RESUMO
Bone marrow (BM) provides chemoprotection for acute lymphoblastic leukemia (ALL) cells, contributing to lack of efficacy of current therapies. Integrin alpha4 (alpha4) mediates stromal adhesion of normal and malignant B-cell precursors, and according to gene expression analyses from 207 children with minimal residual disease, is highly associated with poorest outcome. We tested whether interference with alpha4-mediated stromal adhesion might be a new ALL treatment. Two models of leukemia were used, one genetic (conditional alpha4 ablation of BCR-ABL1 [p210(+)] leukemia) and one pharmacological (anti-functional alpha4 antibody treatment of primary ALL). Conditional deletion of alpha4 sensitized leukemia cell to nilotinib. Adhesion of primary pre-B ALL cells was alpha4-dependent; alpha4 blockade sensitized primary ALL cells toward chemotherapy. Chemotherapy combined with Natalizumab prolonged survival of NOD/SCID recipients of primary ALL, suggesting adjuvant alpha4 inhibition as a novel strategy for pre-B ALL.
Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Resistencia a Medicamentos Antineoplásicos , Proteínas de Fusão bcr-abl/fisiologia , Integrina alfa4/química , Neoplasia Residual/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamento farmacológico , Animais , Medula Óssea/efeitos dos fármacos , Medula Óssea/metabolismo , Medula Óssea/patologia , Adesão Celular , Criança , Citometria de Fluxo , Humanos , Integrases/metabolismo , Integrina alfa4/genética , Integrina alfa4/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Natalizumab , Neoplasia Residual/metabolismo , Neoplasia Residual/mortalidade , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/mortalidade , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Estromais/efeitos dos fármacos , Células Estromais/metabolismo , Células Estromais/patologiaRESUMO
Natural killer (NK) cells are innate lymphocytes with cytotoxic activity. Understanding the factors regulating cytotoxicity is crucial for improving NK-cell adoptive therapies. Here, we studied a previously unknown role of p35 (CDK5R1), a coactivator of cyclin-dependent kinase 5 (CDK5) in NK-cell function. p35 expression was thought to be neuronal-specific and the majority of studies are still focused on neuronal cells. Here, we show that CDK5 and p35 are expressed in NK cells and are kinase-active. NK cells from p35 knockout mice were analyzed and showed significantly increased cytotoxicity against murine cancer cells, while they did not show any differences in cell numbers or maturation stages. We confirmed this using human NK cells transduced with p35 short hairpin RNA (shRNA), showing similar increase in cytotoxicity against human cancer cells. Overexpression of p35 in NK cells resulted in moderate decrease in cytotoxicity, while expressing a kinase-dead mutant of CDK5 displayed increased cytotoxicity. Together, these data suggest that p35 negatively regulates NK-cell cytotoxicity. Surprisingly, we found that TGFß, a known negative regulator of NK-cell cytotoxicity, induces p35 expression in NK cells. NK cells cultured with TGFß exhibit reduced cytotoxicity, while NK cells transduced with p35 shRNA or mutant CDK5 expression exhibited partial reversal of this inhibitory effect pointing to an interesting hypothesis that p35 plays an important role in TGFß-mediated NK-cell exhaustion. Significance: This study reports a role for p35 in NK-cell cytotoxicity and this might help to improve NK-cell adoptive therapy.
Assuntos
Proteínas do Tecido Nervoso , Fator de Crescimento Transformador beta , Animais , Humanos , Camundongos , Células Matadoras Naturais/metabolismo , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Fosfotransferases/metabolismo , RNA Interferente Pequeno , Fator de Crescimento Transformador beta/genéticaRESUMO
Natural killer (NK) cells mediate killing of malignant and virus-infected cells, a property that is explored as a cell therapy approach in the clinic. Various cell intrinsic and extrinsic factors affect NK cell cytotoxic function, and an improved understanding of the mechanism regulating NK cell function is necessary to accomplish better success with NK cell therapeutics. Here, we explored the role of O-GlcNAcylation, a previously unexplored molecular mechanism regulating NK cell function. O-GlcNAcylation is a post-translational modification mediated by O-GlcNAc transferase (OGT) that adds the monosaccharide N-acetylglucosamine to serine and threonine residues on intracellular proteins and O-GlcNAcase (OGA) that removes the sugar. We found that stimulation of NK cells with the cytokines interleukin-2 (IL-2) and IL-15 results in enhanced O-GlcNAcylation of several cellular proteins. Chemical inhibition of O-GlcNAcylation using OSMI-1 was associated with a decreased expression of NK cell receptors (NKG2D, NKG2A, NKp44), cytokines [tumor necrosis factor (TNF)-α, interferon (IFN-γ)], granulysin, soluble Fas ligand, perforin, and granzyme B in NK cells. Importantly, inhibition of O-GlcNAcylation inhibited NK cell cytotoxicity against cancer cells. However, increases in O-GlcNAcylation following OGA inhibition using an OGA inhibitor or shRNA-mediated suppression did not alter NK cell cytotoxicity. Finally, we found that NK cells pretreated with OSMI-1 to inhibit O-GlcNAcylation showed compromised cytotoxic activity against tumor cells in vivo in a lymphoma xenograft mouse model. Overall, this study provides the seminal insight into the role of O-GlcNAcylation in regulating NK cell cytotoxic function.
Assuntos
Acetilglucosamina , Processamento de Proteína Pós-Traducional , Acetilglucosamina/metabolismo , Animais , Citocinas/metabolismo , Humanos , Células Matadoras Naturais/metabolismo , Camundongos , Serina/metabolismoRESUMO
B cell-activating factor (BAFF) binds the three receptors BAFF-R, BCMA, and TACI, predominantly expressed on mature B cells. Almost all B cell cancers are reported to express at least one of these receptors. Here we develop a BAFF ligand-based chimeric antigen receptor (CAR) and generate BAFF CAR-T cells using a non-viral gene delivery method. We show that BAFF CAR-T cells bind specifically to each of the three BAFF receptors and are effective at killing multiple B cell cancers, including mantle cell lymphoma (MCL), multiple myeloma (MM), and acute lymphoblastic leukemia (ALL), in vitro and in vivo using different xenograft models. Co-culture of BAFF CAR-T cells with these tumor cells results in induction of activation marker CD69, degranulation marker CD107a, and multiple proinflammatory cytokines. In summary, we report a ligand-based BAFF CAR-T capable of binding three different receptors, minimizing the potential for antigen escape in the treatment of B cell cancers.
Assuntos
Fator Ativador de Células B/genética , Receptor do Fator Ativador de Células B/genética , Antígeno de Maturação de Linfócitos B/genética , Linfoma de Célula do Manto/terapia , Mieloma Múltiplo/terapia , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Proteína Transmembrana Ativadora e Interagente do CAML/genética , Animais , Antígenos CD/genética , Antígenos CD/imunologia , Antígenos de Diferenciação de Linfócitos T/genética , Antígenos de Diferenciação de Linfócitos T/imunologia , Fator Ativador de Células B/imunologia , Receptor do Fator Ativador de Células B/imunologia , Antígeno de Maturação de Linfócitos B/imunologia , Linfócitos B/imunologia , Linfócitos B/patologia , Linhagem Celular Tumoral , Técnicas de Cocultura , Citotoxicidade Imunológica , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Lectinas Tipo C/genética , Lectinas Tipo C/imunologia , Ativação Linfocitária , Linfoma de Célula do Manto/genética , Linfoma de Célula do Manto/imunologia , Linfoma de Célula do Manto/patologia , Proteína 1 de Membrana Associada ao Lisossomo/genética , Proteína 1 de Membrana Associada ao Lisossomo/imunologia , Masculino , Camundongos , Mieloma Múltiplo/genética , Mieloma Múltiplo/imunologia , Mieloma Múltiplo/patologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/imunologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Ligação Proteica , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/imunologia , Transdução de Sinais , Linfócitos T/imunologia , Linfócitos T/transplante , Proteína Transmembrana Ativadora e Interagente do CAML/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Natural Killer (NK) cells are cytotoxic lymphocytes critical to the innate immune system. We found that germline deficiency of NF-κB c-Rel results in a marked decrease in cytotoxic function of NK cells, both in vitro and in vivo, with no significant differences in the stages of NK cell development. We found that c-Rel binds to the promoters of perforin and granzyme B, two key proteins required for NK cytotoxicity, and controls their expression. We generated a NK cell specific c-Rel conditional knockout to study NK cell intrinsic role of c- Rel and found that both global and conditional c-Rel deficiency leads to decreased perforin and granzyme B expression and thereby cytotoxic function. We also confirmed the role of c-Rel in perforin and granzyme B expression in human NK cells. c-Rel reconstitution rescued perforin and granzyme B expressions in c-Rel deficient NK cells and restored their cytotoxic function. Our results show a previously unknown role of c-Rel in transcriptional regulation of perforin and granzyme B expressions and control of NK cell cytotoxic function.
Assuntos
Citotoxicidade Imunológica , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas c-rel/metabolismo , Animais , Células Cultivadas , Citocinas/biossíntese , Citotoxicidade Imunológica/genética , Granzimas/metabolismo , Humanos , Melanoma Experimental , Camundongos , Camundongos Knockout , Modelos Biológicos , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Proteínas Proto-Oncogênicas c-rel/genéticaRESUMO
Mantle cell lymphoma (MCL) is an aggressive form of B cell non-Hodgkin's lymphoma and remains incurable under current treatment modalities. One of the main reasons for treatment failure is the development of drug resistance. Accumulating evidence suggests that B cell activating factor (BAFF) and BAFF receptor (BAFF-R) play an important role in the proliferation and survival of malignant B cells. High serum BAFF levels are often correlated with poor drug response and relapse in MCL patients. Our study shows that BAFF-R is expressed on both MCL patient cells and cell lines. BAFF-R knockdown leads to MCL cell death showing the importance of BAFF-R signaling in MCL survival. Moderate knockdown of BAFF-R in MCL cells did not affect its viability, but sensitized them to cytarabine treatment in vitro and in vivo, with prolonged mice survival. Anti-BAFF-R antibody treatment promoted drug-induced MCL cell death. Conversely, the addition of recombinant BAFF (rhBAFF) to MCL cells protected them from cytarabine-induced apoptosis. We tested the efficacy of a humanized defucosylated ADCC optimized anti-BAFF-R antibody in killing MCL. Our data show both in vitro and in vivo efficacy of this antibody for MCL therapy. To conclude, our data indicate that BAFF/BAFF-R signaling is crucial for survival and involved in drug resistance of MCL. Targeting BAFF-R using BAFF-R antibody might be a promising therapeutical strategy to treat MCL patients resistant to chemotherapy.
Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Receptor do Fator Ativador de Células B , Linfoma de Célula do Manto , Animais , Apoptose , Fator Ativador de Células B/genética , Receptor do Fator Ativador de Células B/genética , Humanos , Linfoma de Célula do Manto/tratamento farmacológico , Camundongos , Recidiva Local de NeoplasiaRESUMO
Systemic lupus erythematosus (SLE) is an autoimmune disease mediated by T and B cells. It is characterized by a variety of autoantibodies and systemic clinical manifestations. A tolerogenic peptide, designated hCDR1, ameliorated the serological and clinical manifestations of SLE in both spontaneous and induced models of lupus. In the present study, we evaluated the status of mature B cells in the bone marrow (BM) of SLE-afflicted mice, and determined the effect of treatment with the tolerogenic peptide hCDR1 on these cells. We demonstrate herein that mature B cells of the BM of SLE-afflicted (New Zealand Black x New Zealand White)F(1) mice were largely expanded, and that treatment with hCDR1 down-regulated this population. Moreover, treatment with hCDR1 inhibited the expression of the pathogenic cytokines [interferon-gamma and interleukin (IL)-10], whereas it up-regulated the expression of transforming growth factor-beta in the BM. Treatment with hCDR1 up-regulated the rates of apoptosis of mature B cells. The latter was associated with inhibited expression of the survival Bcl-xL gene and of IL-7 by BM cells. Furthermore, the addition of recombinant IL-7 abrogated the suppressive effects of hCDR1 on Bcl-xL in the BM cells and resulted in elevated levels of apoptosis. Hence, the down-regulated production of IL-7 contributes to the hCDR1-mediated apoptosis of mature B cells in the BM of SLE-afflicted mice.
Assuntos
Anticorpos Monoclonais/imunologia , Subpopulações de Linfócitos B/imunologia , Células da Medula Óssea/imunologia , Interleucina-7/antagonistas & inibidores , Lúpus Eritematoso Sistêmico/imunologia , Fragmentos de Peptídeos/imunologia , Animais , Apoptose/imunologia , Modelos Animais de Doenças , Regulação para Baixo/imunologia , Feminino , Tolerância Imunológica/imunologia , Interferon gama/biossíntese , Interleucina-10/biossíntese , Interleucina-7/imunologia , Camundongos , Camundongos Endogâmicos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Baço/imunologia , Fator de Crescimento Transformador beta/biossíntese , Regulação para Cima/imunologiaRESUMO
Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by dysregulated immune responses mediated by T and B cells. A tolerogenic peptide, designated hCDR1, ameliorated the serological and clinical manifestations of SLE in mouse models of lupus. We investigated the role of B-cell activating factor (BAFF) in the beneficial effects of hCDR1. BAFF production was reduced in hCDR1-treated mice in association with diminished production of dsDNA-specific autoantibodies and proteinuria levels. In addition, IFN-gamma and IL-10, which induce BAFF secretion, were down-regulated in hCDR1-treated mice. The reduced levels of BAFF correlated with a lower rate of maturation and differentiation of B cells, and with a decrease in integrin expression and anti-apoptotic gene expression by B cells. Moreover, BAFF signaling through the NF-kB pathways was inhibited in hCDR1-treated mice. Thus, down-regulation of BAFF plays a role in the mechanism of action by which hCDR1 ameliorates lupus manifestations.
Assuntos
Autoantígenos/farmacologia , Fator Ativador de Células B/metabolismo , Lúpus Eritematoso Sistêmico/fisiopatologia , Proteínas do Tecido Nervoso/farmacologia , Peptídeos/farmacologia , Animais , Autoantígenos/genética , Fator Ativador de Células B/sangue , Linfócitos B/citologia , Linfócitos B/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Ensaio de Imunoadsorção Enzimática , Feminino , Integrinas/metabolismo , Interferon gama/metabolismo , Interleucina-10/metabolismo , Lúpus Eritematoso Sistêmico/tratamento farmacológico , Camundongos , Camundongos Endogâmicos BALB C , Proteínas do Tecido Nervoso/genética , Peptídeos/genética , Peptídeos/uso terapêutico , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Baço/efeitos dos fármacos , Baço/metabolismo , Análise de SobrevidaRESUMO
Dietary lectins are carbohydrate-binding proteins found in food sources. We used a panel of seven dietary lectins to analyze cytotoxicity against hematological cancers. Wheat germ agglutinin (WGA), even at low doses, demonstrated maximum toxicity toward acute myeloid leukemia (AML) cells. Using AML cell lines, we show time- and dose-dependent killing by WGA. We also show that low doses of WGA kills primary patient AML cells, irrespective of subtype, with no significant toxicity to normal cells. WGA caused AML cell agglutination, but failed to agglutinate RBC's at this dose. WGA, primarily, binds to N-acetyl-D-glucosamine (GlcNAc) and is also reported to interact with sialic-acid-containing glycoconjugates and oligosaccharides. After neuraminidase pre-treatment, which catalyzes the hydrolysis of terminal sialic acid residues, AML cells were less sensitive to WGA-induced cell death. AML cells were also not sensitive to succinyl-WGA, which does not react with sialic acid. Incubation with LEL lectin, which recognizes GlcNAc or SNA, which binds preferentially to sialic acid attached to terminal galactose in α-2,6 and to a lesser degree α-2,3 linkage, did not alter AML cell viability. These data indicate that WGA-induced AML cell death is dependent on both GlcNAc binding and interaction with sialic acids. We did not observe any in vitro or in vivo toxicity of WGA toward normal cells at the concentrations tested. Finally, low doses of WGA injection demonstrated significant in vivo toxicity toward AML cells, using xenograft mouse model. Thus, WGA is a potential candidate for leukemia therapy.
RESUMO
Drug-resistant acute lymphoblastic leukemia (ALL) patients do not respond to standard chemotherapy, and an urgent need exists to develop new treatment strategies. Our study exploited the presence of B-cell activating factor receptor (BAFF-R) on the surface of drug-resistant B-ALL cells as a therapeutic target. We used anti-BAFF-R (VAY736), optimized for natural killer (NK) cell-mediated antibody-dependent cellular cytotoxicity (ADCC), to kill drug-resistant ALL cells. VAY736 antibody and NK cell treatments significantly decreased ALL disease burden and provided survival benefit in vivo However, if the disease was advanced, the ADCC efficacy of NK cells was inhibited by microenvironmental transforming growth factor-beta (TGFß). Inhibiting TGFß signaling in NK cells using the TGFß receptor 1 (R1) inhibitor (EW-7197) significantly enhanced VAY736-induced NK cell-mediated ALL killing. Our results highlight the potential of using a combination of VAY736 antibody with EW-7197 to treat advance-stage, drug-resistant B-ALL patients.
Assuntos
Compostos de Anilina/farmacologia , Anticorpos Monoclonais Humanizados/farmacologia , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células Matadoras Naturais/efeitos dos fármacos , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Triazóis/farmacologia , Adulto , Animais , Apoptose , Receptor do Fator Ativador de Células B/antagonistas & inibidores , Proliferação de Células , Quimioterapia Combinada , Feminino , Seguimentos , Humanos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Leucemia-Linfoma Linfoblástico de Células Precursoras/imunologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Prognóstico , Receptor do Fator de Crescimento Transformador beta Tipo I/antagonistas & inibidores , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
In vitro and in vivo studies implicate a series of cytokines in regulation of lymphohematopoiesis. However, direct indications for a local role of most of these cytokines within the bone marrow is lacking. In the present study, we aimed to test the contribution of a specific cytokine, activin A, a member of the transforming growth factor-beta (TGF-beta) family, to lymphohematopoiesis in mouse bone marrow. We show that mouse embryonic fibroblasts (MEFs) are indistinguishable from multipotent stromal cells (MSCs). Such MEFs overexpressing activin A, supported in vitro myelopoiesis in long-term bone marrow cultures as effectively as control MEFs. In contrast, activin A-overexpressing MEFs interfered with the in vitro generation of B lineage cells in such cultures. Thus, excessive expression in vitro of activin A, by supportive stromal cells, causes preferential maturation of myeloid rather than lymphoid cells. Moreover, the activin A-overexpressing MEFs caused an increased incidence in vivo of relatively immature B lineage cells; upon transplantation through the spleen route, MEFs engrafted the bone marrow specifically. Activin A-overexpressing MEFs accumulated in the bone marrow compartment and slowed down the progression of B cell precursors along the differentiation pathway, while sparing the myeloid population. The assay system described in this paper provides a means to assess the contribution of a wide range of molecules to hematopoiesis without perturbing the constitution of other organs.
Assuntos
Ativinas/genética , Linfócitos B/citologia , Medula Óssea , Linfopoese/genética , Células Estromais/metabolismo , Ativinas/fisiologia , Animais , Técnicas de Cultura de Células , Diferenciação Celular , Linhagem da Célula , Células Cultivadas , Células-Tronco Embrionárias , Feminino , Fibroblastos , Expressão Gênica , Camundongos , Células-Tronco MultipotentesRESUMO
Treatment for acute myeloid leukemia (AML) has remained unchanged for past 40 years. Targeting cell metabolism is a promising avenue for future cancer therapy. We found that enzymes involved in metabolic hexosamine biosynthetic pathway (HBP) are increased in patients with AML. Inhibiting GFAT (the rate-limiting enzyme of HBP) induced differentiation and apoptosis in AML cells, sparing normal cells. UDP-GlcNAc, the end product of HBP, is the substrate for O-GlcNAcylation, a posttranslational modification. O-GlcNAc transferase (OGT) is the enzyme which transfers GlcNAc from UDP-GlcNAc to target proteins. Inhibition of O-GlcNAcylation, using OGT inhibitors as well as genetic knockdown of OGT, also led to cell differentiation and apoptosis of AML cells. Finally, HBP inhibition in vivo reduced the tumor growth in a subcutaneous AML xenograft model and tumor cells showed signs of differentiation in vivo A circulating AML xenograft model also showed clearance of tumor load in bone marrow, spleen, and blood, after HBP inhibition, with no signs of general toxicity. This study reveals an important role of HBP/O-GlcNAcylation in keeping AML cells in an undifferentiated state and sheds light into a new area of potential AML therapy by HBP/O-GlcNAc inhibition. Mol Cancer Ther; 17(10); 2226-37. ©2018 AACR.
Assuntos
Vias Biossintéticas/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Hexosaminas/biossíntese , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Morte Celular , Linhagem Celular Tumoral , Modelos Animais de Doenças , Humanos , Camundongos , Fosforilação , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
STUDY OBJECTIVE: To investigate the immunohistochemical localization of betaA subunit of activin A in human term placenta, as a marker for placental infection/inflammation and elevated temperature, in parturients laboring during two analgesic regimens. DESIGN: Prospective, randomized controlled study. SETTING: Delivery room. PATIENTS: 56 healthy, ASA physical status I and II primiparous women in labor. INTERVENTIONS: Parturients were assigned to receive patient-controlled epidural analgesia (PCEA) with 0.2% ropivacaine or patient-controlled intravenous analgesia PCA with meperidine. MEASUREMENTS: Histologic and immunohistochemical placental evaluation for white blood cell infiltration and activin betaA staining were made. Maternal temperature elevation above 37.6 degrees C and leukocytosis above 15,000/microL were recorded. MAIN RESULTS: Temperature was not significantly increased in parturients receiving PCEA over those who received (PCA) with meperidine (31% vs 11%, respectively; P = 0.1). There was also no association between temperature elevation during epidural analgesia and increased white blood cell count (>15,000/microL) or presence of polymorphonuclear and/or lymphocyte aggregation in the placenta. Immunohistochemical staining with antisera against the betaA subunit of activin was present mainly in the placental cytotrophoblast, syncytiotrophoblast, and vascular endothelium, and was not associated with an increase in maternal temperature. No significant difference was noted between the two analgesic techniques with regard to maternal temperature elevation. Intrapartum temperature elevation was not associated with histologic signs of placental inflammation or with expression of activin betaA in the placenta. CONCLUSION: Other mechanisms may be involved in the etiology of temperature elevation during labor.
Assuntos
Analgesia Epidural/efeitos adversos , Analgesia Obstétrica/efeitos adversos , Corioamnionite/diagnóstico , Febre/etiologia , Subunidades beta de Inibinas/análise , Placenta/química , Adulto , Analgesia Controlada pelo Paciente/efeitos adversos , Analgésicos Opioides , Biomarcadores/análise , Corioamnionite/patologia , Feminino , Humanos , Meperidina , Placenta/patologia , Gravidez , Estudos ProspectivosRESUMO
The nuclear factor-κB protein c-Rel plays a critical role in controlling autoimmunity. c-Rel-deficient mice are resistant to streptozotocin-induced diabetes, a drug-induced model of autoimmune diabetes. We generated c-Rel-deficient NOD mice to examine the role of c-Rel in the development of spontaneous autoimmune diabetes. We found that both CD4(+) and CD8(+) T cells from c-Rel-deficient NOD mice showed significantly decreased T-cell receptor-induced IL-2, IFN-γ, and GM-CSF expression. Despite compromised T-cell function, c-Rel deficiency dramatically accelerated insulitis and hyperglycemia in NOD mice along with a substantial reduction in T-regulatory (Treg) cell numbers. Supplementation of isogenic c-Rel-competent Treg cells from prediabetic NOD mice reversed the accelerated diabetes development in c-Rel-deficient NOD mice. The results suggest that c-Rel-dependent Treg cell function is critical in suppressing early-onset autoimmune diabetogenesis in NOD mice. This study provides a novel natural system to study autoimmune diabetes pathogenesis and reveals a previously unknown c-Rel-dependent mechanistic difference between chemically induced and spontaneous diabetogenesis. The study also reveals a unique protective role of c-Rel in autoimmune diabetes, which is distinct from other T-cell-dependent autoimmune diseases such as arthritis and experimental autoimmune encephalomyelitis, where c-Rel promotes autoimmunity.
Assuntos
Diabetes Mellitus Tipo 1/metabolismo , NF-kappa B/deficiência , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas c-rel/deficiência , Proteínas Proto-Oncogênicas c-rel/metabolismo , Animais , Linfócitos T CD4-Positivos/metabolismo , Proliferação de Células/genética , Proliferação de Células/fisiologia , Citocinas/metabolismo , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/imunologia , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Knockout , NF-kappa B/genética , Pâncreas/metabolismo , Proteínas Proto-Oncogênicas c-rel/genética , Reação em Cadeia da Polimerase em Tempo Real , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismoRESUMO
Natural killer cells from acute myeloid leukaemia patients (AML-NK) show a dramatic impairment in cytotoxic activity. The exact reasons for this dysfunction are not fully understood. Here we show that the glycogen synthase kinase beta (GSK3ß) expression is elevated in AML-NK cells. Interestingly, GSK3 overexpression in normal NK cells impairs their ability to kill AML cells, while genetic or pharmacological GSK3 inactivation enhances their cytotoxic activity. Mechanistic studies reveal that the increased cytotoxic activity correlates with an increase in AML-NK cell conjugates. GSK3 inhibition promotes the conjugate formation by upregulating LFA expression on NK cells and by inducing ICAM-1 expression on AML cells. The latter is mediated by increased NF-κB activation in response to TNF-α production by NK cells. Finally, GSK3-inhibited NK cells show significant efficacy in human AML mouse models. Overall, our work provides mechanistic insights into the AML-NK dysfunction and a potential NK cell therapy strategy.
Assuntos
Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Células Matadoras Naturais/imunologia , Leucemia Mieloide Aguda/imunologia , Aminofenóis/química , Aminofenóis/farmacologia , Animais , Microambiente Celular , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Compostos Heterocíclicos com 3 Anéis/química , Compostos Heterocíclicos com 3 Anéis/farmacologia , Imunoterapia , Molécula 1 de Adesão Intercelular/metabolismo , Maleimidas/química , Maleimidas/farmacologia , Camundongos , NF-kappa B/metabolismo , Tiadiazóis/química , Tiadiazóis/farmacologia , Fator de Necrose Tumoral alfaRESUMO
The mesenchyme plays a crucial regulatory role in organ formation and maintenance. However, comprehensive molecular characterization of these cells is lacking. We found unexpectedly that primary mesenchyme, as well as mesenchymal cell clones, express T cell receptor (TCR)alphabeta mRNAs, lacking the variable region. Immunological and genetic evidence support the expression of a corresponding TCRbeta protein. Additionally, mRNAs encoding TCR complex components including CD3 and zeta chain are present. A relatively higher expression of the mesenchymal TCRbeta mRNA by cultured mesenchymal cell clones correlates with fast growth, whereas poorly expressing cells are slow growers and are contact inhibited. The clones that express relatively higher amount of the TCR mRNA exhibit an increased capacity to form tumors in nude mice. However, the expression of this mRNA in the mesenchyme is not per se leading to tumorigenesis, as demonstrated by primary mesenchyme that does not form tumors in mice while expressing moderate amounts of the TCR transcripts. The expression of mesencymal TCRbeta was confined to the G2/M phases of the cell cycle in the MBA-13 mesenchymal cell line. This cell cycle dependent expression, considered together with the correlation between growth properties and the level of TCR expression by cell clones, implies association of mesenchymal TCR with cell growth control.
Assuntos
Mesoderma/citologia , Mesoderma/metabolismo , Receptores de Antígenos de Linfócitos T/genética , Animais , Ciclo Celular , Divisão Celular , Linhagem Celular , Citometria de Fluxo , Perfilação da Expressão Gênica , Células HeLa , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Transplante de Neoplasias , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Células Tumorais CultivadasRESUMO
The negative control of B cell generation is only partially resolved. We assessed the role of activin A in regulation of B lymphopoiesis in view of its specific inhibitory effects on tumor B lineage cells. Activin A is constitutively expressed in mouse hemopoietic organs and in cultured mesenchymal cell lines. We observed an inverse relationship between activin A titer and B lineage cell production. In the spleen, the red pulp exhibited a relatively higher abundance of the protein as compared with the lymphoid follicles, wherein B cell accumulation occurs. Furthermore, a specific shut off in activin A expression was observed in bone marrow and spleen following in vivo induction of B lymphocyte polyclonal activation. We further substantiated these in vivo observations by in vitro studies of primary bone marrow cultures, in which the expression of functional activin A was found to be diminished prior to the onset of B lymphopoiesis. The reduction in functional activin A is shown to concomitantly occur with spontaneous induction of the expression of activin A specific inhibitors. We therefore propose that the mesenchymal organ stroma expresses activin A that negatively controls B cell lymphopoiesis.
Assuntos
Ativinas/metabolismo , Linfócitos B/citologia , Subunidades beta de Inibinas/metabolismo , Linfopoese , Células Estromais/metabolismo , Ativinas/genética , Animais , Medula Óssea/metabolismo , Células Cultivadas , Feminino , Citometria de Fluxo , Perfilação da Expressão Gênica , Subunidades beta de Inibinas/genética , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB C , Especificidade de Órgãos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Baço/metabolismo , Fatores de TempoRESUMO
B-cell activating factor receptor (BAFF-R) is expressed on precursor B acute lymphoblastic leukemia (pre-B ALL) cells, but not on their pre-B normal counterparts. Thus, selective killing of ALL cells is possible by targeting this receptor. Here, we have further examined therapeutic targeting of pre-B ALL based on the presence of the BAFF-R. Mouse pre-B ALL cells lacking BAFF-R function had comparable viability and proliferation to wild-type cells, but were more sensitive to drug treatment in vitro. Viability of human pre-B ALL cells was further reduced when antibodies to the BAFF-R were combined with other drugs, even in the presence of stromal protection. This indicates that inhibition of BAFF-R function reduces fitness of stressed pre-B ALL cells. We tested a novel humanized anti-BAFF-R monoclonal antibody optimalized for FcRγIII-mediated, antibody-dependent cell killing by effector cells. Antibody binding to human ALL cells was inhibitable, in a dose-dependent manner, by recombinant human BAFF. There was no evidence for internalization of the antibodies. The antibodies significantly stimulated natural killer cell-mediated killing of different human patient-derived ALL cells. Moreover, incubation of such ALL cells with these antibodies stimulated phagocytosis by macrophages. When this was tested in an immunodeficient transplant model, mice that were treated with the antibody had a significantly decreased leukemia burden in bone marrow and spleen. In view of the restricted expression of the BAFF-R on normal cells and the multiple anti-pre-B ALL activities stimulated by this antibody, a further examination of its use for treatment of pre-B ALL is warranted.
Assuntos
Anticorpos Monoclonais Humanizados/administração & dosagem , Receptor do Fator Ativador de Células B/imunologia , Imunoterapia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamento farmacológico , Animais , Receptor do Fator Ativador de Células B/antagonistas & inibidores , Linhagem Celular Tumoral , Humanos , Fragmentos Fc das Imunoglobulinas/administração & dosagem , Fragmentos Fc das Imunoglobulinas/imunologia , Camundongos , Terapia de Alvo Molecular , Leucemia-Linfoma Linfoblástico de Células Precursoras B/imunologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologiaRESUMO
The development of resistance to chemotherapy is a major cause of relapse in acute lymphoblastic leukemia (ALL). Though several mechanisms associated with drug resistance have been studied in detail, the role of carbohydrate modification remains unexplored. Here, we investigated the contribution of 9-O-acetylated N-acetylneuraminic acid (Neu5Ac) to survival and drug resistance development in ALL cells. A strong induction of 9-O-acetylated Neu5Ac including 9-O-acetyl GD3 was detected in ALL cells that developed resistance against vincristine or nilotinib, drugs with distinct cytotoxic mechanisms. Removal of 9-O-acetyl residues from Neu5Ac on the cell surface by an O-acetylesterase made ALL cells more vulnerable to such drugs. Moreover, removal of intracellular and cell surface-resident 9-O-acetyl Neu5Ac by lentiviral transduction of the esterase was lethal to ALL cells in vitro even in the presence of stromal protection. Interestingly, expression of the esterase in normal fibroblasts or endothelial cells had no effect on their survival. Transplanted mice induced for expression of the O-acetylesterase in the ALL cells exhibited a reduction of leukemia to minimal cell numbers and significantly increased survival. This demonstrates that Neu5Ac 9-O-acetylation is essential for survival of these cells and suggests that Neu5Ac de-O-acetylation could be used as therapy to eradicate drug-resistant ALL cells.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Ácido N-Acetilneuramínico/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Acetilação , Animais , Antineoplásicos Fitogênicos/uso terapêutico , Antineoplásicos Fitogênicos/urina , Sobrevivência Celular , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Masculino , Camundongos , Ácido N-Acetilneuramínico/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Pirimidinas/uso terapêutico , Vincristina/uso terapêuticoRESUMO
B-lineage acute lymphoblastic leukemia (ALL) arises by transformation of a progenitor (pre-B) cell. Cure rates in adults remain low and treatment is complicated by support provided by the microenvironment to the leukemic cells, indicating an urgent need to better understand the factors that promote their survival. B-cell-activating factor (BAFF) and its receptor BAFF-R are important for survival and growth of mature normal and malignant B cells but are not expressed on pre-B cells. Unexpectedly, all cells in the primary Philadelphia chromosome (Ph)-positive and Ph-negative ALL samples tested were positive for high BAFF-R cell surface expression. BAFF-R was fully competent to bind BAFF, and stimulation of the receptor activated both the classic and the noncanonical NF-kappaB pathways. Recombinant BAFF supported survival of the ALL cells in the absence of stroma, and it significantly attenuated the rate of apoptosis caused by exposure to nilotinib, a drug used therapeutically to treat Ph-positive ALLs. Surprisingly, BAFF mRNA and protein were also expressed in the same cells but BAFF was not shed into the medium. Our report is the first showing universal expression of BAFF-R by pre-B ALL cells and opens the possibility of blocking its function as an adjuvant therapeutic strategy.