fibroblasts resulting from upregulation of JunB, Fra-1 and cyclin D1 protein levels may indicate a potential risk associated with the HOXB4 overexpression approach. This prompted us to investigate the proliferation rate, differentiation and expression of cell cycle regulators directly in bone marrow cultures overexpressing HOXB4. Here we show that in comparison to neo-transduced control bone marrow cultures, HOXB4-overexpressing cultures had a more homogenous morphology and increased numbers of haematopoietic progenitor cells capable to generate primitive colonies in vitro. In contrast, neo-transduced bone marrow cells in long-term cultures showed hallmarks of myeloid differentiation and a reduced secondary colony forming activity. We further show that multilineage repopulating activity in vivo, which was present only in HOXB4 long-term cultures, declined over time. HOXB4 overexpression in vitro did not result in an increase but in a stabilization of the proliferation rate (1.4-1.8 cell divisions per day), while the proliferation rate of control neo-transduced bone marrow cultures gradually declined. Correspondingly, increased HOXB4 expression was paralleled by decreased expression levels of cyclins, CDKs and AP-1 family members. These results suggest that the growth rate of HOXB4- compared to neo-transduced bone marrow cells remains constant in long-term cultures along with a suppression of myeloid differentiation. In contrast to HOXB4 overexpression in fibroblasts, bone marrow cells engineered to overexpress HOXB4 do not upregulate AP-1 complex members or cyclins indicating that HOXB4 acts in a cell type-specific way.


Assuntos
Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Proteínas de Homeodomínio/metabolismo , Animais , Transplante de Medula Óssea , Diferenciação Celular/genética , Proliferação de Células , Células Cultivadas , Quinases Ciclina-Dependentes/genética , Ciclinas/genética , Proteínas de Homeodomínio/genética , Camundongos , Fator de Transcrição AP-1/genética , Fatores de Transcrição , Transcrição Gênica , Transdução Genética
5.
Mech Ageing Dev ; 127(7): 600-9, 2006 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-16620920

RESUMO

BCL-2 functions as a death repressor molecule in an evolutionary conserved cell death pathway. Inactivation of bcl-2 in mice results in pleiotropic effects including postnatal growth retardation, massive apoptosis in lymphoid tissues, polycystic kidney disease (PKD) and shortened lifespan. To evaluate the influence of the affected bcl-2 deficient kidneys on the postnatal development and lifespan of bcl-2 knockout mice we used "the rescue of (n-1) affected tissues" strategy. According to this strategy bcl-2 heterozygous animals were crossed with H2K-hbcl-2 transgenic mice expressing human BCL-2 in most tissues and organs excluding the kidney. Overexpression of hBCL-2 in bcl-2-/- mice rescues growth retardation, normalizes and protects the hematolymphoid system from gamma-radiation. However, the hbcl-2 transgene is not expressed in kidneys and the rescued mice have PKD and a shortened lifespan. Thus, our results indicated that PKD is the main reason of early mortality in bcl-2 deficient mice. Moreover, we have created mouse model, similar to the kidney specific knockout of bcl-2. Such models can be useful to study the influence of bcl-2 or other gene deficiency in individual organs (or tissues) on development and ageing of whole organism.


Assuntos
Apoptose/genética , Longevidade/genética , Doenças Renais Policísticas/genética , Proteínas Proto-Oncogênicas c-bcl-2/deficiência , Insuficiência Renal/genética , Animais , Cruzamentos Genéticos , Transtornos do Crescimento/genética , Transtornos do Crescimento/patologia , Heterozigoto , Humanos , Camundongos , Camundongos Knockout , Doenças Renais Policísticas/patologia , Insuficiência Renal/patologia
6.
Blood ; 107(8): 3350-8, 2006 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-16397132

RESUMO

Macrophage actin-associated tyrosine phosphorylated protein (MAYP)/PSTPIP2, a PCH protein, is involved in the regulation of macrophage motility. Mutations in a closely related gene, PSTPIP1/CD2BP1, cause a dominantly inherited autoinflammatory disorder known as PAPA syndrome. A mutant mouse obtained by chemical mutagenesis exhibited an autoinflammatory disorder characterized by macrophage infiltration and inflammation, leading to osteolysis and necrosis in paws and necrosis of ears. Positional cloning of this recessive mutation, termed Lupo, identified a T to A nucleotide exchange leading to an amino acid substitution (I282N) in the sequence of MAYP. Mayp(Lp/Lp) disease was transferable by bone marrow transplantation and developed in the absence of lymphocytes. Consistent with the involvement of macrophages, lesion development could be prevented by the administration of clodronate liposomes. MAYP is expressed in monocytes/macrophages and in a Mac1+ subfraction of granulocytes. LPS stimulation increases its expression in macrophages. Because of the instability of the mutant protein, MAYP expression is reduced 3-fold in Mayp(Lp/Lp) macrophages and, on LPS stimulation, does not rise above the level of unstimulated wild-type (WT) cells. Mayp(Lp/Lp) mice expressed elevated circulating levels of several cytokines, including MCP-1; their macrophages exhibited altered cytokine production in vitro. These studies suggest that MAYP plays an anti-inflammatory role in macrophages.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Substituição de Aminoácidos , Doenças Autoimunes/genética , Movimento Celular/genética , Proteínas do Citoesqueleto/genética , Macrófagos/metabolismo , Mutação Puntual , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Doenças Autoimunes/tratamento farmacológico , Doenças Autoimunes/metabolismo , Doenças Autoimunes/patologia , Conservadores da Densidade Óssea/administração & dosagem , Transplante de Medula Óssea/métodos , Células Cultivadas , Ácido Clodrônico/administração & dosagem , Citocinas/metabolismo , Proteínas do Citoesqueleto/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Genes Recessivos/genética , Granulócitos/metabolismo , Granulócitos/patologia , Inflamação/tratamento farmacológico , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Lipopolissacarídeos/farmacologia , Linfócitos/metabolismo , Linfócitos/patologia , Antígeno de Macrófago 1/metabolismo , Macrófagos/patologia , Camundongos , Camundongos Mutantes , Mutagênese , Osteólise/genética , Osteólise/metabolismo , Osteólise/patologia , Síndrome
7.
EMBO J ; 24(3): 554-66, 2005 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-15660132

RESUMO

Modifications of DNA and chromatin are fundamental for the establishment and maintenance of cell type-specific gene expression patterns that constitute cellular identities. To test whether the developmental potential of fetal brain-derived cells that form floating sphere colonies (neurospheres) can be modified by destabilizing their epigenotype, neurosphere cells were treated with chemical compounds that alter the acetylation and methylation patterns of chromatin and DNA. Intravenous infusion of bulk or clonally derived neurosphere cells treated with a combination of trichostatin A (TSA) plus 5-aza-2'-deoxycytidine (AzaC) (TSA/AzaC neurosphere cells) yielded long-term, multilineage and transplantable neurosphere-derived haematopoietic repopulation. Untreated neurosphere cells exhibited no haematopoietic repopulation activity. The neurosphere-derived haematopoietic cells showed a diploid karyotype, indicating that they are unlikely to be products of cell fusion events, a conclusion strengthened by multicolour fluorescence in situ hybridization. Our results indicate that altering the epigenotype of neurosphere cells followed by transplantation enables the generation of neurosphere-derived haematopoietic cells.


Assuntos
Azacitidina/análogos & derivados , Azacitidina/farmacologia , Cromatina/efeitos dos fármacos , Hematopoese/efeitos dos fármacos , Ácidos Hidroxâmicos/farmacologia , Animais , Decitabina , Diploide , Genes bcl-2 , Proteínas de Fluorescência Verde/genética , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Técnicas In Vitro , Cariotipagem , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células-Tronco Multipotentes/citologia , Células-Tronco Multipotentes/efeitos dos fármacos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Esferoides Celulares
8.
Immunity ; 22(4): 451-65, 2005 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15845450

RESUMO

The identification of specific genetic loci that contribute to inflammatory and autoimmune diseases has proved difficult due to the contribution of multiple interacting genes, the inherent genetic heterogeneity present in human populations, and a lack of new mouse mutants. By using N-ethyl-N-nitrosourea (ENU) mutagenesis to discover new immune regulators, we identified a point mutation in the murine phospholipase Cg2 (Plcg2) gene that leads to severe spontaneous inflammation and autoimmunity. The disease is composed of an autoimmune component mediated by autoantibody immune complexes and B and T cell independent inflammation. The underlying mechanism is a gain-of-function mutation in Plcg2, which leads to hyperreactive external calcium entry in B cells and expansion of innate inflammatory cells. This mutant identifies Plcg2 as a key regulator in an autoimmune and inflammatory disease mediated by B cells and non-B, non-T haematopoietic cells and emphasizes that by distinct genetic modulation, a single point mutation can lead to a complex immunological phenotype.


Assuntos
Autoimunidade , Cálcio/metabolismo , Inflamação/genética , Mutação Puntual , Fosfolipases Tipo C/genética , Animais , Artrite Experimental/genética , Artrite Experimental/imunologia , Linfócitos B/metabolismo , Sequência de Bases , Células da Medula Óssea/citologia , Dermatite/genética , Dermatite/imunologia , Masculino , Camundongos , Dados de Sequência Molecular , Fosfolipase C gama , Fosfolipases Tipo C/metabolismo , Regulação para Cima
9.
Immunol Rev ; 187: 9-21, 2002 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-12366679

RESUMO

Stem cell systems represent an effective and powerful approach for tissue development and regeneration of diverse tissue types. Common and defining features of these exceptional cells are the capacity for self-renewal and the potential for differentiation into multiple mature cell types. Recently, surprising new observations have indicated that stem cells isolated from one adult tissue can also give rise to mature cells of other cell lineages, irrespective of classical germ layer designations. This discovery has resulted in quantum leaps in both scientific knowledge and the potential applications of stem cells. The new findings contradict central dogmas of commitment and differentiation of stem and progenitor cells. However, the true potential of somatic stem cells is just emerging and the new findings have to be defined more fully and integrated into a unifying model of stem cell potential and behavior. Here we analyze the developmental potential of hematopoietic stem cells of mouse and man following their injection into the murine preimplantation blastocyst, an environment that allows the development of all cell lineages. In addition, we discuss the emerging lines of evidence of the developmental plasticity of hematopoietic and other somatic stem cells and consider how cellular memory of transcriptional states is established and may be potentially involved in this phenomenon.


Assuntos
Diferenciação Celular , Células-Tronco Hematopoéticas/citologia , Animais , Linhagem da Célula , Embrião de Mamíferos/citologia , Regulação da Expressão Gênica no Desenvolvimento , Transplante de Células-Tronco Hematopoéticas , Humanos , Transdução de Sinais
10.
J Biol Chem ; 279(6): 4894-902, 2004 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-14615479

RESUMO

ADAR1 (adenosine deaminase acting on RNA-1) is widely expressed in mammals, but its biological role is unknown. We show here by gene targeting that ADAR1 selectively edits in vivo two of five closely spaced adenosines in the serotonin 5-hydroxytryptamine subtype 2C receptor pre-mRNA of nervous tissue; and hence, site-selective adenosine-to-inosine editing is indeed a function of ADAR1. Remarkably, homozygosity for two different null alleles of ADAR1 caused a consistent embryonic phenotype appearing early at embryonic day 11 and leading to death between embryonic days 11.5 and 12.5. This phenotype manifests a rapidly disintegrating liver structure, along with severe defects in definitive hematopoiesis, encompassing both erythroid and myeloid/granuloid progenitors as well as spleen colony-forming activity from the aorta-gonad-mesonephros region and fetal liver. Probably as a consequence of these developmental impairments, ADAR1-deficient embryonic stem cells failed to contribute to liver, bone marrow, spleen, thymus, and blood in adult chimeric mice. Thus, ADAR1 subserves critical steps in developing non-nervous tissue, which are likely to include transcript editing.


Assuntos
Adenosina Desaminase/deficiência , Fígado/enzimologia , Fígado/patologia , Adenosina Desaminase/genética , Alelos , Animais , Sequência de Bases , DNA Complementar/genética , Feminino , Morte Fetal/enzimologia , Morte Fetal/genética , Morte Fetal/patologia , Camundongos , Camundongos Knockout , Fenótipo , Gravidez , Edição de RNA , Precursores de RNA/genética , Proteínas de Ligação a RNA , Receptor 5-HT2C de Serotonina/genética
11.
Cells Tissues Organs ; 171(1): 77-89, 2002.
Artigo em Inglês | MEDLINE | ID: mdl-12021493

RESUMO

Like many other animals, mammals develop from fertilized oocytes - the ultimate stem cells. As embryogenesis proceeds, most cells lose developmental potential and eventually become restricted to a specific cell lineage. The result is the formation of a complete and structured mature organism with complex organs composed of a great variety of mature, mostly mitotically quiescent effector cells. However, along the way, some exceptional cells, known as somatic stem cells (SSCs) are set aside and maintain a high proliferation and tissue-specific differentiation potential. SSCs, in contrast to embryonic stem (ES) cells, which are able to give rise to all cell types of the body, have been regarded as being more limited in their differentiation potential in the sense that they were thought to be committed exclusively to their tissue of origin. However, recent studies have demonstrated that somatic stem cells from a given tissue can also contribute to heterologous tissues and thus show a broad nontissue restricted differentiation potential. The question arises: how plastic are somatic stem cells? To provide a tentative answer, we describe and review here recent investigations into the developmental potentials of two somatic stem cell types, namely hematopoietic and neural stem cells.


Assuntos
Diferenciação Celular/fisiologia , Células-Tronco Hematopoéticas/fisiologia , Neurônios/fisiologia , Animais , Biomarcadores , Linhagem da Célula , Separação Celular , Embrião de Mamíferos/anatomia & histologia , Embrião de Mamíferos/fisiologia , Camundongos , Camundongos Transgênicos
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