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1.
PLoS Pathog ; 15(2): e1007583, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30726291

RESUMO

CD8 T cells are necessary for the elimination of intracellular pathogens, but during chronic viral infections, CD8 T cells become exhausted and unable to control the persistent infection. Programmed cell death-1 (PD-1) blockade therapies have been shown to improve CD8 T cell responses during chronic viral infections. These therapies have been licensed to treat cancers in humans, but they have not yet been licensed to treat chronic viral infections because limited benefit is seen in pre-clinical animal models of chronic infection. In the present study, we investigated whether TLR4 triggering could improve PD-1 therapy during a chronic viral infection. Using the model of chronic lymphocytic choriomeningitis virus (LCMV) infection in mice, we show that TLR4 triggering with sublethal doses of lipopolysaccharide (LPS) followed by PD-1 blockade results in superior improvement in circulating virus-specific CD8 T cell responses, relative to PD-1 blockade alone. Moreover, we show that the synergy between LPS and PD-1 blockade is dependent on B7 costimulation and mediated by a dendritic cell (DC) intrinsic mechanism. Systemic LPS administration may have safety concerns, motivating us to devise a safer regimen. We show that ex vivo activation of DCs with LPS, followed by adoptive DC transfer, results in a similar potentiation of PD-1 therapy without inducing wasting disease. In summary, our data demonstrate a previously unidentified role for LPS/TLR4 signaling in modulating the host response to PD-1 therapy. These findings may be important for developing novel checkpoint therapies against chronic viral infection.


Assuntos
Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor 4 Toll-Like/metabolismo , Transferência Adotiva , Animais , Linfócitos T CD8-Positivos/fisiologia , Doença Crônica , Células Dendríticas , Feminino , Lipopolissacarídeos/farmacologia , Ativação Linfocitária , Coriomeningite Linfocítica , Vírus da Coriomeningite Linfocítica/imunologia , Vírus da Coriomeningite Linfocítica/patogenicidade , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptor de Morte Celular Programada 1/imunologia , Transdução de Sinais , Receptor 4 Toll-Like/imunologia , Viroses/imunologia
2.
Immunology ; 155(4): 458-466, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30014618

RESUMO

Antiretroviral therapy (ART) for the treatment of human immunodeficiency virus (HIV) infection represents a major breakthrough in the treatment of HIV/acquired immune-deficiency syndrome. However, it remains unclear how ART influences virus-specific immune responses and understanding this is important for developing novel cure and eradication interventions for HIV-1. In the present study, we evaluate how ART impacts T-cell and antibody responses in simian immunodeficiency virus (SIV) -infected rhesus macaques. We evaluated CD4 and CD8 T-cell responses by multiparameter flow cytometry, viral loads by quantitative RT-PCR by a two-step process using SIV-specific primers and antibody neutralization function by luciferase-based TZM-bl assays. We demonstrate that macaques treated with ART exhibit phenotypic and qualitative effects on T-cell and antibody responses. Macaques on ART exhibited low numbers of virus-specific T-cell responses, and these responses appeared to be partially biased towards central memory subsets. More importantly, there were significantly reduced neutralizing antibody responses in macaques treated with ART. Collectively, these data improve the understanding of how virus-specific immune responses are generated during ART, and suggest the potential importance of therapeutic vaccines to maintain adaptive immunity during treated infection.


Assuntos
Antirretrovirais/uso terapêutico , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/tratamento farmacológico , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Subpopulações de Linfócitos T/imunologia , Animais , Diferenciação Celular/imunologia , Feminino , Ativação Linfocitária/imunologia , Macaca mulatta , Masculino , Vírus da Imunodeficiência Símia/efeitos dos fármacos , Vírus da Imunodeficiência Símia/imunologia , Subpopulações de Linfócitos T/citologia , Carga Viral/imunologia
3.
AIDS Res Hum Retroviruses ; 34(5): 430-438, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29620933

RESUMO

Bone marrow transplantation is a treatment for various cancers and genetic diseases, and the only case of a cured HIV infection involved the use of this clinical procedure, highlighting the potential use of this therapy for curing many chronic diseases. However, little is known about how chronic viral infection influences lymphocyte reconstitution after bone marrow transplantation. To address this, we infected mice with chronic lymphocytic choriomeningitis virus, and performed bone marrow transplantation to assess lymphocyte reconstitution. Interestingly, we observed that adoptively transferred marrow cells exhibited preferential B cell differentiation in chronically infected mice. Moreover, donor marrow cells that were adoptively transferred into chronically infected mice differentiated into virus-specific CD8 T cells that were able to expand after PD-L1 blockade. Taken together, our data show that chronic viral infection induces a biased differentiation of bone marrow stem cells into B cells, and that exhausted virus-specific CD8 T cells generated de novo in this setting are rescuable by PD-1 blockade. These data contribute to the understanding of how chronic viral infection impacts lymphocyte reconstitution, and may provide valuable information to improve current hematopoietic transplantation regimens in chronically infected hosts.


Assuntos
Transplante de Medula Óssea , Linfócitos/imunologia , Coriomeningite Linfocítica/terapia , Vírus da Coriomeningite Linfocítica/imunologia , Animais , Linfócitos B/imunologia , Linfócitos T CD8-Positivos/imunologia , Diferenciação Celular , Modelos Animais de Doenças , Feminino , Camundongos Endogâmicos C57BL , Células-Tronco/fisiologia , Resultado do Tratamento
4.
Immunology ; 152(2): 328-343, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28582800

RESUMO

CD4 T cells help immune responses, but knowledge of how memory CD4 T cells are regulated and how they regulate adaptive immune responses and induce immunopathology is limited. Using adoptive transfer of virus-specific CD4 T cells, we show that naive CD4 T cells undergo substantial expansion following infection, but can induce lethal T helper type 1-driven inflammation. In contrast, memory CD4 T cells exhibit a biased proliferation of T follicular helper cell subsets and were able to improve adaptive immune responses in the context of minimal tissue damage. Our analyses revealed that type I interferon regulates the expansion of primary CD4 T cells, but does not seem to play a critical role in regulating the expansion of secondary CD4 T cells. Strikingly, blockade of type I interferon abrogated lethal inflammation by primary CD4 T cells following viral infection, despite that this treatment increased the numbers of primary CD4 T-cell responses. Altogether, these data demonstrate important aspects of how primary and secondary CD4 T cells are regulated in vivo, and how they contribute to immune protection and immunopathology. These findings are important for rational vaccine design and for improving adoptive T-cell therapies against persistent antigens.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Proliferação de Células , Memória Imunológica , Inflamação/imunologia , Ativação Linfocitária , Coriomeningite Linfocítica/imunologia , Vírus da Coriomeningite Linfocítica/imunologia , Imunidade Adaptativa , Transferência Adotiva , Animais , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/transplante , Linfócitos T CD4-Positivos/virologia , Citotoxicidade Imunológica , Modelos Animais de Doenças , Genótipo , Interações Hospedeiro-Patógeno , Imunidade Humoral , Inflamação/metabolismo , Inflamação/virologia , Interferon Tipo I/imunologia , Interferon Tipo I/metabolismo , Coriomeningite Linfocítica/genética , Coriomeningite Linfocítica/metabolismo , Coriomeningite Linfocítica/virologia , Vírus da Coriomeningite Linfocítica/patogenicidade , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fenótipo , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Fatores de Tempo
5.
Immunology ; 151(3): 340-348, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28295248

RESUMO

T regulatory (Treg) cells are critical for preventing autoimmunity and suppressing immune responses during cancer and chronic infection. However, the role of Treg cells in the generation of vaccine-induced immune memory remains ill-defined. Using the mouse model of lymphocytic choriomeningitis virus (LCMV) infection, we demonstrate that transient absence of Treg cells during effector to memory CD8 T-cell transition results in a permanent impairment in the maintenance, function and recall capacity of CD8 T cells. Memory CD8 T cells in mice that were transiently depleted of Treg cells exhibited defective up-regulation of memory markers with a significant decrease in polyfunctionality. However, Treg-depleted mice showed no significant change in CD4 T-cell responses, and antibody levels relative to control. Altogether, this study evaluates the role of Treg cells in the formation of immune memory and demonstrates an important role for Treg cells in promoting memory CD8 T-cell differentiation and vaccine-induced immune protection against intracellular pathogens.


Assuntos
Imunidade Adaptativa/efeitos dos fármacos , Antígenos Virais/administração & dosagem , Linfócitos T CD8-Positivos/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ativação Linfocitária/efeitos dos fármacos , Coriomeningite Linfocítica/prevenção & controle , Vírus da Coriomeningite Linfocítica/efeitos dos fármacos , Vírus da Imunodeficiência Símia/imunologia , Vacinas Virais/administração & dosagem , Animais , Anticorpos Antivirais/sangue , Antígenos Virais/imunologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Chlorocebus aethiops , Toxina Diftérica/administração & dosagem , Toxina Diftérica/imunologia , Modelos Animais de Doenças , Feminino , Memória Imunológica/efeitos dos fármacos , Coriomeningite Linfocítica/imunologia , Coriomeningite Linfocítica/virologia , Vírus da Coriomeningite Linfocítica/imunologia , Camundongos Transgênicos , Linfócitos T Reguladores/imunologia , Fatores de Tempo , Células Vero , Vacinas Virais/imunologia
6.
Oncotarget ; 7(43): 69321-69336, 2016 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-27732568

RESUMO

Constitutive fibroblast activation is responsible for organ fibrosis in fibrotic disorders including systemic sclerosis (SSc), but the underlying mechanisms are not fully understood, and effective therapies are lacking. We investigated the expression of the mitochondrial deacetylase sirtuin 3 (SIRT3) and its modulation by hexafluoro, a novel fluorinated synthetic honokiol analogue, in the context of fibrosis. We find that augmenting cellular SIRT3 by forced expression in normal lung and skin fibroblasts, or by hexafluoro treatment, blocked intracellular TGF-ß signaling and fibrotic responses, and mitigated the activated phenotype of SSc fibroblasts. Moreover, hexafluoro attenuated mitochondrial and cytosolic reactive oxygen species (ROS) accumulation in TGF-ß-treated fibroblasts. Remarkably, we found that the expression of SIRT3 was significantly reduced in SSc skin biopsies and explanted fibroblasts, and was suppressed by TGF-ß treatment in normal fibroblasts. Moreover, tissue levels of acetylated MnSOD, a sensitive marker of reduced SIRT3 activity, were dramatically enhanced in lesional skin and lung biopsies from SSc patients. Mice treated with hexafluoro showed substantial attenuation of bleomycin-induced fibrosis in the lung and skin. Our findings reveal a cell-autonomous function for SIRT3 in modulating fibrotic responses, and demonstrate the ability of a novel pharmacological SIRT3 agonist to attenuate fibrosis in vitro and in vivo. In light of the impaired expression and activity of SIRT3 associated with organ fibrosis in SSc, pharmacological approaches for augmenting SIRT3 might have therapeutic potential.


Assuntos
Pulmão/enzimologia , Escleroderma Sistêmico/enzimologia , Sirtuína 3/metabolismo , Pele/enzimologia , Adulto , Idoso , Animais , Bleomicina , Células Cultivadas , Ativação Enzimática/efeitos dos fármacos , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Fibrose/induzido quimicamente , Fibrose/prevenção & controle , Humanos , Hidrocarbonetos Fluorados/farmacologia , Pulmão/patologia , Masculino , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Interferência de RNA , Escleroderma Sistêmico/genética , Escleroderma Sistêmico/patologia , Sirtuína 3/genética , Pele/patologia
7.
Mol Cell Biol ; 34(17): 3244-58, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24958101

RESUMO

The Fbw7 ubiquitin ligase critically regulates hematopoietic stem cell (HSC) function, though the precise contribution of individual substrate ubiquitination pathways to HSC homeostasis is unknown. In the work reported here, we used a mouse model in which we introduced two knock-in mutations (T74A and T393A [changes of T to A at positions 74 and 393]) to disrupt Fbw7-dependent regulation of cyclin E, its prototypic substrate, and to examine the consequences of cyclin E dysregulation for HSC function. Serial transplantation revealed that cyclin E(T74A T393A) HSCs self-renewed normally; however, we identified defects in their multilineage reconstituting capacity. By inducing hematologic stress, we exposed an impaired self-renewal phenotype in cyclin E knock-in HSCs that was associated with defective cell cycle exit and the emergence of chromosome instability (CIN). Importantly, p53 deletion induced both defects in self-renewal and multilineage reconstitution in cyclin E knock-in HSCs with serial transplantation and CIN in hematopoietic stem and progenitor cells. Moreover, CIN was a feature of fatal T-cell malignancies that ultimately developed in recipients of cyclin E(T74A T393A); p53-null HSCs. Together, our findings demonstrate the importance of Fbw7-dependent cyclin E control to the hematopoietic system and highlight CIN as a characteristic feature of HSC dysfunction and malignancy induced by deregulated cyclin E.


Assuntos
Instabilidade Cromossômica/fisiologia , Ciclina E/genética , Ciclina E/metabolismo , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Células-Tronco Hematopoéticas/fisiologia , Linfoma de Células T/etiologia , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Substituição de Aminoácidos , Animais , Linhagem da Célula , Instabilidade Cromossômica/genética , Proteína 7 com Repetições F-Box-WD , Genes p53 , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/patologia , Linfoma de Células T/genética , Linfoma de Células T/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Estabilidade Proteica , Proteólise , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
8.
Cancer Biol Ther ; 7(10): 1630-40, 2008 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-18769124

RESUMO

ErbB2 (or Her2/Neu) overexpression in breast cancer signifies poorer prognosis, yet it has provided an avenue for targeted therapy as demonstrated by the success of the humanized monoclonal antibody Trastuzumab (Herceptin). Resistance to Trastuzumab and eventual failure in most cases, however, necessitate alternate ErbB2-targeted therapies. HSP90 inhibitors such as 17-allylaminodemethoxygeldanamycin (17-AAG), potently downregulate the cell surface ErbB2. While the precise mechanisms of Trastuzumab or 17-AAG action remain unclear, ubiquitinylation-dependent proteasomal or lysosomal degradation of ErbB2 appears to play a substantial role. As Trastuzumab and 17-AAG induce the recruitment of distinct E3 ubiquitin ligases, Cbl and CHIP respectively, to ErbB2, we hypothesized that 17-AAG and Trastuzumab combination could induce a higher level of ubiquitinylation and downregulation of ErbB2 as compared to single drug treatments. We present biochemical and cell biological evidence that combined 17-AAG and Trastuzumab treatment of ErbB2-overexpressing breast cancer cell lines leads to enhanced ubiquitinylation, downregulation from the cell surface and lysosomal degradation of ErbB2. Importantly, combined 17-AAG and Trastuzumab treatment induced synergistic growth arrest and cell death specifically in ErbB2-overexpressing but not in ErbB2-low breast cancer cells. Our results suggest the 17-AAG and Trastuzumab combination as a mechanism-based combinatorial targeted therapy for ErbB2-overexpressing breast cancer patients.


Assuntos
Anticorpos Monoclonais/administração & dosagem , Benzoquinonas/administração & dosagem , Neoplasias da Mama/metabolismo , Lactamas Macrocíclicas/administração & dosagem , Lisossomos/metabolismo , Receptor ErbB-2/metabolismo , Ubiquitina/química , Anticorpos Monoclonais Humanizados , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Morte Celular , Linhagem Celular Tumoral , Citometria de Fluxo , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Microscopia de Fluorescência , Modelos Biológicos , Estrutura Terciária de Proteína , Trastuzumab
9.
Infect Immun ; 76(10): 4677-85, 2008 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-18678662

RESUMO

Enteric bacteria and their products play an important role in intestinal inflammation; however, the complete mechanisms are not elucidated yet. Tumor suppressor Foxo3a regulates gene expression in the nucleus, and its translocation to the cytosol leads to inactivation. Proximally, Foxo3a is regulated by different pathways including the phosphoinositide 3-kinase (PI3K) pathway. The aim of this study was to determine the effect of bacterial infection on Foxo3a in intestinal epithelial cells and to examine the contribution of Foxo3a in intestinal inflammation. Bacterial lipopolysaccharide (LPS) and infection with mouse pathogen Citrobacter rodentium induce translocation of the nuclear Foxo3a into the cytosol, where it degrades in human HT-29 and mouse CMT-93 cells. In colonic epithelia of healthy mice, Foxo3a is localized in the epithelia at the bottom of the crypts in both the nucleus and the cytosol, while in C. rodentium-infected colon Foxo3a is expressed along the crypts and located mainly in the cytosol, suggesting its inactivation. LPS utilized the PI3K pathway to inhibit Foxo3a. Additionally, inhibition of PI3K attenuated LPS-induced proinflammatory interleukin-8 (IL-8). LPS-induced IL-8 is increased in HT-29 cells with silenced Foxo3a. Moreover, in HT-29 cells with silenced Foxo3a, the amount of IkappaBalpha, an NF-kappaB inhibitor, is decreased. In conclusion, LPS and bacterial infection inactivate Foxo3a in intestinal epithelia via the PI3K pathway and inactivated Foxo3a leads to the upregulation of IL-8 by suppressing inhibitory IkappaBalpha.


Assuntos
Citrobacter rodentium/imunologia , Fatores de Transcrição Forkhead/imunologia , Interleucina-8/biossíntese , Lipopolissacarídeos/imunologia , Animais , Colo/imunologia , Colo/microbiologia , Células Epiteliais/imunologia , Células Epiteliais/microbiologia , Proteína Forkhead Box O3 , Fatores de Transcrição Forkhead/metabolismo , Células HT29 , Humanos , Mucosa Intestinal/patologia , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Fosfatidilinositol 3-Quinases/imunologia , Fosfatidilinositol 3-Quinases/metabolismo
10.
J Neurocytol ; 33(3): 331-43, 2004 May.
Artigo em Inglês | MEDLINE | ID: mdl-15475688

RESUMO

Individual neurons dissected from immunohistochemically stained paraffin sections of the developing rat geniculate (VIIth cranial) ganglion were assayed for their content of mRNA of the neurotrophin receptor genes, p75 , trkA , trkB and trkC. Fetal and postnatal rats, from the 13th embryonic day (E13) until the 20th postnatal day (P20), were used. Single cells were subjected to RNA amplification, followed by treatment with reverse transcriptase and DNA amplification by the polymerase chain reaction (PCR). The identity of the PCR products was verified by subcloning and sequencing. A total of 227 neurons were examined, of which 212 (93%) gave a PCR signal for at least one neurotrophin receptor. We found: (1) Approximately half of the neurons expressed more than one receptor. (2) A truncated version of trkB , possessing the ligand-binding region but lacking the tyrosine kinase domain, occurred quite frequently, often in combination with the full-length trkB, with trkA or both. (3) The pattern of staining for trkB-like immunoreactivity was usually predictive that either its full length or truncated mRNA would be present. This was not the case for trkC-like immunoreactivity. Western blots on E15 brain tissue showed no band for full-length trkC ( approximately 150 kDa), suggesting the antibody may have been immunoreactive with a truncated ( approximately 120 kDa) but not a full-length version of the trkC receptor. (4) The pattern of neurotrophin receptor gene expression changed during development. (5) p75 expression occurred infrequently--in only 7 of the 212 neurons that gave a signal for any receptor.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento/genética , Gânglio Geniculado/metabolismo , Neurônios/metabolismo , RNA Mensageiro/metabolismo , Receptores de Fator de Crescimento Neural/genética , Animais , Animais Recém-Nascidos , Gânglio Geniculado/embriologia , Gânglio Geniculado/crescimento & desenvolvimento , Imuno-Histoquímica , Peso Molecular , Mutação/genética , Neurônios/citologia , Estrutura Terciária de Proteína/genética , Ratos , Receptor de Fator de Crescimento Neural , Receptor trkA/genética , Receptor trkA/metabolismo , Receptor trkB/genética , Receptor trkB/metabolismo , Receptor trkC/genética , Receptor trkC/metabolismo , Receptores de Fator de Crescimento Neural/metabolismo
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